International Review of Research in Mental Retardation: Neurotoxicity and Developmental Disabilities

Neurodevelopmental Effects of Alcohol THOMAS M. BURBACHER AND KIMBERLY S. GRANT DEPARTMENT OF ENVIRONMENTAL AND OCCUPATI...

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Neurodevelopmental Effects of Alcohol THOMAS M. BURBACHER AND KIMBERLY S. GRANT DEPARTMENT OF ENVIRONMENTAL AND OCCUPATIONAL HEALTH SCIENCES SCHOOL OF PUBLIC HEALTH AND COMMUNITY MEDICINE WASHINGTON NATIONAL PRIMATE RESEARCH CENTER AND CENTER ON HUMAN DEVELOPMENT AND DISABILITY UNIVERSITY OF WASHINGTON SEATTLE, WASHINGTON

I.

INTRODUCTION

The goal of this chapter is to provide an overview of the developmental eVects of prenatal exposure to alcohol in the forms of ethanol and methanol. All alcohols have a similar chemical structure. The three most commonly known alcohols are ethyl alcohol (ethanol), methyl alcohol (methanol), and isopropyl alcohol (isopropanol). Isopropanol is better known as rubbing alcohol, a common item in most American homes. Human exposures to toxic levels of isopropanol are uncommon and have not been reported in pregnant women. Health eVects data from animal studies suggest that isopropanol has low acute and chronic toxicity and is not a teratogen or developmental neurotoxicant (Kapp et al., 1996). Most research on the fetal eVects of maternal alcohol exposure has focused on the compounds ethanol and methanol. The physical chemical properties of these two agents are displayed in Table I. Methanol (H3C–OH), a component of many products, including alternative motor fuels, antifreeze, glass cleaner, paints, and varnishes, is the simplest alcohol with a chain consisting of a carbon atom with three hydrogen atoms attached. Ethanol (H3C–CH2–OH), the psychoactive ingredient in alcoholic beverages that results in intoxication, has a chain that is two times as long. While similar exposure scenarios exist for these two compounds (occupational exposure, intentional ingestion), the primary routes of exposure that are related to developmental eVects are quite diVerent. Ethanol is an ancient drug that is widely accepted throughout the world, consumed in the form of alcoholic beverages to achieve a pleasant state of euphoria or relaxation. Although women working in professions such as nurses, assemblers, janitors, INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0074-7750(05)30001-2

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Thomas M. Burbacher and Kimberly S. Grant TABLE I PHYSICAL CHEMICAL PROPERTIES a AND METHANOL

OF

ETHANOL

Name

Methanol

Ethanol

Synonyms Structure Formula CAS RN Molecular weight Melting point Boiling point Flash point Physical appearance

Methyl alcohol, wood alcohol H3C–OH CH4O 67‐56‐1 32.04 98 C 64.6 C 12 C Clear, colorless, very mobile, flammable liquid with pungent, slightly alcoholic odor Miscible with water and most other organic solvents Industrial and laboratory solvent, to denature ethanol, chemical reagent, antifreeze octane booster in gasoline, requisite for hydrogen fuel cell technology

Ethyl alcohol, alcohol H3CCH2OH C2H6O 64‐17‐5 46.07 114 C 78.5 C 13 C Clear, colorless, very mobile, flammable liquid with pleasant alcoholic odor and burning taste Miscible with water and most other organic solvents Alcoholic beverages Industrial and laboratory solvent In pharmaceutical preparations and perfumes Antiseptic Octane booster in gasoline

Miscibility Principal uses

a From ‘‘The Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals’’ (2001). Merck & Co., Inc, Whitehouse Station, New Jersey.

clinical lab technicians, and housekeepers can also be exposed to ethanol (Seta et al., 1988), a recent review found that low blood alcohol levels resulting from occupational exposure do not represent a risk to pregnant women (Irvine, 2003). In contrast, exposure to methanol occurs almost exclusively in industrial and laboratory settings. Women working as assemblers, janitors, clinical laboratory technicians, machine operators, and mechanics can be exposed to methanol via dermal absorption and inhalation (Seta et al., 1988). Since 1988, methanol has received attention as a low‐emission, high‐performance motor fuel and the primary fuel source for vehicles powered by hydrogen‐based fuel cell technology (Gold & Moulis, 1988; Fuller et al., 1997). If methanol‐based fuels were used on a widespread basis in the future, there would be public exposure on streets, refueling stations, and garages. This chapter draws from both the human and animal literature to explore the consequences of prenatal exposure to ethanol and methanol on the behavioral development of exposed

NEURODEVELOPMENTAL EFFECTS OF ALCOHOL

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oVspring. For ethanol, the weight of evidence to determine the risk of adverse eVects on development from prenatal exposure comes largely from prospective, longitudinal studies of human maternal–infant pairs. While a large volume of supportive studies using animal models does exist for ethanol, a comprehensive review of these studies was considered beyond the scope of this chapter. Excellent reviews of the teratogenic eVects of alcohol using animal models are available (see Guerri & Hannigan, 1996). For methanol, few reports are available describing exposure eVects in human infants. The weight of evidence to determine the risk of adverse eVects on development from prenatal exposure to methanol, therefore, comes largely from studies using animal models.

II.

ETHANOL

Ethanol (EtOH) is a small molecule compound that is soluble in water and lipids, easily passing through cell membranes in the body (Ramchandani et al., 2001). Ethanol selectively concentrates in highly vascularized organs such as the lungs and the brain and these organs have higher EtOH concentrations after exposure. Metabolism of EtOH is dependent on enzymes in the liver that initiate its metabolic breakdown. Ethanol is metabolized by the enzyme alcohol dehydrogenase (ADH) to acetaldehyde, which, in turn, is metabolized by the enzyme, aldehyde dehydrogenase, to acetic acid, and ultimately to water and carbon dioxide. Women metabolize EtOH diVerently from men and have higher blood levels of EtOH due to higher fat content, smaller body size, and less gastric‐ADH activity (Frezza et al., 1990; Seitz et al., 1993). In general, women are at increased risk for EtOH‐induced brain injury (see review by Prendergast, 2004). Female alcoholics show measurable brain shrinkage after shorter periods of EtOH exposure than male subjects (Mann et al., 1992). The enhanced sensitivity of women to the adverse eVects of alcoholism is not limited to the brain but includes higher rates of advanced liver disease and other EtOH‐related disorders (Morgan & Sherlock, 1977). Prenatal exposure to EtOH is the leading cause of preventable birth defects and mental retardation in the United States, if not the world. The consumption of beer, wine, or spirits during pregnancy can have a profound impact on the processes of normal child development. The eVects of EtOH are dose‐dependent and children born to alcoholic or EtOH‐abusing mothers are at the highest risk for poor developmental outcome (Stratton et al., 1996). Developmental eVects are widespread and range from structural malformations (skeleton, heart, kidney) to delays in physical growth and deficits in neurobehavioral development (learning, memory, language,

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Thomas M. Burbacher and Kimberly S. Grant

and social behavior) (American Academy of Pediatrics, 2000; Mattson & Riley, 1998; Mattson et al., 2001). The United States has made great strides in educating women of reproductive age about the dangers of drinking EtOH during pregnancy. The rate of drinking among pregnant women in the United States declined from 16.3% in 1995 to 12.8% in 1999 (MMWR, 2002). The rate of binge‐drinking and frequent EtOH abuse rates, however, remained stable during the same period (2.7 and 3.3%, respectively). A.

Historical Perspective

Throughout history, maternal drinking during pregnancy has been suspected of causing adverse eVects in exposed oVspring. Mentioned by the Hebrews in the Book of Judges 13:4, the harmful eVects of drinking have been known since biblical times when couples were forbidden to drink wine on their wedding night so that aVected infants would not be conceived (Haggard & Jellinek, 1942). A report on drunkenness to the British House of Commons in the 1800s indicated that oVspring of alcoholic women were frequently ‘‘born weak and silly . . . shriveled and old, as though they had numbered many years’’ (Goodacre, 1965). In 1900, a published report noted that alcoholic women had increased rates of spontaneous miscarriage and delivery of stillborn infants and exposed oVspring were at high risk for epilepsy (Sullivan, 1900). Although historical writings warned of the dangers of drinking during pregnancy, physicians during the post‐prohibition era in the United States dismissed these concerns as moralism and interest in the subject sharply declined (Warner & Rosett, 1975). In the late 1960s, a team of French investigators published a report indicating that children born to alcoholic mothers shared a number of distinguishing physical characteristics (Lemoine et al., 1968). International attention was not, however, directed at this constellation of birth defects until the clinical observations of Jones and Smith were published in 1973. These investigators coined the term ‘‘fetal alcohol syndrome’’ (FAS) to describe the prenatal and postnatal growth deficiencies and physical malformations observed in infants born to alcoholic mothers. The death of one of the study subjects allowed for the first necropsy of an FAS infant and neuropathology results confirmed that there was severe damage to both neuronal and glial cells as well as absence of the corpus callosum. The pioneering work of these Seattle dysmorphologists triggered new clinical and scientific interest in EtOH as a serious teratogen and prospective research programs were initiated in the cities of Seattle, Detroit, Cleveland, Atlanta, and Pittsburgh (Streissguth et al., 1981; Jacobson et al., 1993a; Greene et al., 1991a; Coles et al., 1991; Day et al., 1989).


B.

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Clinical Features of Fetal Alcohol Syndrome and Related Disorders

The most serious outcome for the children of drinking mothers is fetal alcohol syndrome (FAS). As detailed in the 1996 report from the Institute of Medicine (IOM) (Stratton et al., 1996), clinical diagnosis of this syndrome is based on three required criteria: (1) a deficiency in prenatal and postnatal physical growth, (2) impairment of the central nervous system (CNS), and (3) specific craniofacial malformations. The diagnostic criteria for FAS and alcohol‐related eVects from the 1996 IOM report are displayed in Table II. The diagnosis of FAS requires that the height, weight, and/or length of the child be less than the 10th percentile. Physical growth in FAS infants is characterized by growth retardation and may include reductions in birth weight, loss of weight over time not associated with nutrition, and body weight that is disproportional to height, length, and head circumference. Damage to the CNS is required for the FAS diagnosis and eVects in oVspring include mental retardation, neurological abnormalities, or developmental delays. The diagnosis of FAS also requires the presence of a distinct facial dysmorphology, often involving abnormalities of the eyes and the nose (see Figs. 1 and 2). In addition to microcephaly, facial features of the syndrome include short palpebral fissures, flattened midface, indistinct philtrum, thin upper lip, and upturned nose (Abel, 1984). Common features of the FAS face that are not required for a formal diagnosis include epicanthal folds, low nasal bridge, minor ear anomalies, and micrognathia. In addition to the diagnostic criteria set forth in the 1996 IOM report, a second set of clinical diagnostic criteria have been published by Astley and Clarren (2000). This system is known as the Washington criteria and is based on a four‐digit code that corresponds to the requisite diagnostic features of FAS. While vastly helpful in accurate diagnoses of aVected infants, a number of weaknesses with both coding schemes have been pointed out (Hoyme et al., 2005). Limitations include failing to adequately integrate the family/genetic history of the child into the criteria, confusing terminology, and inadequate definitions of clinical diagnoses (encephalopathy, neurobehavioral disorder). Hoyme and colleagues (2005) have proposed a clarification of the 1996 IOM criteria to enhance the identification and treatment of children with fetal alcohol spectrum disorders. These changes allow for more accurate diagnoses in routine clinical settings. Many children born to mothers who drink heavily during pregnancy do not meet the criteria for a formal diagnosis of FAS. It is now well recognized that there can be significant behavioral changes in children born to drinking

6 DIAGNOSTIC CRITERIA

Thomas M. Burbacher and Kimberly S. Grant TABLE II* FETAL ALCOHOL SYNDROME (FAS) RELATED EFFECTS (IOM, 1996)

FOR

AND

ALCOHOL‐

Fetal Alcohol Syndrome 1. FAS with confirmed maternal alcohol exposurea A. Confirmed maternal alcohol exposurea B. Evidence of a characteristic pattern of facial anomalies that includes features such as short palpebral fissures and abnormalities in the premaxillary zone (flat upper lip, flattened philtrum, and flat midface) C. Evidence of growth retardation, as in at least one of the following: ‐ low birth weight for gestational age ‐ decelerating weight over time not due to nutrition ‐ disproportional low weight to height D. Evidence of CNS neurodevelopmental abnormalities, as in at least one of the following: ‐ decreased cranial size at birth ‐ structural brain abnormalities (microcephaly, partial or complete agenesis of the corpus callosum, cerebellar hypoplasia) ‐ neurological hard or soft signs (as age‐appropriate), such as impaired fine motor skills, neurosensory hearing loss, poor tandem gait, poor eye–hand coordination 2. FAS without confirmed maternal alcohol exposure B, C, and D as previously stated 3. Partial FAS with confirmed maternal alcohol exposure A. Confirmed maternal alcohol exposurea B. Evidence of some components of the pattern of characteristic facial anomalies Either C or D or E C. Evidence of growth retardation, as in at least one of the following: ‐ low birth weight for gestational age ‐ decelerating weight over time not due to nutrition ‐ disproportional low weight to height D. Evidence of CNS neurodevelopmental abnormalities, as in: ‐ decreased cranial size at birth ‐ structural brain abnormalities (microcephaly, partial or complete agenesis of the corpus callosum, cerebellar hypoplasia) ‐ neurological hard or soft signs (as age‐appropriate), such as impaired fine motor skills, neurosensory hearing loss, poor tandem gait, poor eye–hand coordination E. Evidence of a complex pattern of behavior or cognitive abnormalities that are inconsistent with developmental level and cannot be explained by familial background or environment alone, such as learning diYculties; deficits in school performance; poor impulse control; problems in social perception; deficits in higher‐level receptive and expressive language; poor capacity for abstraction or metacognition; specific deficits in mathematical skills; or problems in memory, attention, or judgment. Alcohol‐Related EVects Clinical conditions in which there is a history of maternal alcohol exposure,a,b and where clinical or animal research has linked maternal alcohol ingestion to an observed outcome. There are two categories, which may co‐occur. If both diagnoses are present, then both diagnoses should be rendered:


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TABLE II (continued ) 4. Alcohol‐related birth defects (ARBD) List of congenital anomalies, including malformations and dysplasias Cardiac Atrial septal defects Aberrant great vessels Ventricular septal defects Tetralogy of Fallot Skeletal Hypoplastic nails Clinodactyly Shortened fifth digits Pectus excavatum and carinatum Radioulnar synostosis Klippel–Feil syndrome Flexion contractures Hemivertebrae Camptodactyly Scoliosis Renal Aplastic, dysplastic, hypoplastic kidneys Ureteral duplications Horseshoe kidneys Hydronephrosis Ocular Strabismus Refractive problems secondary to small globes Retinal vascular anomalies Auditory Conductive hearing loss Neurosensory hearing loss Other Virtually every malformation has been described in some patient with FAS. The etiologic specificity of most of these anomalies to alcohol teratogenesis remains uncertain. 5. Alcohol‐related neurodevelopmental disorder (ARND) Presence of: A. Evidence of CNS neurodevelopmental abnormalities, as in any one of the following: ‐ decreased cranial size at birth ‐ structural brain abnormalities (microcephaly, partial or complete agenesis of the corpus callosum, cerebellar hypoplasia) ‐ neurological hard or soft signs (as age‐appropriate), such as impaired fine motor skills, neurosensory hearing loss, poor tandem gait, poor eye–hand coordination and/or: B. Evidence of a complex pattern of behavior or cognitive abnormalities that are inconsistent with developmental level and cannot be explained by familial background or environment alone, such as learning diYculties; deficits in school performance; poor impulse control; problems in social perception; deficits in higher‐level receptive and expressive language; poor capacity for abstraction or metacognition; specific deficits in mathematical skills; or problems in memory, attention, or judgment. a

A pattern of excessive intake characterized by substantial, regular intake, or heavy episodic drinking. Evidence of this pattern may include frequent episodes of intoxication, development of tolerance or withdrawal, social problems related to drinking, legal problems related to drinking, engaging in physically hazardous behavior while drinking, or alcohol‐related medical problems, such as hepatic disease. b As further research is completed and as, or if, lower quantities or variable patterns of alcohol use are associated with ARBD or ARND, these patterns of alcohol use should be incorporated into the diagnostic criteria. *Reprinted with permission from ‘‘Fetal Alcohol Syndrome: Diagnosis, Epidemiology, Prevention, and Treatment’’ (1996) by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC.

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FIG. 1. Facial features associated with fetal alcohol syndrome in the young child. Reprinted with permission from Streissguth, A. P., & Little, R. E. (1994). ‘‘Unit 5 Alcohol, Pregnancy, and Fetal Alcohol Syndrome: Second Edition’’ of the Project Cork Institute Medical School Curriculum (slide lecture series) on Biomedical Education: Alcohol Use and Its Medical Consequences, produced by Dartmouth Medical School.

FIG. 2. Infants and young children with fetal alcohol syndrome. Reprinted with permission from Streissguth, A. P., Landesman‐Dwyer, S., Martin, D. C., & Smith, D. W. (1980). Teratogenic eVects of alcohol in humans and laboratory animals. Science, 209(18), 353–361.

mothers without the full expression of FAS. These children may have mild to severe impairments in attention, memory, and adaptive behavior but lack the dysmorphic facial features required for a formal diagnosis of FAS. This condition has been historically referred to as fetal alcohol eVects (FAE). In 1996, the IOM proposed the terms alcohol‐related neurodevelopmental disorder (ARND) and alcohol‐related birth defects (ARBD) to describe non‐FAS children with a history of maternal EtOH exposure and poor developmental outcomes that have been clinically or scientifically linked to in utero EtOH exposure (Stratton et al., 1996). In an attempt


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to unify the diverse terms being used in the EtOH literature, the term fetal alcohol spectrum disorder (FASD) was proposed by Barr and Streissguth (2001) to denote all clinical and pathological manifestations of EtOH teratogenicity. Sampson and colleagues evaluated the incidence of FAS and ARND using two longitudinal U.S. studies (in Seattle and Cleveland) and a large, prospective study of infants born in Roubaix, France (Sampson et al., 1997). Results from the Seattle cohort of primarily white, married, middle‐class women yielded an FAS incidence of 2.8 per 1000 live births. The incidence of FAS in the Cleveland study with a low‐SES, inner‐city sample was 4.6 per 1000 while the incidence of FAS in Roubaix was 2.3 per 1000 live births. The authors noted that mothers of children diagnosed with FAS in Seattle, Cleveland, and Roibaix were likely to be alcoholic and poor. The prevalence of ARND was also calculated for the Seattle cohort, yielding a combined rate of FAS and ARND of 9.1/1000 live births. This finding implies that nearly one child in every 100 births is aVected by maternal EtOH consumption, a disquieting figure that underscores the magnitude of this public health problem. C.

Neurodevelopmental Effects of Ethanol Consumption During Pregnancy

It is now well established that prenatal exposure to EtOH can lead to a continuum of neurodevelopmental eVects in infants, children, and adolescents (Mattson & Riley, 1998). In keeping with the landmark principles of teratology laid out by Wilson (1977), the eVects of prenatal EtOH exposure are dependent on dose, timing, and conditions (e.g., age of mother) of exposure. Fetal alcohol syndrome is typically associated with heavy drinking or alcoholism during pregnancy. A drink is defined as 0.5 oz absolute alcohol (AA), whether it is in the form of beer, wine, or spirits. Although definitions vary, light drinking is usually defined as approximately one drink per day, moderate drinking as two drinks per day, and heavy drinking as 3.5 or more drinks per day (Abel et al., 1998). 1. PATTERN OF CONSUMPTION

Children born to alcoholic women may vary in their developmental outcome based on the pattern of maternal EtOH consumption. Peak blood EtOH levels of alcoholics diVer, depending on whether drinks are steadily consumed throughout the day or consumed in a single binge episode (Gladstone et al., 1996). The binge pattern of drinking refers to the consumption of 5 to 6 drinks on some occasions and is typically seen in women who may drink heavily on the weekend but abstain during the week. The

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increased threat that binge drinking poses to normal fetal development was demonstrated in a study of moderate to heavy drinking in a large, inner‐city sample of pregnant women in Detroit (Jacobson et al., 1998a). This study found that the most serious eVects of EtOH were observed in children whose mothers consumed more than five drinks per occasion at least once a week, indicating that a pattern of heavy, intermittent consumption placed the fetus at greatest risk for poor developmental outcome. Other cohort studies have also demonstrated that the sudden and significant increases in blood EtOH associated with binge drinking pose a greater danger to the developing fetus than do lower‐dose, chronic exposure scenarios (Streissguth et al., 1994b). A recent study of binge drinking during pregnancy found that exposed children were 1.7 times more likely to have an IQ in the range of mental retardation and 2.5 times more likely to have delinquent behavior in the classroom (Bailey et al., 2004). Data from comparative studies of EtOH exposure and pregnancy outcome have also demonstrated that in mice, rats, and monkeys, a single binge exposure or a series of binge exposures can result in significant physical and neurobehavioral abnormalities in exposed oVspring (Clarren et al., 1992; Goodlett & Eilers, 1997; Goodlett et al., 1990; Sulik et al., 1986). These results indicate that maternal peak blood EtOH values and not total volume consumed is most predictive of the teratogenic risk associated with prenatal drinking. Based on the available data, the intermittent binge pattern of heavy drinking with its attendant peaks in blood EtOH is associated with the greatest expression of neurotoxicity in exposed oVspring. 2. TIMING OF EXPOSURE

Timing of exposure is another important variable in defining the risk that maternal drinking poses to oVspring development. The groundbreaking work by Sulik (1984, 2005) demonstrated that the critical period for the induction of EtOH‐induced facial malformations in the mouse occurs very early in gestation (day 7) and lasts for a brief period of time (a few hours). As illustrated in Fig. 3, when EtOH exposure occurs on day 7 in gestation, the mouse fetus exhibits facial characteristics that are similar to children with FAS (small head, short palpebral fissures, and long upper lip with deficient philtrum). The limited time period during which the craniofacial area is sensitive to the eVects of EtOH may be one of the factors that explain why most women who are chronic alcoholics do not give birth to infants with facial dysmorphia. Research using macaque monkeys as an animal model of FAS found evidence that a critical period for craniofacial defects may exist in nonhuman primates as well (Astley et al., 1999). The greatest number of craniofacial alterations in young pigtailed macaques exposed to EtOH occurred when exposure took place on gestation day 19 or 20 (average gestation length is


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FIG. 3. Facial characteristics of fetal mouse exposed to ethanol. Reprinted with permission from Dr. Kathy Sulik, Department of Cell and Developmental Biology and Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill.

175 days). Ethanol‐induced skeletal changes were diYcult to detect at birth, increased substantially at 6 months, and then gradually lessened as the animals reached 12 and 24 months of age. The authors note that studies in two animal species (monkey and mouse) support the notion of a critical period for induction of facial dysmorphia resulting from prenatal EtOH exposure. 3. NEUROBEHAVIORAL PROFILES ASSOCIATED WITH PRENATAL ETHANOL EXPOSURE

Not every child exposed to EtOH during prenatal development will display developmental deficits. Those that are affected can express deficits across a broad range of neurobehavioral domains (Jacobson & Jacobson, 2002; Mattson & Riley, 1998; Mattson et al., 2001). In the first published report on FAS, the investigators noted tremors and a weak sucking reflex in one infant while a second infant displayed marked developmental retardation (Jones & Smith, 1973). A third infant became cyanotic within 5 hours of birth, dying within 5 days. This report provided the first clear demonstration that infants with a history of high‐level gestational exposure can be accurately classified at birth. Data from early studies on maternal drinking and oVspring development indicated that a number of parameters were adversely aVected by exposure. Little and colleagues found that the birth weight of infants born to alcoholic women who drank during pregnancy was, on average, 493 grams less than that of control infants (Little et al., 1980).

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Published in the same year, data from a small study using the Bayley Scales of Infant Development indicated that EtOH‐exposed infants had significantly lower scores on scales of mental and motor development (Golden et al., 1982). A number of prospective, longitudinal research studies have provided the lion’s share of the data available on prenatal alcohol exposure and neurodevelopmental disability. To provide a perspective that emphasizes the long‐ term nature of alcohol neurotoxicity in exposed children, over years and even over decades, the general findings of larger cohort studies are examined in the following section. a. Seattle. In 1974, the Seattle Longitudinal Prospective Study on Alcohol and Pregnancy was initiated to study the eVects of socially acceptable levels of maternal drinking on pregnancy outcome and infant development in Washington state (Streissguth et al., 1981). A group of 1529 women, primarily white, married, and middle‐class, were identified from local hospitals and screened for participation in the study. Two cohorts of infants were examined. One cohort consisted of 163 infants with mothers who drank, on average, 2 or more drinks per day or who had a history of binge drinking (5 or more drinks per occasion). Study infants were examined by dysmorphologists and two newborns in this sample were diagnosed with full FAS (Hanson et al., 1978). There was a significant relationship between level of prenatal exposure to EtOH and the presence of physical features compatible with FAS. Infants with features compatible with FAS did not receive a formal FAS diagnosis but did express some of the growth anomalies associated with this syndrome (fetal growth retardation, microcephaly, and facial dysmorphia). The diagnosis of ‘‘features compatible with FAS’’ was found in 19% of infants with mothers who drank 4 or more drinks per day and 11% of children whose mothers reported drinking 2 to 4 drinks per day. The second Seattle cohort consisted of approximately 500 infants who were selected based on maternal traits, eVectively dividing the group between infants born to heavier drinkers (two or more drinks/day) and infants born to women who drank infrequently or abstained (Streissguth et al., 1981). Close to 80% of the women in this cohort reported drinking at some point during pregnancy and infants born to mothers consuming more than two drinks per day were overrepresented in the sample (36%). A binge style of drinking (five or more drinks on any one occasion) was reported by 39% of women in early pregnancy and 25% continued this pattern during midpregnancy. Only eight women in this cohort reported a significant problem with alcohol, emphasizing that most subjects in this group were not drinking at levels associated with chronic alcohol abuse and alcoholism. Increased EtOH consumption during pregnancy was related to decreased neonatal growth patterns, particularly for birth weight, length, and head circumference


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(Martin et al., 1980). While eVects on early postnatal growth were related to prenatal alcohol exposure in a dose‐dependent fashion, these diVerences were not present after 8 months of age (Streissguth et al., 1980). Subsequent data analyses revealed a lack of association between physical growth parameters and prenatal alcohol exposure from 8 months through 14 years of age, suggesting that the early size eVects documented in this cohort were transient and physical growth was not permanently disrupted (Sampson et al., 1994). The Brazelton Neonatal Assessment Scale (BNAS), commonly used in clinical and research settings, measures a range of abilities, including reflex strength, behavioral state, and general reactivity. On day 1 of life, Seattle investigators found alcohol use during midpregnancy was related to poorer habituation and increased levels of low arousal on the BNAS (Streissguth et al., 1983). The authors note that the levels of drinking associated with diminished performance on the Brazelton were clearly within the socially acceptable range (2–4 drinks per day). It is interesting to note that in this cohort, 474 behavioral outcomes were studied across the first 7 years of life and habituation to light in early infancy showed the strongest relationship with maternal drinking (Streissguth et al., 1993). On day 2 of life, head turning and sucking behaviors were evaluated using operant test procedures to study newborn conditioning (a form of simple learning) (Martin et al., 1977). Although maternal EtOH exposure alone was not related to performance, the combination of maternal drinking and smoking did significantly impact early learning abilities in exposed infants. Mental and psychomotor processing was evaluated in this cohort at 8 months of age with the Bayley Scales of Infant Development (Streissguth et al., 1980). Prenatal EtOH exposure had significant eVects on behavioral development when mothers drank, on average, 4 or more drinks per day while infants born to women drinking 2 to 4 drinks per day were not adversely aVected. These data suggest that the threshold of drinking associated with adverse behavioral eVects in infancy is somewhere between 2 and 4 drinks per day. At 4 years of age, decrements in IQ were documented in the EtOH‐ exposed children from the Seattle cohort (Streissguth et al., 1989). Results support a threshold relationship where 3 drinks per day during pregnancy was associated with a 5‐point drop in IQ. Assessment of fine and gross motor development revealed that more errors, longer performance time, longer latency to correct errors, and poorer balance were associated with maternal EtOH consumption (Barr et al., 1990). Children exposed to approximately one drink per day during early pregnancy made more errors on a test of manual dexterity while time to completion was increased in children exposed to approximately three drinks per day. Reaction time and attention

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were measured with a vigilance test where children were asked to press a button when a small cat appeared on a screen (Streissguth et al., 1984). Alcohol‐exposed children made more errors of omission and commission on this task and frequently took longer to respond than did unexposed controls, suggesting a fundamental slowing of mental processing speed. At 7.5 years, further vigilance testing revealed attentional decrements and slower reaction times in children with a history of prenatal EtOH exposure (Streissguth et al., 1986). Errors of commission (responding when no stimulus was present) was the performance variable most highly correlated with prenatal EtOH exposure. Drinking during pregnancy was also related to longer reaction times, further emphasizing decrements in speed of cognitive operations. In general, the magnitude of the EtOH eVect on childhood attention increased with higher levels of exposure and a pattern of maternal binge drinking. Further testing at 7.5 years focused on the assessment of IQ, learning, and classroom behavior (Streissguth et al., 1990). Although most children were performing in the range of normal intelligence, consumption of two drinks or more per day during midpregnancy was associated with a 7‐point decrement in IQ. The IQ subtests most aVected by EtOH exposure were Arithmetic, Digit Span, and Block Design. On tests of academic achievement, arithmetic emerged as the area most aVected by a history of prenatal EtOH exposure but reading was also negatively impacted. A binge pattern of maternal drinking (greater than five drinks on any one occasion) was associated with a 1‐ to 3‐month delay in learning math and reading by the end of the first grade. Ratings of classroom behavior revealed that prenatal EtOH exposure was related to distractibility, diYculty with retention and recall, and poor organizational skills. Deficits in problem‐solving, the ability most strongly aVected by gestational EtOH exposure, and speed of information processing were primarily associated with a binge‐style drinking pattern. Findings from this study suggest that heavy gestational exposure to alcohol aVects memory, attention, and abstract problem solving and that by school age; these children are having diYculties with learning and adaptive classroom behavior. Eighty‐two percent of the original Seattle cohort was evaluated on measures of school achievement, adaptive behavior, and social competence in early adolescence (Olson et al., 1997). Study results demonstrated a subtle but significant relationship between maternal drinking and antisocial behavior, school problems, and negative self‐perception. Behavioral dysfunction was most strongly related to a pattern of maternal binge drinking and the level of drinking before recognition of pregnancy, not the level maintained during midpregnancy. At a 14‐year follow‐up, young adults were also


15

evaluated using a number of psychometric tests measuring attention, memory, reading, and arithmetic (Streissguth et al., 1994a,b). Maternal binge or massed drinking was the pattern of EtOH exposure most highly associated with deficits in reading proficiency and numerical problem solving during adolescence. Although not all exposed subjects were aVected, maternal drinking during pregnancy was linked to problems with response inhibition, focused and sustained attention, and spatial learning in a dose‐dependent manner. The pattern of drinking associated with the highest risk to exposed oVspring was one in which drinks were clustered or massed. Scores on tests of arithmetic and phonological processing were also related to prenatal EtOH exposure in a dose‐dependent fashion. The attentional/memory deficits observed in these adolescent subjects were correlated (0.67) with neurobehavioral eVects at 7 years of age. For children born to heavy drinkers, 91% of those who scored poorly in arithmetic at 7 years continued to score poorly on arithmetic at 14 years. Results from the latest published study of this cohort indicate that at 21 years of age, a history of prenatal EtOH exposure has placed these young adults at risk for experiencing EtOH‐related problems (Baer et al., 2003). Subjects were given the Alcohol Dependency Scale (ADS), and items such as passing out, blackouts, and physical illness were related to prenatal EtOH exposure. In contrast, actual alcoholic beverage consumption rates were not related to maternal drinking. These findings suggest that prenatal EtOH exposure is associated with negative consequences of heavy drinking in adulthood but is not related to patterns of behavior that reflect compulsive use or addiction. b. Detroit. A prospective study of prenatal EtOH exposure and cognitive development was conducted in Detroit, Michigan, with a sample of primarily poor, inner‐city women who received obstetrical care from a local maternity hospital (Jacobson et al., 1991). Recruitment of pregnant women took place from 1986 to 1989 and enrollment into the study was based on alcohol consumption during pregnancy. The cohort consisted of approximately 416 infants who were born to women enrolled in the study. Fifty‐one percent of women enrolled in the study were drinking around the time of conception and 10% continued during pregnancy. Sixteen percent of the mothers enrolled (n ¼ 67) abstained from alcohol while 74% (n ¼ 308) were light drinkers (less than one drink per day). Five percent of the mothers (n ¼ 22) were moderate drinkers (one to two drinks per day) while 3% (n ¼ 12) were heavy (two to four drinks per day) and 2% (n ¼ 7) were very heavy (over 4 drinks per day) drinkers. Birth weight and crown–rump length data were obtained from this cohort to evaluate the eVects of gestational EtOH exposure on fetal growth (Jacobson et al., 1994a). Heavily exposed infants weighed an

16


average of 509 grams less at birth and were 4 cm shorter (crown–rump length) than unexposed controls. The eVects of ETOH exposure on fetal growth were primarily limited to those women who drank in excess of 4 drinks per day, supporting a threshold for prenatal EtOH eVects on early growth. Infants from this cohort were tested on visual recognition memory at 6.5 and 12 months and cross‐modal transfer at 12 months (a measure of memory that involves the transfer of sensory information between two modalities) (Jacobson et al., 1993b). The results indicated that basic recognition memory, both within and across sensory modalities, was normal in EtOH‐exposed infants. Although maternal drinking did not adversely impact early memory, a closer examination of the data revealed that the duration of visual fixations was longer in exposed subjects on both tasks. These findings suggest that ethanol‐exposed infants may be processing information more slowly than unexposed controls, requiring longer periods of time to encode test stimuli in memory. Infants born to mothers who drank at least two drinks per day were twice as likely to show deficits in speed of processing information. The threshold for an alcohol eVect on early information processing speed was approximately one or more drinks/day. Additional testing at 6.5 months utilized a visual expectancy test paradigm that relies on the shifting of visual gazes from one physical location to another (Jacobson et al., 1994b). Prenatal ethanol exposure was associated with slower response times and a reduction in the number of fast responses, corroborating initial results that suggested a delay or deficit in mental processing speed within this cohort of EtOH‐exposed infants. Slowed response times and fewer fast responses (measures of early cognitive dysfunction) were evident in infants born to mothers who averaged at least one drink per day. At 1 year of age, infants from this cohort were tested using the Bayley Scales of Infant Development to evaluate early mental and psychomotor development (Jacobson et al., 1993a). Higher levels of maternal drinking were associated with poorer scores on the Bayley and no threshold for safe drinking was ascertained. Substandard scores on the Bayley (1 SD below the sample mean) were primarily related to maternal consumption of 4 or more drinks per day around conception or one drink or more per day during pregnancy. The threshold for an alcohol eVect on early mental performance was approximately one drink per day. Functional impairment during infancy, when examined over all test measures, was not present in the oVspring of women who consumed less than one drink per day during pregnancy (Jacobson et al., 1998c). Deficits were greatest in exposed infants born to heavy drinkers who were older than 30 years of age and those born


17

to mothers with a binge pattern of EtOH consumption (five drinks per occasion at least once a week). Additional neurobehavioral evaluations were undertaken with this cohort when the children reached 7 years of age. Subjects were tested on measures of learning and memory and preliminary data suggest that greater levels of prenatal EtOH exposure were related to deficits in executive functioning, focused attention, and flexible problem solving (Jacobson et al., 1998a). From a psychosocial perspective, EtOH‐exposed children frequently lacked interpersonal social skills and acted aggressively in the classroom, emphasizing the toll of maternal drinking on social behavior in exposed oVspring (Jacobson et al., 1998b). A 2004 report published on this cohort focused on the relationship between prenatal EtOH exposure and IQ at 7.5 years, with a special emphasis on maternal age, alcohol abuse history, and home environment as moderating variables (Jacobson et al., 2004). Overall results indicated that while there was no EtOH eVect on full‐scale IQ, specific deficits in attention, arithmetic, and working memory were evident. Certain subgroups of children emerged from the analysis as more vulnerable to the adverse eVects of EtOH exposure because of advanced maternal age, greater maternal alcohol abuse, and/or being raised in a home lacking intellectual stimulation. c. Atlanta. In a prospective study in Atlanta, 103 subjects were recruited from a prenatal care clinic that primarily served low‐SES, inner‐city women from 1980 to 1986 (Coles et al., 1985). Data were collected on maternal drinking behavior around conception and during pregnancy so that the eVects of drinking cessation on developmental outcome could be examined. Women were recruited into one of three experimental groups: those who reported consuming an average of 3.5 drinks/day during pregnancy (n ¼ 26), those who drank, on average, four drinks/day but stopped by midpregnancy (n ¼ 22), and those who never drank at all (n ¼ 55). Infants were weighed, measured, examined for dysmorphia, and evaluated for neurobehavioral eVects during the neonatal period. Three of the infants in this study received dysmorphia scores that classified them as FAE but there were no eVects of prenatal EtOH exposure on measures of physical growth. The results from the Brazelton Neonatal Behavioral Assessment Scale indicated that infants exposed to EtOH at any point during gestation exhibited abnormal reflexes, immature motor skills, and greater activity levels when compared to unexposed controls. Infants born to women who stopped alcohol consumption during midpregnancy had better scores on measures of state control, need for stimulation, motor tonicity, tremulousness, and asymmetries in reflexive behavior than did infants born to mothers who continued to drink. The data support the concept that abstinence in later pregnancy is associated with behavioral gains in exposed newborns and that infants with a history of chronic fetal exposure are at the greatest risk for behavioral eVects in the

18


neonatal period. A subset of the group tested in early infancy was reexamined at 6 months of age using the Bayley Scales of Infant Development (Coles et al., 1987). Poor scores from early infancy measures on the Brazelton (days 1–3) were associated with reduced mental and psychomotor scores on the Bayley. The findings indicate that abnormal behavior is present from the first days of postnatal life in EtOH‐exposed newborns and that changes in neonatal behavior may be predictive of later deficits in cognitive and motor behaviors. A subset of the original cohort from Atlanta was retested at approximately 6 years of age to evaluate facial dysmorphia, physical growth parameters, measures of cognition, academic progress, and adaptive behavior (Coles et al., 1991). There was a high correlation between neonatal dysmorphia scores, including microcephaly, and results from a physical exam at 6 years of age. Results from cognitive testing indicated that academic achievement scores were lower in children with a history of prenatal EtOH exposure but the greatest performance deficits were associated with a pattern of maternal drinking that persisted throughout pregnancy. Math and reading skills were significantly lower in both experimental groups (continued‐to‐drink and stopped‐drinking) but children from the continued‐to‐drink group also showed deficits in sequential processing and general intellectual functioning. Ratings of adaptive behavior were similar between exposed and unexposed children. The overall results suggest that EtOH‐induced dysmorphias are persistent over early childhood and mothers who continue to drink throughout pregnancy place their oVspring at the highest risk for neurodevelopmental eVects. At 7 years of age, impulsivity, attention, and internalizing/externalizing behaviors were evaluated in these children (Brown et al., 1991). Hyperactivity and impulsive behavior were not observed in the general cohort but children in the continued‐to‐drink group had diYculty with mental concentration (a measure of sustained attention) and were more often described by teachers as having attentional and behavioral problems in the classroom. Externalizing behaviors (aggression, destructiveness, inattention) were significantly linked to prenatal EtOH exposure but internalizing behaviors such as anxiety and depression were only weakly associated with maternal drinking history. The authors suggest that current levels of maternal EtOH use and the quality of the caretaking environment are more influential on internalizing behaviors than prenatal EtOH exposure. Study results once again support the idea that mothers who quit drinking during midpregnancy confer significant developmental advantages to their oVspring when compared to mothers who continue to drink throughout pregnancy. The latest published reports from the Atlanta cohort describe studies performed on a subset of children at approximately 15 years of age (Coles


19

et al., 2002; Riley et al., 2003). Alcohol‐exposed children with dysmorphia were compared to alcohol‐exposed children without dysmorphia and children not exposed to alcohol prenatally. Subjects were tested to examine the eVects of prenatal EtOH exposure on growth, cognition/sustained attention, and behavioral problems during adolescence. Facial dysmorphia persisted in ETOH‐exposed teenagers but there was no evidence of continuing deficits in aspects of physical growth such as weight and height. Subjects with a history of prenatal exposure but no dysmorphia performed as well as did unexposed subjects on tests of sustained attention and IQ. In contrast, dysmorphic individuals had lower IQ scores and more diYculty solving arithmetic problems. A similar pattern was found on tests of sustained attention where errors of omission (failing to detect a stimulus when presented) were particularly high in the dysmorphic group, suggesting a specific deficit in visual perception. Neonatal scores on the dysmorphia exam and the Brazelton were predictive of IQ, achievement scores (including math), and sustained attention during adolescence in this cohort. Study results support the diagnostic sensitivity of neonatal dysmorphia exams and infant behavior scales in predicting long‐term intellectual and cognitive outcomes in EtOH‐exposed individuals. d. Cleveland. The Cleveland Prospective Alcohol‐in‐Pregnancy Study was launched in 1980 to evaluate the rate of alcoholism in a general population of pregnant women and to study the reproductive eVects of EtOH on pregnancy outcome (Sokol et al., 1981). Data from 2913 low‐SES women seeking prenatal care from a city hospital were collected during the first year of the study. This sample was eVectively divided into two experimental groups based on scores from the Michigan Alcoholism Screening Test (MAST) given at the initial visit. This test is a composite of 25 questions that are primarily related to the psychosocial aspects of problem drinking and scores of 5 or more are considered indicative of EtOH abuse. Women with MAST scores of 5 or more were selected to represent heavy drinkers (MAST positive) and women with MAST scores lower than 5 were enrolled to represent light drinkers (MAST negative). Drawn from the larger prospective study, a cohort of infants from MAST‐ positive pregnancies (n ¼ 176) and MAST‐negative pregnancies (n ¼ 183) were examined for the presence of physical anomalies during the neonatal period (Ernhart et al., 1987). Twenty‐six percent of the mothers enrolled in the infancy study (n ¼ 95) abstained from alcohol while 45% (n ¼ 161) were light/moderate drinkers (fewer than two drinks per day). Seventeen percent of the mothers (n ¼ 60) drank 4 or fewer drinks per day, 6% (n ¼ 23) drank six or fewer drinks per day, and 5% of the women (n ¼ 20) drank more than six drinks per day. Teratogenic eVects were dose‐dependent and the highest counts of congenital anomalies and facial dysmorphia were seen in children

20


exposed to EtOH during the embryonic period. The most frequently noted items were ptosis, cleft palate, and cleft lip. The greatest risk for facial dysmorphia was related to maternal drinking of 6 or more drinks per day around the time of conception. No clear threshold could be identified for craniofacial abnormalities since these eVects were observed in infants born to women who consumed fewer than 6 drinks per day. The Cleveland cohort was evaluated several times during infancy and early childhood (6 mo, 1 yr, 2 yr, 3 yr, 4 yr 10 mo) by trained observers who visited each child in their home (Boyd et al., 1991; Greene et al., 1991a). Tallies of dysmorphia obtained during the neonatal period were strongly related to indices of maternal drinking during pregnancy. Birth weight was also aVected by maternal EtOH consumption during pregnancy, although this relationship was less robust. Tests of mental development, IQ, and sustained attention were used to evaluate neurobehavioral outcome in these children. Excluding the 1 confirmed case of FAS, multiple analyses relating maternal EtOH use and childhood outcome did not find evidence of neurobehavioral deficits on any test measure. Heavily exposed children performed as well as children born to light drinkers on test measures of cognition and intelligence. These results suggest that prenatal EtOH exposure, at levels not resulting in FAS, does not impair the development of normal childhood intelligence. A longitudinal analysis of physical growth in this cohort found evidence of an alcohol eVect on size at birth but this eVect was attenuated in the preschool years, providing little evidence of persistent growth deficits in EtOH‐exposed children (Greene et al., 1991b). No reports have been published for this cohort since 1991. e. Pittsburgh. The Pittsburgh study was designed to examine the eVects of prenatal ETOH and marijuana use on infant growth and development. The sample consisted of approximately 650 low‐SES women participating in the Maternal Health Practices and Child Development Project from 1983 to 1985 (Day et al., 1989). During the first trimester, 31% of the mothers enrolled (n ¼ 199) abstained from alcohol while 37% (n ¼ 240) were light drinkers ( >2.9 drinks per week). Eight percent of the mothers (n ¼ 49) were moderate drinkers (three to six drinks per week) while 24% (n ¼ 154) were heavy drinkers (one or more drinks per day). Rates of heavy drinking decreased from 24% during the first trimester to 5% during the last trimester. Within 2 days of delivery, oVspring from live‐born singleton births (n ¼ 595) were measured, weighed, and examined by trained nurses for physical anomalies. Prenatal EtOH exposure was related to an increased risk of low birth weight, length, and head circumference under the 10th percentile, and the presence of minor physical malformations. The average birth weight of infants exposed to EtOH throughout pregnancy was 815 grams lower than the birth weight of unexposed control infants. These adverse eVects were


21

primarily associated with drinking during the first trimester, particularly during months 1 and 2, and illustrate the increased vulnerability of the fetus to EtOH exposure during early pregnancy. At 8 months of age, infants from this cohort were weighed and measured again to examine eVects of exposure on physical development (Day et al., 1990). There was a significant relationship between EtOH use throughout pregnancy and weight and length of infants at this age. Growth and morphology deficits remained persistent in exposed infants, particularly in subjects whose mothers drank continuously or during the last 2 trimesters of pregnancy. A month later (9 months of age), infants were tested on the Bayley Scales of Infant Development. Results from the Bayley did not indicate an association between rates of maternal drinking and delays in mental or psychomotor development (Richardson et al., 1995). At 3 years of age, children prenatally exposed to EtOH remained smaller on measures of weight, height, and head circumference and these eVects were dose‐dependent (Day et al., 1991). Growth eVects were primarily associated with 1 or more drinks/day during the second and third trimester. The number of minor physical malformations was also elevated in children who were exposed to 1 or more drink/day during the first trimester of pregnancy. The authors note that while the growth eVects associated with gestational alcohol exposure are modest and lack clinical significance for the individual child, they suggest the long‐term disruption of physical growth in this population. Children from the Pittsburgh cohort were tested at ages 6 and 10 years on measures of IQ and academic achievement (Goldschmidt et al., 1996, 2004). At age 6 years, consumption of one or more drinks per day during the second trimester was associated with reduced scores in arithmetic, reading, and spelling. Follow‐up at 10 years found that teachers rated children with first‐ and second‐trimester EtOH exposure more poorly on measures of classroom performance than unexposed controls. Binge drinking (four or more drinks per occasion) during midpregnancy was associated with lower reading recognition and comprehension and poorer teacher ratings of academic achievement. Additional testing at this age was undertaken to provide information on learning and memory, abstract reasoning, mental flexibility, eye–hand coordination, and information processing (Richardson et al., 2002). Prenatal EtOH use was associated with significantly poorer performance on tests of design and story memory as well as verbal learning. These eVects were associated primarily with levels of maternal drinking at four or more drinks per day during the first and second trimesters of pregnancy. Exposed oVspring at age 10 were physically smaller than their unexposed counterparts, expressing a pattern of growth deficits that has been consistent since birth (Day et al., 1999). Prenatal exposure to EtOH during the first trimester was related to reduced height and head circumference and exposure

22


during the second trimester was associated with lighter body weight. The data suggest that the period of greatest vulnerability for EtOH eVects on oVspring size is during early gestation when fetal cells are rapidly multiplying. Reports from the Pittsburgh cohort have described results from examinations of children at 14 years of age. Weight, height, and head circumference continued to be aVected by prenatal EtOH exposure (Day et al., 2002). On average, the oVspring of heavy drinkers were 16 lbs lighter than the offspring of abstainers. EVects were dose‐dependent and found at levels of exposure less than 1 drink/day. Results of cognitive tests show that prenatal EtOH exposure was associated with deficits in learning and memory that were specific to the verbal domain (Willford et al., 2004). These results are consistent with the cognitive findings at the 10‐year follow‐up and suggest long‐term losses in the intellectual potential of children exposed to light (< 0.4 drinks/day) to moderate (30 yrs) are more likely to have an aVected infant. □ EVects during infancy associated with 1 or more drinks/day during gestation. Atlanta Physical Growth Yes Yes No Behavioral Development Yes Yes Yes □ Abstinence from drinking during later pregnancy reduces risk of aVected infant. □ Only subjects with facial dysmorphia have persistent neurobehavioral deficits. Cleveland Physical Growth Yes Yes No data Behavioral Development Yes Yes No data □ 6 or more drinks/day around conception associated with greatest risk for facial dysmorphia. □ Levels of exposure that do not produce FAS do not result in neurobehavioral deficits. Pittsburgh Physical Growth Yes Yes Yes Behavioral Development No Yes Yes □ Drinking during the first trimester poses greatest overall risk to the fetus. □ Light to moderate drinking during early pregnancy associated with long‐term eVects on physical and mental development.

mature, deficits in social behavior become more pronounced and are often expressed in the form of aggression in the classroom, impaired social judgments, and antisocial/delinquent behavior. A history of prenatal exposure may place young adults at risk for abusing EtOH themselves and experiencing EtOH‐related dependency problems (Baer et al., 2003), underscoring the intergenerational nature of EtOH abuse and the long‐term consequences of prenatal exposure.


D.

25

Ethanol‐Induced Brain Injury

Although prenatal ethanol exposure can have adverse consequences on multiple organ systems, the most dramatic eVects are clearly on the central nervous system. Autopsy reports of aVected children have demonstrated that serious, widespread brain damage is frequently attendant to gestational EtOH exposure. Since the identification of the FAS 30 years ago, there have been many eVorts to identify the areas of the brain most sensitive to alcohol during gestation and the mechanisms that may be responsible for the disruption of neural development. Although no specific pattern of malformations can be identified, adverse eVects on brain size, corpus callosum, basal ganglia, cerebellum, and neural glial cells have been documented (Clarren, 1986; Roebuck et al., 1998). Since 1995, the persistent eVects of prenatal alcohol exposure on the brain have been studied with magnetic resonance imaging (MRI), a quantitative tool that allows structural examination of the size and volume of brain structures in living subjects (reviewed by Mattson et al., 2001; Riley et al., 2004). The most consistent finding in these studies has been a reduction in the size of the cranial vault (microcephaly) of children with a history of heavy prenatal EtOH exposure. Consistent with behavioral observations that indicate EtOH‐exposed children have balance and motor impairments, reductions in cerebellar volume have been documented. The corpus callosum, the fiber tract that connects the two hemispheres of the brain, may be the neural structure most aVected by EtOH exposure. The absence or thinning of the corpus callosum is a common neuroanatomical defect associated with intrauterine EtOH exposure and may significantly contribute to problems with cognition, motor skills, and bimanual coordination. In a study of adults with FAS or FAE, a thinning of the corpus callosum was associated with functional motor losses while a thickened callosum was linked to deficits in executive functioning (the ability to plan and execute problem‐solving strategies) (Bookstein et al., 2002). The basal ganglia, important for limb and eye movements and cognition, are also vulnerable to gestational EtOH exposure and show marked size reductions in EtOH‐exposed children. New whole‐brain analytic techniques have been applied to imaging data from children with a history of heavy prenatal EtOH exposure. Results indicate changes in the shape and size of the corpus callosum, a disproportionate reduction in temporal lobe size, changes in the symmetry of the gray matter in parts of the temporal lobe, and increased gray matter/decreased white matter in the perisylvian cortices of the temporal and parietal lobes. Significant abnormalities in whole brain shape, such as narrowing of the temporoparietal regions and reduced growth of the frontal lobes, have also been documented. These research findings indicate that EtOH‐induced brain

26


malformations may be regionally specific and suggest that neurobehavioral deficits characteristic of FAS/FAE children may be tied to distinct areas of neural injury. Groundbreaking research has demonstrated that EtOH may interfere with the glutamate and gamma‐aminobutyric (GABA) neurotransmitter systems by triggering widespread cell death in the developing rat brain (Ikonomidou et al., 1999, 2000). The greatest period of vulnerability for neuronal loss coincides with the period of synaptogenesis, also known as the brain growth spurt. This research suggests that EtOH exposure during the brain growth spurt in humans, from approximately the sixth month of gestation to several years after birth, can cause the loss of millions of developing nerve cells. The authors postulate that these losses may explain the diverse range of neurobehavioral deficits and reduced brain size commonly observed in children aVected by prenatal alcohol exposure. Work published by Olney and colleagues (2001) has demonstrated that a single intoxicating dose of EtOH, lasting for several hours, is suYcient to trigger a massive loss of brain cells in developing rats and mice. Ethanol has the potential to trigger widespread apoptotic neurodegeneration through blockade of NMDA glutamate receptors and excessive activation of GABA (A) receptors. This massive cell death, induced by EtOH, may be an important mechanism in the etiology of FAS and may play a defining role in the expression of neurobehavioral deficits in FAE and related disorders (Olney et al., 2002).

III.

METHANOL

Methanol is one of the most commonly used chemicals in American industry (National Library of Medicine, Toxicology Information Program). Also referred to as methyl alcohol and wood alcohol, methanol (MeOH) is an important industrial solvent that is necessary in the production of consumer goods such as solid fuels (Sterno), antifreeze, and photocopying fluids. It is also used in the pharmaceutical and agricultural industries and in the manufacture of ethylene glycol, methyl halides, methacrylates, and methylamines (Von‐Burg, 1994). As described in the federal Hazardous Substances Data Bank, MeOH is required to produce chemical intermediates such as formaldehyde and acetic acid and is used in products as diverse as paints, plastic bottles, and contemporary fabrics (http://toxnet.nlm.nih.gov). Within the last two decades, the utilization of MeOH as an alternative motor fuel has been explored in both public and private sectors. Recent attention has focused on the use of MeOH as a primary fuel source for vehicles powered by hydrogen‐based fuel cell technology (Fuller et al., 1997).


27

One of the most important characteristics of MeOH is that it is a low‐ emission, high‐performance combustible motor fuel. Increased use of MeOH as a fuel source could lead to improved air quality by reducing the hydrocarbon emissions responsible for increased atmospheric ozone concentrations and, ultimately, global warming (Gold & Moulis, 1988). If methanol is used in the production of new fuels, there could be widespread exposure to the public, primarily from inhalation of MeOH vapors (Carson et al., 1987). While the eVects of acute high‐dose MeOH poisoning in adults have been well chronicled, little is known about eVects of chronic, low‐dose exposure, particularly in sensitive subgroups such as pregnant women (International Programme on Chemical Safety, 1997). The eVects of acute high‐dose MeOH exposure have been characterized from clinical cases of human poisoning and comparative toxicology work with rodents and monkeys. The time course and progression of MeOH toxicity in humans have been documented in detail (Bennett et al., 1953; Tephly & McMartin, 1984). In brief, the individual typically experiences a short period of intoxication, followed by a period in which no symptoms of intoxication or toxicity are noted. This asymptomatic period is followed by symptoms of poisoning, such as headache, nausea, vomiting, loss of equilibrium, severe abdominal pain, and diYculty in breathing. These symptoms can be followed by coma and death. Neurological abnormalities, including focal cranial nerve deficits, optic atrophy, and a Parkinsonian‐like syndrome, usually involving symptoms such as rigidity, tremor, and impaired balance, have been reported (Guggenheim et al., 1971; Ley & Gali, 1983; Riegel & Wolf, 1966). Findings from occupational health research indicate that workplace exposure to MeOH may have links to the immediate or delayed onset of this Parkinsonian‐like disorder (Tanner, 1992). In 2002, this syndrome was reported in a physicist after chronic exposure to MeOH in the laboratory (Finkelstein & Vardi, 2002). There are numerous reports on the metabolism and disposition of MeOH in rodents and nonhuman primates (see reviews by Tephly, 1991; Tephly & McMartin, 1984). Data on human subjects are limited to clinical observations in cases of MeOH poisoning and experimental exposure studies in healthy volunteers. The absorption of MeOH is rapid following oral ingestion, inhalation of MeOH vapor, or dermal contact. Once absorbed, MeOH distributes readily to all organs and tissues roughly in proportion to their water content (Yant & Schrenk, 1937). Metabolism is the predominant route of elimination at low or moderately high doses of MeOH. Methanol is first converted to formaldehyde which rapidly undergoes oxidation to formate (formic acid). Formate then enters the folate biochemical pathway and is eventually oxidized to carbon dioxide (CO2). Formate is considered the toxic metabolite of MeOH, responsible for disturbances of the visual system and

28


metabolic acidosis, the cause of death in highly exposed subjects (Jacobsen & McMartin, 1986).

A.

Exposure Scenarios for Pregnant Women

In the context of MeOH as an alternative motor fuel, most exposures of pregnant women would take place on highways, urban streets, during refueling, and in private garages. For each scenario involving MeOH‐based fuel, calculations have been made as to the extent of predicted MeOH exposure for the average adult (Gold & Moulis, 1988). Even in situations likely to result in the highest exposures, such as refueling (23–38 ppm) and hot‐soak emissions in private garages (192–383 ppm), the length of exposure is relatively brief and anticipated levels of MeOH exposure fall below those associated with clinical neurotoxicity. The current threshold limit value for MeOH for an 8‐hour work day, 40 hours per week, is a time‐weighted average (TWA) of 230 ug/m3 or 200 ppm (American Conference of Governmental Industrial Hygienists, 1990). In addition to maternal inhalation exposure, prenatal exposure to MeOH can also occur through the maternal consumption of adulterated alcoholic beverages, fruit, vegetables, and food/ drinks that have been artificially sweetened with aspartame. In the case of tainted alcohol, it is not possible to disentangle the eVects of MeOH from ethanol.

B.

Neurodevelopmental Effects of Exposure

1. HUMAN INFANTS

Until recently, there were no cases in the published medical literature of developmental neurotoxicity in human infants associated with prenatal MeOH exposure. In 2004, the first case report was described in which an infant was exposed to MeOH during gestation through maternal exposure (Belson & Morgan, 2004). The level of blood MeOH was tested in the newborn (61.6 mg/dL) and death due to severe intraventricular bleeding occurred on postnatal day 4. The mother remained in a state of metabolic acidosis despite treatment and died 10 days after delivery. Postnatal exposure was reported in an infant who was fed a mixture of formula and windshield cleaner that contained MeOH. The infant was hospitalized and appeared to recover without long‐term neurological damage (Brent et al., 1991). In Egypt, a cluster of infant deaths following immunization were due to metabolic acidosis from MeOH poisoning (Darwish et al., 2002). These deaths and possibly other infant deaths in this farming community were due to the excessive topical application of MeOH following immunization.


29

Methanol is an eVective anti‐inflammatory and antipyretic agent and concern over adverse reactions to vaccines led to the misuse of MeOH by well‐ meaning medical personnel. 2. STUDIES WITH THE RODENT ANIMAL MODEL

Laboratory studies of prenatal MeOH exposure using rodent animal models have reported numerous signs of teratogenicity in MeOH‐exposed pups. Pregnant rats exposed by inhalation to either MeOH or ethanol, in concentrations ranging from 5000 to 20,000 ppm, delivered oVspring with an increased number of malformations (primarily cervical ribs and urinary or cardiovascular defects) (Nelson et al., 1985). At equivalent doses, the treatment‐related eVects were more pronounced in litters exposed to MeOH than those exposed to EtOH. Bolon and colleagues exposed pregnant mice to 5000, 10,000, or 15,000 ppm MeOH vapor and at the two higher doses, near‐term fetuses showed an increased number of resorptions, fetal malformations such as neural/ocular defects, cleft palate, hydronephrosis, and dysmorphic limbs as well as reduced fetal weights (Bolon et al., 1993). EVects were dependent on when exposure occurred during embryonic development. Exposure during gestation days 7 through 9, coinciding with neural tube development and closure, resulted in neural tube defects and ocular lesions. In contrast, exposure during gestation days 9 through 11, the period of likely neural tube reopening, was associated with malformations of the paw and digits. No MeOH‐related eVects were observed in the group exposed to 5000 ppm. Rogers et al. (1993) exposed pregnant mice to concentrations of MeOH vapor ranging from 1000 to 15,000 ppm on days 6 through 15 of gestation. In pups exposed to 5000 ppm MeOH or higher, an increase in the number of exencephalies and cleft palate was observed while pups exposed to 7500 ppm MeOH or higher exhibited increases in embryo/fetal death. At 10,000 ppm and above, reduced fetal weight was documented. A significant increase in the proportion of fetuses per litter with cervical ribs was noted at 2000 ppm, providing evidence that the 2000 ppm dose was the Lowest Observed Adverse EVect level (LOAEL). Based on these data, the No Observed Adverse EVect Level (NOAEL) would be 1000 ppm. Subsequent work from this investigative team has demonstrated that in the mouse, gastrulation and early organogenesis are periods in which the fetus is particularly sensitive to the teratogenic eVects of inhaled MeOH (Rogers & Mole, 1997). A study of oral MeOH exposure in rats (1.6, 0.9, 0.6% v/v) indicated significant eVects on litter size and neonatal and postnatal mortality (Abel & Bilitzke, 1992). The rate of postnatal deaths in exposed pups was very high and reached 100% in the highest dose group (1.6%). In a separate study of oral MeOH exposure, a single dose resulted in fetal growth deficits and a

30


dose‐dependent increase in congenital malformation such as undescended testes, exophthalmia, and anophthalmia (Youssef et al., 1997). Neurobehavioral evaluations of rodents developmentally exposed to MeOH are few in number. In a study by Infurna and Weiss (1986), pregnant rats were exposed to MeOH in the drinking water at 20,000 ppm on either gestational day (GD) 15–17 or GD 17–19. Exposed pups displayed diYculties in suckling behavior and in finding nesting material from the home‐cage. Stanton et al. (1995) exposed pregnant rats to MeOH at a concentration of 15,000 ppm, via inhalation, on GD 7–19. A broad‐based battery of behavioral tests was given to the oVspring including motor activity, T‐maze learning, olfactory learning, acoustic startle, and passive avoidance learning. The exposed pups did not show MeOH‐related impairments on any of the neurobehavioral measures. The only adverse eVect observed in this study was reduced birth weight. Weiss et al. (1996) exposed pregnant rats to either 4500 ppm MeOH or 0 ppm via inhalation for 6 hours daily. Maternal exposure began on GD 6 and both pups and dams were exposed until postnatal day 21. Suckling behavior, odor discrimination, and motor activity were measured prior to weaning. Adult functioning was measured with two operant procedures, the fixed‐ratio wheel‐running procedure and a stochastic spatial discrimination task. While the eVects in exposed oVspring were subtle, MeOH exposure influenced a number of neurobehavioral endpoints. In the preweaning phase of testing, a treatment‐related eVect was observed on the motor activity test. On the operant measures, both tests showed evidence of performance decrements due to MeOH exposure but the diVerences were not robust. 3. METHANOL EXPOSURE FROM ASPARTAME (ARTIFICIAL SWEETENER)

As previously noted, the consumption of aspartame‐sweetened food products and beverages results in MeOH exposure (Butchko et al., 2002). The release of MeOH occurs when aspartame is absorbed and metabolized during digestion (Stegink et al., 1983). In a review chapter of aspartame ingestion during pregnancy, the authors were unable to definitively comment on the extent of prenatal MeOH loading from maternal ingestion but it is generally regarded to be insignificant (Pitkin, 1984). In an attempt to investigate the eVects of postnatal aspartame exposure, infant stumptail macaque monkeys were fed high levels of aspartame in their formula for 9 months (Reynolds et al., 1984). Aspartame exposure was unrelated to developmental parameters such as physical growth, serum chemistry, urinalysis, hematology, and brain wave patterns. These monkeys were also evaluated on tests of hearing and cognition and no deficits in performance were reported (Suomi, 1984).

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4. METHANOL RESEARCH IN A NONHUMAN PRIMATE MODEL

A series of studies conducted in our laboratory were designed to characterize MeOH metabolism, blood clearance, and distribution kinetics prior to and during pregnancy, and to evaluate subsequent reproductive and oVspring developmental outcome, after long‐term, low‐dose maternal exposure to MeOH vapor in nonhuman primates (Burbacher et al., 1999, 2004a,b). The two‐cohort study design used adult female Macaca fascicularis monkeys exposed to 0, 200, 600, or 1800 ppm MeOH vapor for 2 hours/day, 7 days/ week prior to and during pregnancy. Female monkeys were bred to nonexposed male breeders and 34 liveborn oVspring were delivered at the Infant Primate Research Laboratory at the University of Washington. The 34 oVspring were evaluated during the first 9 months of life using a test battery that included procedures largely adapted from studies with human infants. As outlined in Table IV, test procedures to evaluate MeOH eVects on fetal mortality and malformations, oVspring size at birth, newborn health, neonatal behavioral responses, visually coordinated reaching, visual acuity, gross motor skills, spatial and visual memory, development of social behavior, learning, and postnatal physical growth were utilized in this study. Maternal exposure to MeOH resulted in peak methanol blood levels of 2 to 10 times above background. Blood methanol levels returned to baseline before 8 hr post‐exposure. MeOH exposure was not associated with overt TABLE IV OFFSPRING TEST BATTERY USED TO EVALUATE DEVELOPMENTAL EFFECTS OF PRENATAL METHANOL EXPOSURE OVspring test

Postnatal age at test

Newborn Size Medical Treatments Newborn Health Exam Neonatal Behavioral Scale Object Retrieval Visual Acuity Motor Milestones Object Permanence Visual Recognition Memory Social Behavior Observations Physical Growth Spatial Discrimination & Reversal Nonmatch‐to‐sample

Birth Birth to 9 months Birth Day 1 to 13 Weeks 2 to 6 Weeks 1 to 12 Weeks 2 to 7 months Weeks 2 to 3½ months Days 190 to 220 (postconception age) Week 2 to 7 months Birth to 9 months Months 5 to 7 Month 8 to 9

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maternal toxicity, reproductive loss, oVspring congenital malformations, or a reduction in the size of the oVspring at birth. Previous studies have reported increases in fetal mortality or malformations following prolonged daily exposures at high concentrations of MeOH vapor: over 10,000 ppm for rats (Infurna & Weiss, 1986; Nelson et al., 1985; Stanton et al., 1995) and over 2000 ppm for mice (Rogers et al., 1993). The 2‐hour exposure period used in the present study was most likely too brief to cause increases in maternal/fetal mortality or malformations, even at the 1800 ppm exposure concentration. Methanol exposures were, however, associated with a reduction in the length of pregnancy. In this study, the duration of pregnancy for all of the MeOH exposure groups (average: 160 day) was significantly shorter than the control group (average: 168 days). These results suggest that MeOH exposure during pregnancy may influence the hormonal control of the onset of labor at exposure concentrations that do not aVect overall fetal growth. The reduced gestation lengths of the MeOH‐exposed infants may reflect the premature activation of the fetal HPA axis that controls timing of birth. The basis for such an eVect is unknown but it could represent the direct action of MeOH on the fetal neuroendocrine system or an indirect action of MeOH on the maternal uterine environment. Independent of the specific biological mechanism, the reduced pregnancy durations of MeOH‐exposed dams suggest a subtle but systematic disturbance in the timing of labor and delivery. On most developmental assessments, the MeOH‐exposed infants performed as well as controls. However, treatment effects were found on two test measures: visually guided reaching and recognition memory. Results indicated that prenatal MeOH exposure is associated with a delay in early sensorimotor development as measured by the infant’s ability to reach for, grasp, and retrieve a small object during the first month of life. As illustrated in Fig. 4, this eVect was only observed in male subjects, suggesting a sex‐ specific eVect on the development of visually directed reaching. The delay for males was dose related and ranged from approximately 9 days for the 200 ppm MeOH subjects to more than 2 weeks for the 600 ppm and 1800 ppm exposure groups. The Fagan Test of Infant Intelligence is used with human infants to study visual recognition memory and provides an early measure of information processing, attention, and memory (Fagan & Detterman, 1992). When familiar (previously seen) stimuli are paired with novel (new) stimuli, normal human and macaque monkeys will typically prefer to view the novel stimuli. Novelty scores are interpreted as evidence of visual recognition memory as some attributes of the familiar stimuli must be encoded in memory for the novelty response to occur. Deficits in visual recognition memory have been reported for groups of infant monkeys at high risk for poor developmental


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FIG. 4. Results of Object Retrieval Test. Infant monkey reaching toward and retrieving a small toy to receive the applesauce reward. The ability to accurately reach for, grasp, and pick up the test objects is scored by trained observers.

outcome (methylmercury exposure; Gunderson et al., 1986, 1988), low birth weight (Gunderson et al., 1989), and failure‐to‐thrive (Gunderson et al., 1987). The results of the Fagan Test were examined by problem type. Methanol‐exposed infants performed as well as controls on problems using abstract geometric patterns, but all MeOH‐exposed groups were unable to solve more diYcult test problems using complex social stimuli (monkey faces). A schematic of the apparatus and the data from the social memory problems are displayed in Fig. 5. Prenatal MeOH exposure did not retard physical growth rates during the first 9 months of life. Our later studies, however, indicated that growth retardation in females may be a delayed eVect of high‐dose MeOH exposure. This eVect was not observed as a general decrease in the growth of females as a group, but as a ‘‘wasting syndrome’’ in two of the seven female oVspring in the 1800 ppm exposure group after 1 year of age. The syndrome was severe and resulted in the euthanasia of both of the females. Results of assays for simian retroviruses, blood chemistry, CBC, liver, kidney, thyroid, and pancreatic function were unremarkable. The results of clinical blood tests and autopsy examinations did not provide evidence as to the etiology of this puzzling syndrome. In summary, the results of our study suggest that maternal inhalation of MeOH may be related to a subtle but systematic disturbance in the timing of labor and delivery in pregnant nonhuman primates. While no clear pattern of adverse eVects was found on neurobehavioral development, visually coordinated reaching and early memory was disrupted in exposed

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FIG. 5. Results of Visual Recognition Memory Assessment using social (faces) stimuli. Infants are presented with visually engaging stimuli and then tested for memory of these images. Visual fixations to the test stimuli (on the right and left) are recorded by trained research staV via foot switches. Visual preferences for novel stimuli are interpreted as evidence of memory for previously seen (i.e., familiar) visual targets.

oVspring. The results from this study demonstrate that chronic in utero MeOH exposure, at subclinical levels, is not associated with frank teratogenic eVects but does alter the course of behavioral development in young monkeys.

IV.

EVALUATING THE RISK FROM PRENATAL EXPOSURE TO ETHANOL AND METHANOL

For ethanol, the weight of evidence to determine the risk of adverse eVects on development from prenatal exposure comes largely from prospective, longitudinal studies of human maternal–infant pairs. The results from these studies support several general conclusions. First, the developmental eVects of EtOH are dose‐dependent. The more a pregnant woman drinks, the greater the likelihood that she will give birth to an aVected infant. In addition, at high levels of exposure, the damage to the fetal nervous system from gestational EtOH exposure can be severe and may result in frank mental retardation and long‐term neurodevelopmental disability. The most serious eVects are generally seen in infants born to women who report consuming an average of four drinks or more per day during pregnancy. At lower levels of exposure, EtOH eVects are subtler and may take the form of small reductions in physical size, behavioral problems in school, and diYculty with mental concentration. These eVects are generally associated with an average of one to two drinks per day during pregnancy, which would result in


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maternal blood alcohol levels above background for only a brief period of time each day. Second, the developmental eVects of EtOH are related to the pattern of maternal drinking. Results indicate that women who exhibit a binge or massed pattern of drinking, particularly in early pregnancy, place their oVspring at the greatest risk for neurobehavioral deficits. Third, there are clear biological advantages to limiting fetal exposure and abstaining from EtOH consumption during late pregnancy, even if drinking occurred during early and midpregnancy. Abstinence during late pregnancy is associated with improved developmental outcomes in exposed oVspring. Not all children who are exposed to EtOH during gestation are equally aVected and many children with histories of heavy maternal exposure appear to be clinically normal. All of the factors that are associated with this range of responses even at high exposure levels are not known but some may have to do with maternal characteristics such as age (there is greater fetal vulnerability in older mothers), nutrition, and other drug use. New data indicate that a positive and stimulating home environment is associated with improved outcome in alcohol‐exposed children (Jacobson et al., 2004). In addition, experimental research with animals suggests that the use of Vitamin C and E may help reduce the adverse eVects of ethanol on the developing fetus (Cohen‐Kerem & Koren, 2003). In terms of understanding the dose–response relationship, the use of average number of drinks per day may poorly characterize the actual risks of maternal drinking. Abel (1998, 1999) has strongly cautioned that the use of such a metric may vastly underestimate the actual exposure of the fetus. Abel argued that most drinking in pregnant women occurs on 1 or 2 days of the week so that the actual consumption per drinking day is significantly greater than what the average would suggest. Given this pattern of drinking, the adverse developmental eVects of EtOH are not the result of one or two drinks per day but, instead, the result of seven to 14 drinks on a drinking day during the week. In 2000, the American Academy of Pediatrics Committee on Substance Abuse and Committee on Children with Disabilities recommended that women who are pregnant or who are planning a pregnancy should abstain from alcohol because ‘‘there is no known safe amount of alcohol consumption during pregnancy.’’ Noted in the Committee’s recommendations were the findings from a large prospective study of the relationship between EtOH intake and birth weight (Mills et al., 1984). Alcohol intake and birth weight data collected on over 30,000 pregnancies indicated a reduction in mean birth weight ranging from 14 grams for women who drank less than 1 drink per day to 165 grams for those who consumed three to five drinks daily. The Committee recommended that significant eVorts should be made to educate pediatricians, health care professionals, and their patients as well as elementary, junior, and high school students concerning the harmful eVects of

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alcohol consumption during pregnancy. The Committee also encouraged pediatricians to assume a leadership role in public education campaigns aimed at ‘‘decreasing the incidence of FAS through reduction in alcohol use by pregnant women’’ (American Academy of Pediatrics, 2000). For methanol, the weight of evidence to determine the risk of adverse eVects on development from prenatal exposure comes largely from studies using animal models. The current Environmental Protection Agency (EPA) risk assessment for methanol is restricted to oral intake (Integrated Risk Information System or IRIS). A no‐observed‐eVect level (NOAEL) of 500 mg/kg/day was calculated based on a 90‐day subchronic study of rats gavaged daily with 0, 100, 500, or 2500 mg/kg/day methanol. Animals were exposed to methanol for 42 to 90 days before sacrifice. Results indicated a reduction in the brain weights of animals in the highest dose group, resulting in a NOAEL at the 500 mg/kg/day dose. Research published in 2004 indicates that a dose of at least 3400 mg/kg/day oral MeOH on GD 7 results in craniofacial abnormalities and skeletal defects in fetal mice (Rogers et al., 2004). As displayed in Fig. 6, malformations in exposed subjects included

FIG. 6. Facial dysmorphia in fetal mice exposed to methanol. Controls are shown in images A and E. Facial malformations induced from maternal oral methanol exposure include holoprosencephaly with single naris and micrognathia (B), cleft lip (C), maxillary and mandibular hypoplasia (B–D, F, H), lateral facial cleft (G), low‐set ears (B–D, F–H), and gross facial dysgenesis (D, F, H). Reprinted with permission from Dr. John Rogers, Reproductive Toxicology Division, National Health and Environmental EVects Laboratory, OYce of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina.


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holoprosencephaly, cleft lip, maxillary and mandibular hypoplasia, facial cleft, low‐set ears, and gross facial dysgenesis. These results are particularly noteworthy as they demonstrate that the craniofacial defects associated with MeOH exposure are strikingly similar to those associated with EtOH exposure. This suggests that the frank teratogenic eVects associated with oral intake may be similar between the two compounds. While no EPA risk assessment is available for inhalation exposure to methanol, the data from our laboratory would indicate that the reference concentration would be quite low. Similar to the results for ethanol, the results from our studies show that exposure to methanol that briefly increases blood methanol levels above background each day is associated with subtle developmental eVects in exposed oVspring. These results indicate that protection of the fetus from chronic exposure to methanol in the environment would require much more stringent exposure standards than those currently in use for occupational exposure. Future studies should focus eVorts on further elucidating the neuroendocrine eVects of methanol that may interfere with full‐term delivery as well as early perceptual–motor and visual memory eVects on oVspring. Studies should use various animal models and focus on the dose–response characteristics of chronic low‐level exposure during pregnancy. ACKNOWLEDGMENTS The authors acknowledge the dedicated assistance of Amy Voltin, Noelle Liberato, and the staV of the Infant Primate Research Laboratory at the University of Washington. We also thank Drs. Ann Streissguth, John Rogers, Kathy Sulik, and Christian EckhoV for providing first‐rate photographic images and assistance with figures and tables. This project was supported by funds from the National Institutes of Health, Grants RO1 ES03745, RO1 ES06673, P51HD02274, P51RR00166, and P30ES07033.

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PCBs and Dioxins HESTIEN J. I. VREUGDENHIL AND NYNKE WEISGLAS‐KUPERUS DEPARTMENT OF PEDIATRICS DIVISION OF NEONATOLOGY, ERASMUS MC–SOPHIA CHILDREN’S HOSPITAL UNIVERSITY MEDICAL CENTER, ROTTERDAM, THE NETHERLANDS

I. A.

NEUROTOXICOLOGY OF PCBs AND DIOXINS

Chemical Properties and Deposition

PCBs and polychlorinated dibenzo‐para‐dioxins (PCDDs) and polychlorinated dibenzo‐furans (PCDFs) (the latter two are summarized as dioxins) are polyhalogenated aromatic hydrocarbons with comparable molecular structures. They consist of a biphenyl ring and, depending on the number and position of chlorine atoms on the two rings, there are 209 theoretically possible PCB discrete chemical compounds, called congeners, and 210 diVerent dioxin congeners (75 PCDDs and 135 PCDFs). Their basic structure is presented in Fig. 1. PCBs were commercially produced as complex mixtures (under trade names such as Aroclor, Clophen, Phenoclor) for a variety of applications, such as dielectric fluids for capacitors and transformers, heat transfer fluids, hydraulic fluids, lubricating and cutting oils, and as additives in pesticides, paints, adhesives, sealants, carbonless copy paper, flame retardants, organic dilutents, and plastics. Their commercial utility was based largely on their chemical and physical stability, including low flammability and their miscibility with organic compounds. The total amount of PCBs produced worldwide from 1929 to the 1980s, when most countries reduced or stopped the production, has been estimated at approximately 1.5 million metric tons (de Voogt & Brinkman, 1989; WHO, 1989). In 1982, it was estimated that 31% had been released to the environment and 65% was still in use or in storage, or deposited in landfills (Tanabe, 1988). Moreover, PCBs can be formed unintentionally as by-products in a variety of chemical processes that contain chlorine and hydrocarbon sources. Dioxins are generally formed as unwanted and often unavoidable byproducts during the synthesis of a wide array of commercial chemical INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus

FIG. 1. Molecular structures of PCBs, PCDDs, and PCDFs.

products, especially those based on chlorinated aromatics, precursors, and intermediates. Moreover, they are formed during various combustion processes, such as burning of solid waste from municipal incinerators. B.

Toxic Mechanisms

The first mechanism that was described for toxic eVects of PCBs and dioxins was, after entering cells, their interaction with a cytoplasmic receptor protein, the aryl‐hydrocarbon (Ah) receptor (Safe & Goldstein, 1989). Depending on the positions of the chlorine atoms on the biphenyl ring

PCBS AND DIOXINS

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structure (ortho, meta, or para position), and consequently the planar shape, the diVerent compounds bind, to a certain extent, to this receptor. Dioxins as well as dioxin-like PCBs (coplanar PCBs; having no chlorine atom on the ortho position) are recognized as potent compounds to interact with the Ah receptor (Safe, 1994). Furthermore, two other groups of PCBs can be distinguished based on the ability to interact with the Ah receptor, mono‐ ortho‐substituted PCBs (weak dioxin-like) and ortho‐substituted PCBs (nondioxin-like PCBs). Mono‐ortho‐substituted congeners have one chlorine atom on the ortho position and are intermediate in their ability to interact with Ah receptors. Ortho‐substituted have more than one ortho‐substitution on the biphenyl ring, which reduces the planarity of the molecule and reduces the ability to interact with the Ah receptor (Kafafi et al., 1993). Both non‐ortho‐substituted (coplanar compounds) and ortho‐substituted PCBs are toxic. Their mechanism of toxicity, however, is likely to be diVerent. As described previously, toxicity of coplanar compounds appears to be mediated by the Ah receptor (Safe, 1994). The toxic potency of a coplanar PCB congener is reflected in a toxic equivalent factor (TEF), based on its ability to bind the Ah receptor relative to the binding ability of the most potent dioxin, TCDD (Safe, 1990; Van den Berg et al., 1998a). For noncoplanar PCBs, the ability of the TEF to predict their neurotoxic potency is low (Giesy & Kannan, 1998; Shain et al., 1991). Since 1995, there is growing evidence that especially non-dioxin-like PCBs and weak dioxin-like PCBs and their metabolites, such as hydroxylated PCBs, may produce a wide spectrum of neurotoxic eVects, while dioxin-like PCBs may have less activity in the central nervous system (CNS) (Fischer et al., 1998; Korach et al., 1988; Shain et al., 1991). C.

CNS Effects

Neurochemical studies have shown that many elements of the CNS, and especially of the developing CNS, are susceptible to exposure to PCBs and dioxins, including cellular and synaptic processes and endocrine systems (Brouwer et al., 1995, 1999; Mariussen & Fonnum, 2001; Tilson & Kodavanti, 1998). These aspects will be further discussed in the following text. At the cellular level, PCBs‐induced alteration in markers for neuronal and glial cell development have been reported in several brain areas in rats that were perinatally exposed to a PCB mixture (Morse et al., 1996a). The levels of these markers for structural and functional brain development were altered in a complex manner, depending on age, sex, or brain region of the animal. The changes were suggestive of neuronal damage or death and were reported in several areas of the brain, including the lateral olfactory tract, striatum, prefrontal cortex, and in the cerebellum and brain stem (Morse et al., 1996a). Perturbations were also reported on intracellular calcium

50


homeostatic mechanisms and second messenger systems that play a role in neuronal growth and normal physiology of cells (Kodavanti & Tilson, 1997; Kodavanti et al., 1993, 1994; Shafer et al., 1996). EVects of exposure to PCBs on synaptic processes included an inhibition of the synaptic transmission assessed in the dendate gyrus of the cerebral cortex in adult rats (Gilbert & Crowley, 1997) as well as in the hippocampus (Niemi et al., 1998) and in the visual cortex in prenatally exposed rats (Altmann et al., 1995). Synaptic transmission can be measured by means of long‐term potentiation, which is a model of synaptic plasticity that is suggested to be related to learning and memory at the synaptic level (McNaughton, 1993). Several brain neurotransmitter systems have been shown to be aVected by exposure to PCBs and dioxin, including dopamine, serotonine, glutamate, GABA, and cholinergic systems (Eriksson et al., 1991; Mariussen & Fonnum, 2001; Mariussen et al., 1999; Morse et al., 1996b; Seegal et al., 1997). EVects of perinatal exposure on dopaminergic systems have been documented most thoroughly. It appeared that in rats, developmental exposure to PCBs can result in opposite alterations in brain dopamine concentrations, depending on the type of the congener. For example, perinatal exposure to ortho‐substituted PCBs led to decreases in brain dopamine, whereas perinatal exposure to a coplanar PCB congener resulted in elevated concentrations of dopamine (Brouwer et al., 1995; Seegal et al., 1997). D.

Other Toxic Effects

PCBs and dioxins, and especially one type of PCB metabolites, the hydroxylated PCB metabolites, are presently known as endocrine disrupters. Multiple PCB congeners may impact upon multiple endocrine systems that may communicate with each other and are involved in fetal CNS development. These complex mechanisms of actions have not been much studied, and their role in developmental neurotoxic PCB and dioxin eVects remains largely unknown. Most information is available on thyroid hormone changes, generally including decreases in plasma thyroid hormone levels in fetal and neonatal rats as well as in plasma of the women of the Dutch cohort and their children, 2 weeks after birth (Brouwer et al., 1995, 1998; Koopman‐Esseboom et al., 1994b). Moreover, interactions with the steroid hormone system are suggested, due to PCB‐ and dioxin‐induced changes in steroid hormone homeostasis or to endocrine-like actions of these contaminants, particularly during development (Golden et al., 1998). Estrogenic (Bitman & Cecil, 1970; Kester et al., 2000; Korach et al., 1988), anti‐estrogenic (Amin et al., 2000; Jansen et al., 1993; Kramer et al., 1997; Moore et al., 1997), and anti‐androgenic (Hany et al., 1999) eVects have been

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51

described in in vivo and in vitro studies, possibly depending on congener type and/or metabolite.

II.

HUMAN EXPOSURE TO PCBs AND DIOXINS

Ninety percent of human exposure to PCBs and dioxins occurs through the diet, with food of animal origin being the predominant source (i.e., background exposure) (Furst et al., 1992). Contamination of food is primarily caused by deposition of emissions of various sources on farmland and water (waste incineration, production of chemicals) followed by bioaccumulation in the food chains, in which they are particularly aYliated with fat. Other sources may include contaminated feed for cattle, chicken, and farmed fish, improper application of sewage sludge, flooding of pastures, and waste eZuents (Furst et al., 1992). Since PCBs and dioxins are lipid‐soluble and are only slowly degraded, with half‐lifetimes in humans ranging from 1.8 to 9.9 years (Steele et al., 1986; Taylor & Lawrence, 1992), these compounds accumulate in adipose tissue. During pregnancy, PCBs and dioxins are transferred through the placenta and are able to cross the blood–brain barrier, exposing the fetus during a vulnerable time of CNS development (Masuda et al., 1978). PCBs have been detected in brain tissue of stillborn babies, exposed to environmental levels of PCBs, from 17 weeks of gestational age onward (Lanting et al., 1998a). A breast‐fed infant is additionally exposed to relatively large amounts of PCBs and dioxins, since these compounds are excreted in breast milk. For example, PCB levels were still approximately four times higher in 42‐month‐old children that were breast‐fed during infancy than in their formula‐fed counterparts that were predominantly prenatally exposed to PCBs and dioxins (Patandin et al., 1997). Since these neurotoxic compounds are able to interact with many processes of the CNS, including neurotransmitters and hormones that mediate brain development, the developing CNS is considered to be especially vulnerable to exposure to these neurotoxic compounds. Hence, prenatally, the CNS may be most vulnerable to harmful eVects of exposure to these compounds. Prenatal exposure can be regarded as chronic exposure of the developing brain. Postnatally, the CNS continues to develop rapidly, doubling in weight in the first year of life, reaching 90% of its adult size by 5 years of age. Much of this increase is due to an increase in neuronal maturation, production of glial cells, outgrowth of dendrites and axons, formation of synapses, and myelination of axons (Dodgson, 1962). Moreover, extensive cell death and synapse elimination takes place postnatally. These postnatal maturation processes

52


may be especially vulnerable to adverse eVects of lactational exposure to PCBs and dioxins. The maturation rates vary for diVerent brain structures. Therefore, lactational exposure to PCBs and dioxins can be hypothesized to cause structure‐related functional diVerences, depending on the time window of exposure. For example, during the first 2 years of life in humans, functional cortical activity increases earliest in the sensorimotor and occipital cortices, before 3 to 6 months, the auditory and visual association cortices from 4 to 7 months, and latest in the frontal cortex, after 6 to 12 months (Chiron et al., 1992; Chugani et al., 1987). Moreover, timing of maximum brain growth, maximum synaptic density, dendritic arborizations, and myelination, all occur first in primary motor and sensory areas and later in the frontal cortex (Barkovich & Kjos, 1988; Barkovich et al., 1988; Becker et al., 1984; Huttenlocher & Dabholkar, 1997; Mrzljak et al., 1990).

A.

Perinatal Exposure to PCBs and Dioxins and Neurodevelopmental Outcomes

1. ACCIDENTAL EXPOSURE

Two accidents (‘‘Yusho,’’ Japan, 1968, and ‘‘Yu Cheng,’’ Taiwan, 1979) clearly showed the neurotoxic potential of prenatal exposure to these compounds. Large populations were accidentally exposed for relatively short periods to rice oil that was contaminated during the manufacturing process with heat transfer fluids containing PCBs, PCDFs, and polychlorinated quarterphenyls (PCQs). Children born to exposed Yusho mothers were described as dull and inactive at 6 years of age and had IQs averaging 70 (Harada, 1976). Cognitive functions were more thoroughly addressed in the Yu Cheng cohort (n ¼ 118), showing consistent cognitive delays of 5 points from 4 to 7 years of age compared to a matched control group (Chen et al., 1992; Lai et al., 1994). In children born up to 6 years after the incident, cognitive abilities were comparably aVected (Chen et al., 1992; Lai et al., 1994). Moreover, in 7‐ to 12‐year‐old Yu Cheng children, latencies and amplitudes of the P300 peak of an auditory event related potential, reflecting CNS mechanisms that evaluate and process relevant stimuli, were respectively longer and decreased in the exposed oVspring compared to their matched controls (Chen & Hsu, 1994). The measured P300 latencies in that study were inversely correlated with IQs. In the Yu Cheng cohort at 6, 7, 8, and 9 years of age, more spatially related cognitive abilities were diVerently aVected in boys and girls. Only the exposed boys scored lower than their nonexposed matched controls (Guo et al., 1995). These results, therefore, may have provided the

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53

first evidence of sex steroid hormone–modulating eVects of PCBs and dioxins on cognitive development in humans. 2. ENVIRONMENTAL EXPOSURE

The neurodevelopmental eVects described in the Yusho and Yu Cheng cohorts leave little doubt that high levels of prenatal exposure to mixtures of PCBs and dioxins result in neurotoxic eVects of these compounds in humans. Subtle neurodevelopmental eVects of perinatal exposure to PCBs and dioxins have also been described in several cohorts of children that were perinatally exposed to environmental levels of PCBs and dioxins (Darvill et al., 2000; Jacobson & Jacobson, 1996; Koopman‐Esseboom et al., 1996; Patandin et al., 1999b; Rogan & Gladen, 1991; Walkowiak et al., 2001). In these cohort studies, neurological, cognitive, and psychomotor aspects have been studied prospectively. The largest PCB cohorts include two cohorts that were selected based on maternal consumption of PCB‐contaminated fish from the North American Great Lakes: the Lake Michigan cohort (n ¼ 313) that was recruited between 1980 and 1981 (Jacobson et al., 1984a,b) and the more recently (1991–1994) recruited Oswego cohort (n ¼ 293) (Lonky et al., 1996). Another large cohort study has been executed in North Carolina, consisting of 912 mother–infant pairs that were recruited from a general population between 1978 and 1982 (Rogan et al., 1986a). In Europe, the main cohort studies include cohorts in Denmark, The Netherlands, and Germany. The two Danish cohorts were recruited in the Faeroe Islands: the first cohort consists of 435 children born between 1986 and 1987 (Grandjean et al., 1992), the second cohort was recruited from 1994 to 1995 (n ¼ 192), as part of a multicenter cohort study in which the Dutch PCB/dioxin study and a German study participated as well. The Danish cohorts are diVerent from other Northern European cohorts, due mainly to local dietary habits that include consumption of pilot whale blubber and whale meat. In these children, PCB levels were higher compared to levels in Northern Europe, whereas dioxin levels were comparable (Grandjean et al., 1995). The Dutch cohort (n ¼ 418) (Koopman‐Esseboom et al., 1994a) and German cohort (n ¼ 171) (Winneke et al., 1998), respectively, recruited between 1990–1992 and 1994–1995, both consist of mother–infant pairs that were drawn from the general population. The cohorts had similar inclusion criteria and used similar neurodevelopmental tests. In the Dutch cohort, however, restrictions were applied on the number of included breast‐fed children to study lactational exposure to PCBs and dioxins more thoroughly. Half of the recruited population had been breast‐fed for at least 6 weeks during infancy and the other half was fed with formula milk in which PCBs and dioxins were not

54


detectable. The formula‐fed children represent children that were exposed mainly prenatally to PCBs and dioxins. The study design, inclusion and exclusion criteria, and PCB and dioxin measurements applied in the Dutch PCB/dioxin study will be presented in more detail. 3. NEURODEVELOPMENTAL EFFECTS

Neonatal neurological eVects of prenatal exposure to PCBs include deficits such as poorer autonomic regulation and more abnormal reflexes (Jacobson et al., 1984b; Lonky et al., 1996), hypotonia (Huisman et al., 1995a; Rogan et al., 1986b), and hyporeflexia (Rogan et al., 1986b). At 18 months of age, prenatal exposure to PCBs was negatively associated with the neurological condition in the Dutch PCB/dioxin cohort (Huisman et al., 1995b); however, this adverse eVect was not seen on the neurological condition in these children at 42 months of age (Lanting et al., 1998b). Assessment of standardized developmental tests, measuring general cognitive and psychomotor abilities, showed negative eVects of prenatal exposure to PCBs on psychomotor abilities until 2 years of age in the North Carolina (Gladen et al., 1988; Rogan & Gladen, 1991) and in the Dutch cohort at 3 months of age (Koopman‐Esseboom et al., 1996). Cognitive eVects of prenatal exposure to PCBs were seen at 7 months of age (Winneke et al., 1998) and more pronounced negative eVects were seen on more matured general cognitive abilities measured at 42 months (Patandin et al., 1999b; Walkowiak et al., 2001) and at 11 years of age (Jacobson & Jacobson, 1996). In the North Carolina study, however, prenatal exposure to PCBs was not related to cognitive and psychomotor abilities at 3, 4, and 5 years of age (Gladen & Rogan, 1991). Adverse eVects of prenatal exposure to PCBs have also been described on more specific cognitive domains, such as processing time, attention, and memory skills (both verbal and numerical auditory memory) in children at 4 years of age (Jacobson et al., 1990, 1992). Moreover, negative relations between prenatal PCB exposure and verbal comprehension skills at 42 months of age (Patandin et al., 1999b) and 11 years of age (Jacobson & Jacobson, 1996) have been described, in addition to verbal IQs and concentration skills (Jacobson & Jacobson, 1996). EVects of lactational or postnatal exposure to PCBs and dioxins have been detected in a few studies. In the Dutch cohort, psychomotor abilities at 7 months of age were decreased in children that were breast‐fed with relatively high concentrations of PCBs and dioxins (Koopman‐Esseboom et al., 1996). At 42 months of age, in the German cohort, negative eVects of postnatal exposure have been described on general cognitive abilities (Walkowiak et al., 2001).

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55

The results of the neurodevelopmental studies from birth to 42 months of age in the Dutch PCB/dioxin cohort are summarized in Table I.

III.

BEHAVIORAL ANIMAL STUDIES

The potential of subtle neurodevelopmental eVects of perinatal exposure to environmental levels of PCBs and dioxins seen in human studies is supported by the results of behavioral animal studies. Perinatal exposure to PCBs and dioxins has been related to several motor deficits, including impaired development of the righting reflex in rats and in mice with impaired ability to remain on a rotating rod (Overmann et al., 1987; Thiel et al., 1994). Moreover, in mice, perinatal exposure to a dioxin-like PCB congener was related with ‘‘spinning’’ behavior, diminished grip strength, and ability to traverse a wire rod (Tilson et al., 1979). Perinatal exposure to a PCB mixture resulted in impairment on several tasks that involve acquisition or recollection of spatial information, including impaired performance on spatial (based on the location of an object) discrimination reversal tasks (Bowman et al., 1978; Schantz et al., 1989, 1991) and decreased accuracy on a spatial delayed alteration task in monkeys (Levin et al., 1988; Schantz et al., 1991). In both tasks, memory and attentional processes are involved. Since the accuracy deficit did not worsen with increasing delay, the eVect was interpreted not as a memory impairment but rather as failure of attentional processes (Schantz et al., 1991). Monkeys that were perinatally exposed to a mixture of PCBs also performed diVerently on a fixed interval scale (Mele et al., 1986). In this task, a range of functions is assessed including inhibitory processes, maximal response rates, and temporal organization of behavior (Rice, 1988). The exposed monkeys showed disruptions in the temporal pattern of responding and slight elevations in their response rate (Mele et al., 1986). It has been suggested that in some of these behavioral deficits, processes related to the prefrontal cortex are involved in the mechanism of neurotoxic action of PCBs, potentially including mesocortical dopaminergic projections that terminate in the prefrontal cortex (Schantz et al., 1989, 1991). The deficit patterns on the discrimination reversal learning task (Schantz et al., 1989, 1991) and on the delayed spatial alteration showed similarities with deficits of monkeys with lesions to the dorsolateral area of the prefrontal cortex (Goldman et al., 1971). However, the current knowledge on brain structure‐related eVects of perinatal exposure to PCBs is too limited to support the hypothesis of prefrontal cortex involvement in the mechanism of eVect.

TABLE I SIGNIFICANT ASSOCIATIONS BETWEEN PERINATAL EXPOSURE TO PCBS AND DIOXINS AND NEURODEVELOPMENTAL OUTCOMES FROM 2 WEEKS TO 42 MONTHS OF AGE IN THE DUTCH COHORT Age

Cohorta

Neurological condition

2 wks

RþG

SPCBbreast milk, Total TEQ

Psychomotor development PDI, Bayley Scales of Infant Abilities Psychomotor development PDI, Bayley Scales of Infant Abilities Neurological condition

3m

R

SPCBmaternal

7m

R

Dioxin TEQ

18 m

RþG

SPCBmaternal/cord

42 m

RþG

SPCBmaternal/cord

42 m

R

SPCBmaternal/cord

Outcome variable

Exposure variable

56 General cognitive abilities K‐ABCc: Cognitive, Sequential & Simultaneous processing scale Verbal comprehension Reynell Developmental Language Scales

EVect description Higher breast milk levels of PCBs and dioxins were related with lower NOSb and higher incidence of hypotonia (Huisman et al., 1995a). Higher prenatal exposure was related with lower PDI scores (Koopman‐Esseboom et al., 1996). The highest exposed BF children (33%) scored lower than the less exposed BF children and comparable to FF children (Koopman‐Esseboom et al., 1996) Higher prenatal exposure was related to lower NOSb (Huisman et al., 1995a). Higher prenatal exposure was related with lower scores on the three scales. EVects were more pronounced in the FF group, lacking significance in the BF group (Patandin et al., 1999b). Higher prenatal exposure was significantly related with lower scores in the FF group, and not in the BF group (Patandin et al., 1999b).

Attentional processes Free play observation

42 m

R

SPCBmaternal/cord

Reaction time and sustained attention Computerized vigilance task

42 m

R

SPCBcord/ SPCB42 months

Problem Behavior Teacher CBCLd

42 m

R

Behavior Groninger Behavioral Scale (GBO)

42 m

R

SPCBmaternal/cord, SPCBbreast milk, Total TEQ SPCB42 months

Higher prenatal exposure was related with less episodes of high-level play, suggestive of less attentional abilities (Patandin et al., 1999c). Prenatal PCB exposure was related with more errors in the beginning of the task, suggestive of less focused attention (Patandin et al., 1999c). SPCB levels at 42 months were related with longer reaction times and a higher slope, suggestive of less sustained attention (Patandin et al., 1999c). Prenatal PCB and dioxin exposure was related to a higher prevalence of Withdrawn/Depressed behavior (Patandin et al., 1999c). SPCB levels at 42 months were related with a higher score on the GBO questionnaire, indicating more hyperactive behavior (Patandin et al., 1999c).

57

SPCB ¼ Sum of PCBs, IUPAC nos. 118, 138, 153, and 180 in maternal plasma measured during pregnancy, in cord plasma, or in breast milk. SPCB42 months ¼ Sum of PCBs, IUPAC nos. 118, 138, 153, and 180, measured in plasma of 42-month old children, representing mainly lactational transfer of maternal PCBs (the breast‐fed group) and in part during gestation (the formula‐fed group) [Patandin, 1997 #59]. TEQ ¼ Toxic Equivalent; Total TEQ ¼ sum of TEQs of 8 dioxin like PCBs (IUPAC nos. 77, 105, 118, 126, 156, and 169) and 17 dioxin congeners measured in breast milk. Shaded cells are results that give evidence of negative eVects of lactational exposure. a R ¼ Rotterdam and G ¼ Groningen cohort. b NOS ¼ Neurological optimality score. c K‐ABC ¼ Dutch Kaufman Assessment Battery for Children. d CBCL ¼ Child Behavior Checklist.

58

Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus IV.

A.

HUMAN NEURODEVELOPMENTAL PCB AND DIOXIN RISK ASSESSMENT

Prenatal Versus Postnatal Exposure to PCBs and Dioxins

Although much larger quantities of PCBs and dioxins are transferred to the child postnatally through lactation than prenatally, human epidemiological studies suggest more pronounced neurodevelopmental eVects of prenatal exposure to PCBs and dioxins compared to postnatal exposure to these compounds. However, several animal studies have shown profound behavioral impairments induced by postnatal exposure to low levels of a mixture of ortho‐substituted PCB mixtures that is representative of the PCB mixture found in human milk. In these monkeys, impaired performance was seen on spatial learning tasks, including impairment in learning a delayed spatial alteration task (Rice, 1998a, 1999) and more perseverative responding (Rice, 1999; Rice & Hayward, 1997). Moreover, slower acquisition of a fixed interval task and inability to inhibit inappropriate responding have been associated with postnatal exposure to PCB mixtures (Rice, 1997, 1999). These impairments suggest a discrimination learning deficit and diYculty in adaptively changing response patterns—deficits that are suggestive of involvement of prefrontal cortex processes in the neurotoxic mechanism of PCBs and dioxins (Rice, 1999). EVects of lactational exposure on these functions need to be addressed more thoroughly in the human studies. In human studies, addressing neurodevelopmental eVects of lactational exposure to PCBs and dioxins is complex. Breast milk contains several substances, such as several long‐chain polyunsaturated fatty acids, that are not available in formula milk. These acids are important constituents of the structural lipids of nonmyelinated cell membranes in the developing nervous system and are essential for growth, function, and integrity (Innis, 1994), and may therefore be important for optimal brain development. A meta‐ analysis of studies that addressed neurodevelopmental benefits of breast‐ feeding provided evidence for enhanced early cognitive development that sustained through childhood and adolescence (Anderson et al., 1999), taking into account a number of studies which suggested that diVerences in cognitive development were attributable to the generally associated diVerences in social economic conditions. The latter aspect forms another complicating feature of assessing neurodevelopmental eVects of lactational exposure: in Western societies, parents who choose to breast‐feed their child are likely to be diVerent in several parental and home environmental conditions. These aspects may influence the susceptibility to harmful eVects of perinatal exposure to PCBs and dioxins. The results of prenatal exposure to PCBs and dioxins on general cognitive abilities at 42 months of age in the Dutch cohort

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may illustrate the complexity of exploring eVects of exposure to PCBs and dioxins in breast‐fed children. At 42 months of age, adverse eVects of prenatal exposure to these compounds were more pronounced in the formula‐fed group compared to the breast‐fed group of children (Patandin et al., 1999b). B.

Sex Steroid–Related Behavioral PCB and Dioxins Effects

Neurotoxic eVects of perinatal exposure to PCBs and dioxins that cause developmental deficits may be mediated by endocrine‐disrupting properties of PCBs and dioxins. For example, steroid hormones play a mediating role in CNS development and influence not only reproductive but also nonreproductive behaviors that show sex diVerences (Fitch & Denenberg, 1998; Matsumoto, 1991). In animals, some eVects of perinatal exposure to PCBs and dioxins on nonreproductive behaviors have been reported. For example, a feminizing eVect on sweet preference was found in male rats that were perinatally exposed to a PCB mixture representative to PCBs found in human milk. In their female counterparts, sweet preference was not aVected (Hany et al., 1999). In contrast, prenatal exposure to a dioxin (TCDD) and coplanar (dioxin-like) PCBs decreased sweet preference in female rats, which can be interpreted as a masculinizing eVect in females. In the exposed males, no change in sweet preference was seen (Amin et al., 2000). The animal studies suggest both feminizing and masculinizing eVects of perinatal exposure to PCBs and dioxins on sex‐specific behavior, which may suggest steroid hormone–mediated eVects of PCB and dioxin exposure. In human studies, eVects of perinatal exposure to PCBs and dioxins on nonreproductive sex‐specific behavior have hardly been addressed. The only study that provided some evidence for steroid hormone–mediated behavioral eVects of prenatal exposure to PCBs and dioxins is the study in the highly exposed children of the Yu Cheng cohort. In this cohort, more spatially related cognitive abilities, which generally show some sex diVerences, were diVerently aVected in boys and girls. Only the exposed boys scored lower than their nonexposed matched controls (Guo et al., 1995). C.

Neurodevelopmental Interstudy Differences

The results of epidemiological studies that address neurodevelopmental eVects of perinatal exposure to environmental levels of PCBs show inconsistencies both between cohorts and within cohorts at diVerent ages. These diVerences in outcome do not necessarily undermine conclusions that prenatal exposure to environmental levels of PCBs is related to subtle harmful eVects on child neurodevelopment. The diVerences could be related to a

60


number of factors, including diVerences in exposure assessment techniques, diVerences in composure of environmental PCB mixtures, and diVerences in exposure levels. Moreover, diVerences in parental and home environmental conditions or the occurrence of other neurotoxic agents, which may confound relationships between exposure and neurodevelopmental outcome, may have led to diVerences in results. Additionally, diVerent neurodevelopmental outcome variables have been used in the cohort studies. Furthermore, addressing neurodevelopmental eVects of perinatal exposure to PCBs and dioxins as well as comparison of eVects assessed by diVerent cohorts at diVerent ages is complicated by the fact that the outcome variables are developmental qualities. EVects of perinatal exposure to PCBs and dioxins may not become evident until further maturation of the child. Some of these issues will be discussed in the following text. 1. EXPOSURE LEVELS

Exposure levels in the cohorts are diYcult to compare since, in the earlier American studies, diVerent assessment techniques have been used compared to the later initiated studies. In an eVort to compare exposure levels of several cohorts, median levels of PCB153 in maternal blood were used for comparison (Longnecker et al., 2003). This congener is always among the PCB congeners present at the highest concentration and constitutes a large proportion of the PCBs mixtures in all studies. That study showed that the median Dutch PCB153 level (0.10 mg/g lipid) was comparable to the median level in the Lake Michigan cohort (0.12 mg/g lipid), the North Carolina cohort (0.08 mg/g lipid), and the German median levels (0.14 mg/g lipid). The median exposure level in the Faeroe Islands cohort (recruited between 1994 and 1995) was 3 to 4 times higher than in these studies (0.45 mg/g lipid). 2. CONFOUNDING VARIABLES AND POTENTIAL DIFFERENCES IN SUSCEPTIBILITY TO EFFECTS OF PCB AND DIOXIN EXPOSURE

In the epidemiological studies, subjects could not be randomly assigned to predetermined levels of exposure or type of feeding during infancy. Samples were based on volunteer mother–infant pairs and parents were free in choosing the type of infant feeding they preferred because of acceptable ethical concerns. Therefore, all cohort studies have made eVorts to assess potential confounding variables, to adjust for these variables when studying the relation between perinatal exposure to PCBs and dioxins and neurodevelopment. In Western societies, the relation between perinatal exposure to PCBs and dioxins and neurodevelopment is often confounded by parental and home environmental conditions. Due to the physical stability and accumulation of PCBs and dioxins in human tissues, PCB and dioxin body burdens are

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61

strongly related to maternal age at birth. Mothers at older age who give birth to a child are often higher educated and have higher IQs than women at younger age who give birth to a child. Maternal age may also reflect other aspects of social economic conditions as well as psychosocial age‐related attributes (Stein, 1985). Child development is a process in which structural changes and environmental experiences influence each other mutually. For example, many cognitive skills, including IQs, verbal and spatial abilities, and perceptual speed (Alarcon et al., 1998; Plomin & Loehlin, 1989; Posthuma et al., 2001), have been shown to be under genetic influences. Environmental or psychosocial aspects, such as intellectual stimulation, organization of the home environment, verbal responsivity of the parents, variability of daily experience, and parental involvement also influence cognitive development (Bradley et al., 2001). In animal studies, environmental aspects influenced cortical diVerentiation and dendritic formation, thereby changing the functional connectivity of the nervous system. Several lines of evidence point toward the relationship between dendritic and synaptic changes and experiences and, more specifically, learning (Devoogd et al., 1985; Moser et al., 1994; Rosenzweig & Bennett, 1996; WolV et al., 1995). Moreover, numerous animal studies showed that environmental enrichment can compensate for and possibly even reverse some of the adverse eVects of developmental insults (Brenner et al., 1985; Diamond et al., 1975; Kolb, 1989). These studies suggest potential for structural and functional recovery throughout the cortical maturation period in animals. In humans, evidence of neural plasticity throughout the maturation period may be supported by results of studies in low birth weight children. These studies reported that in children at high biological risk, favorable early parental and home characteristics could compensate for or mask developmental delays (Landry et al., 1997; PfeiVer & Aylward, 1990; Weisglas‐ Kuperus et al., 1993). Hence, these genetic and environmental conditions are important predictors of cognitive development and can additionally be important in determining the vulnerability of an individual child or a given population to the eVects of neurotoxicants. Favorable parental and home environmental conditions may protect some groups against negative neurodevelopmental eVects of perinatal PCB and dioxin exposure. Evidence for this hypothesis is seen in the Dutch study showing less pronounced eVects of prenatal exposure to PCBs in breast‐fed children compared to formula‐fed children at 42 months of age (Patandin et al., 1999b). A reanalysis in the Lake Michigan cohort similarly showed that prenatal exposure to PCBs was related with lower IQs at 11 years of age in predominantly the group of formula‐fed children (n ¼ 56, 31%) (Jacobson & Jacobson, 2001). In the North Carolina cohort, prenatal exposure to PCBs

62


was not related to later cognitive and motor development from 3 to 5 years of age, in contrast to the Lake Michigan and Dutch study. In the North Carolina cohort, a relatively high proportion of the population was breast‐ fed during infancy (88%). Moreover, the average years of college education in the North Carolina cohort was 3 years and in the Lake Michigan cohort 1 year. In the Dutch cohort, 40% of the mothers have finished high school and 30% of them finished professional and university training. Some of the diVerences in neurodevelopmental eVects of PCB exposure between study centers can therefore be hypothesized to be related to cohort diVerences in the levels of these conditions that are important to child development. 3. CONFOUNDING BY OTHER NEUROTOXIC COMPOUNDS

Due to the correlational feature of the epidemiological studies addressing eVects of perinatal exposure to PCBs and dioxins, relations between these compounds and outcome are potentially related to exposure to other neurotoxic compounds, such as methyl mercury and lead. For example, in the Faeroe study, in which the local diet consists predominantly of fish and fish products, PCB and dioxin levels were seen to be relatively high compared to other European studies, as were the levels of methyl mercury. Significant relations between prenatal exposure to PCBs and reaction time and (semantic) memory skills appeared to be mainly attributable to prenatal exposure to methyl mercury compounds (Grandjean et al., 2001). However, in children exposed to high levels of methyl mercury, eVects of prenatal exposure to PCBs on these outcome variables were more pronounced than in children exposed to lower levels of methyl mercury, suggesting a potential interaction between these neurotoxic compounds in their neurodevelopmental eVects. In the Lake Michigan cohort, mothers were selected based on their diet history on Lake Michigan PCB‐contaminated fish. Fish and other aquatic species often form the source of exposure to PCBs as well as other neurotoxic compounds, such as methyl mercury. The relations between neurodevelopment and prenatal PCB exposure as described in the Lake Michigan studies, therefore, may have been confounded by exposure to this compound. However, based on the congruence between the results of animal studies and several human cohort studies, it has been suggested that the deficits observed in the Lake Michigan studies result, at least in part, from PCB exposure (Rice, 1995). In contrast to the Lake Michigan study, the North Carolina, Dutch, and German cohorts were recruited from the general public which may reduce the risk of confounding by methyl mercury. In the Netherlands, PCB and dioxin exposure occurs mainly through dietary intake of predominantly dairy products, as well as processed food and meat and fish products (Patandin et al., 1999a). In the Dutch PCB/dioxin cohort, lead and

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cadmium levels in blood samples drawn from 18-month old children (n ¼ 151) were relatively low (Weisglas‐Kuperus et al., 2000) and not related to cognitive outcome at 42 months of age (Patandin et al., 1999b). 4. NEURODEVELOPMENTAL TESTS AND THE DEVELOPMENT OF COGNITIVE ABILITIES

The cohort studies also show diVerences in the neurodevelopmental testing protocols that were used to explore neurotoxic eVects of perinatal exposure to PCBs and dioxins. Neurodevelopmental assessment has occurred at diVerent ages using diVerent test materials, which complicates comparison of the diVerent cohorts, especially due to the developmental nature of cognitive and motor abilities and since some aVected functions may not become apparent until a more mature age. Most prospective longitudinal studies have assessed general cognitive and motor abilities by means of developmental tests that were reassessed repeatedly through childhood. Performance on the developmental tests reflects, especially at a more mature age, a broad range of domains of function, including memory, visuospatial abilities, verbal and quantitative reasoning, and attentional aspects. Although general cognitive development seems to be the most relevant outcome variable in risk assessment studies, because of its predictive feature for later outcome, general cognitive ability indices may be too general to assess subtle eVects of exposure to neurotoxic compounds. The general cognitive score can obscure important individual diVerences in specific cognitive profiles, since children with diVerent cognitive profiles can have comparable scores on this outcome variable. Moreover, it can be reasoned that general cognitive scores reflect the product of learning, which is strongly related to social economic aspects, rather than processes of learning. The development of general cognitive abilities may progress at diVerent rates. Reported negative eVects of prenatal exposure to PCBs at diVerent assessment times within one cohort do not resolve the question of whether the same children are aVected in their abilities at the diVerent assessment periods. Consequently, risk assessment studies into eVects of perinatal exposure to PCBs and dioxins on cognitive and motor abilities in children may benefit from addressing the level and course of the development of these abilities. Early PCB and dioxin exposure may induce changes in brain structures that continue to influence neurodevelopment during maturation, resulting in delayed eVects of functions that develop later in childhood. Especially when eVects of lactational exposure to PCBs and dioxins are addressed, structure‐ related functional diVerences potentially, depending on the time window of exposure, can be hypothesized due to diVerences in maturation rates of diVerent brain structures. The exploration of neurotoxic eVects of perinatal exposure to neurotoxic agents therefore should address more specific

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domains of cognitive functioning that can be assessed at a more mature age. These domains are not suYciently measured by developmental or IQ tests, since most domains are indicated by too few items to provide reliable measurement of domain‐specific performance.

V.

THE DUTCH PCB AND DIOXIN STUDY AT SCHOOL AGE

Children enrolled in the Dutch PCB/dioxin cohort (Rotterdam and Groningen cohort) were invited to participate in follow‐up assessment at 6 to 7 years of age and half of the Rotterdam cohort was invited at 9 years of age as well. General cognitive and motor abilities, gender role‐play behavior, and neuropsychological functions were assessed. A neurophysiological assessment was also included. A.

Aims of the Study at School Age

The general aim of this study was to describe neurodevelopmental eVects of perinatal exposure to environmental levels of PCBs and dioxins in normal Dutch children at school age, as well as on the development of general cognitive and motor abilities from 3 to 84 months of age. In addition, the goal was to gain more insight into potential compensating eVects of parental and home environmental conditions and breast‐feeding, as well as into neurotoxic mechanisms of eVects of perinatal exposure to these compounds. B.

Subjects and Inclusion and Exclusion Criteria

A total of 418 healthy mother–infant pairs were recruited from June 1990 to June 1991. Half of the study population was recruited in Rotterdam (n ¼ 207), a highly industrialized and densely populated area, and the other half in Groningen (n ¼ 211), a semi‐urban area in The Netherlands. Healthy pregnant women were asked by their obstetrician or midwife to participate in a prospective neurodevelopmental study. The cohort consists of Caucasian mother–infant pairs. Pregnancy and delivery had been without complications; instrumental deliveries or caesarian sections were excluded. Only first or second at term‐born infants (37–42 weeks of gestation) who had no congenital anomalies or diseases were included. Because of these criteria, the cohort of children can be presumed to be at relatively low risk for neurodevelopmental deficits. To study the eVects of prenatal as well as postnatal PCB and dioxin exposure, it was aimed to include an equal number of women who intended to breast‐feed their child for at least 6 weeks (BF) and women who intended to

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use formula‐feeding (FF). All infants in the FF group received formula from a single batch (Almiron M2, Nutricia NV, Zoetermeer, The Netherlands) from birth until 7 months of age. In this formula, concentrations of both PCBs and dioxins were below the detection limit. The medical ethics committee of the University Hospital Rotterdam/ Sophia Children’s Hospital, and the Academical Hospital Groningen approved the study design and the parents gave informed consent. C.

Exposure Measurements

The exposure variables that were used in these studies included PCB levels in maternal and cord plasma. Maternal plasma samples were collected from the mothers during the last month of pregnancy and cord plasma samples were collected directly after birth. These samples were analyzed by means of gas chromatography with electron capture detection (GC‐ECD) for four PCB congeners, International Union for Pure and Applied Chemistry (IUPAC) numbers 118, 138, 153, and 180 (Burse et al., 1989; Koopman‐ Esseboom et al., 1994a). Two weeks after delivery a 24‐hour representative breast milk sample was collected from the mothers who were breast‐feeding their children. These samples were analyzed for 17 most abundant dioxins (PCDDs and PCDFs) and three dioxin-like PCBs (IUPAC nos. 77, 126, 169) by means of gas chromatography–high‐resolution mass spectometry (GC‐HRMS). In these samples, 23 non-dioxin-like PCBs (IUPAC nos. 28, 52, 66, 70, 99, 101, 105, 118, 128, 137, 138, 141, 151, 153, 156, 170, 177, 180, 183, 187, 194, 195, and 202) were measured by GC‐ECD (Koopman‐Esseboom et al., 1994a). Toxic potency of the mixture of dioxins and dioxin-like PCBs was expressed by using the toxic equivalent factor approach (Van den Berg et al., 1998b). Prenatal exposure to PCBs is defined as the sum of the concentrations of the four PCB congeners measured in maternal plasma and in cord plasma. PCB and dioxin concentrations in breast milk were assessed shortly after birth and form an indirect measure of prenatal exposure (Rogan et al., 1986a). Postnatal exposure to PCBs and dioxins through lactation was estimated in the BF group by multiplying breast milk levels of PCBs, dioxin-like PCB TEQs (dioxin toxic equivalents), and dioxin TEQs with the numbers of weeks of breast‐feeding. D.

Cognitive and Motor Abilities at School Age

At school age, the Dutch version of the McCarthy Scales of Children’s Abilities (Van der Meulen & Smirkovsky, 1985) was used to assess the general cognitive abilities (General Cognitive Index) and memory and motor

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skills in children of the Rotterdam and Groningen cohort (n ¼ 418). From the original cohort, 90% (n ¼ 376) were willing to participate in this follow‐ up (mean age 6.7 years þ 0.3). The data of four children were excluded from the data analysis because of potential confounding pathology. Prenatal PCB and dioxin levels were comparable for the nonparticipating and participating children. Multiple linear regression analysis showed that, adjusted for confounding variables, prenatal exposure to PCBs and dioxins was not significantly related to cognitive and motor development at school age. Moreover, eVects of prenatal exposure on cognitive and motor abilities were not statistically diVerent for the two feeding groups. However, it appeared that eVects of prenatal exposure to PCBs and dioxins on cognitive and motor abilities were modified by parental and home environmental conditions (maternal age, parental education level and verbal IQ, and HOME score). The parental and home environmental conditions were strongly related to each other. Older maternal age was related to a higher parental education level and verbal IQ and higher scores on the HOME questionnaire, conditions that are considered to be relatively more favorable to child development. The impact of adverse eVects of prenatal exposure to PCBs and dioxins on cognitive and motor abilities was suggested to increase as parental and home environmental conditions were lower. In children raised in relatively more favorable parental and home environmental conditions, subtle eVects of prenatal exposure to PCBs and dioxins were not detectable. EVect modifications of PCB and dioxin exposure by maternal age, parental education level and verbal IQ, and HOME scores could not be explored simultaneously in one regression analysis due to the problem of multicollinearity. The results did not show evidence of negative eVects of lactational exposure to PCBs and dioxins on cognitive and motor outcome, nor of eVect modification of lactational exposure by parental and home environmental characteristics. We concluded that neurotoxic eVects of prenatal exposure to environmental levels of PCBs and dioxins may persist into school age and may result in subtle cognitive and motor delays. The results of this study suggest that parental and home environmental conditions influence the consequences of the neurotoxic eVects on cognitive and motor development (Vreugdenhil et al., 2002a). E.

The Development of General Cognitive and Motor Abilities from 3 to 84 Months of Age

A disadvantage of studying relations between perinatal exposure to PCBs and dioxins and cognitive and motor abilities at a certain age is that the developmental course of these abilities is not captured. Therefore, eVects of perinatal exposure to PCBs, measured in maternal plasma, on

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the development of cognitive and motor abilities, as assessed in the Rotterdam cohort at 3, 7, 18, 42, and 84 months of age, were described using the method of random regression modeling (RRM). Moreover, important predictors of general cognitive and motor development from 3 to 84 months of age were identified in this study. Data on cognitive and motor abilities were available for all analyzable children (excluding children with potential confounding pathology, n ¼ 3). In the initial RRM models, all selected variables of potential relevance to cognitive and motor development were included. In these models, higher levels of prenatal PCB exposure were significantly related to a lower level of cognitive and motor development from 3 to 84 months of age. In this study, the problem of multicollinearity when including the four previously described interaction variables of prenatal PCB exposure and parental and home environmental variables simultaneously in the regression model was solved by centering these variables as well as their main terms. Simultaneous inclusion of these variables showed that eVects of prenatal exposure to PCBs on the level of cognitive development were significantly modified by maternal age, overruling eVect modification by the other parental and home environmental conditions. In children born to younger mothers, eVects of prenatal exposure to PCBs on cognitive development were suggested to be more pronounced than in children born to older mothers, a condition that is likely to reflect more favorable parental and home environmental conditions for child development. Prenatal PCB levels, and modification by maternal age, along with parental education level and verbal IQ and HOME scores, were important determinants of the level of cognitive development. Motor development was eYciently estimated by prenatal PCB levels, including its modification by HOME scores along with parental education levels. EVects of prenatal PCB exposure on motor development were more pronounced when the HOME scores were lower. The results provided no evidence of negative eVects of lactational exposure to PCBs on cognitive or motor development and neither were maternal (prenatal) thyroid hormone levels related to these outcomes. These results provided evidence of adverse eVects of prenatal exposure to PCBs on the level of cognitive and motor development, eVects that may be modified by conditions that are favorable to child development. Compared to the large positive eVects of more optimal parental and home environmental conditions, the negative eVects of prenatal PCB exposure on cognitive development from 3 to 84 months of age were relatively small. EVects of prenatal exposure were more pronounced for motor than for cognitive development. Motor development may therefore be a more sensitive outcome to detect prenatal exposure to PCBs and related compounds than is cognitive development (Vreugdenhil et al., 2002c).

68 F.

Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus Sex‐Specific Play Behavior

As part of the first follow‐up study at school age, play behavior was assessed by means of the Pre‐School Activity Inventory (PSAI) in the Rotterdam cohort (Golombok & Rust, 1993). The PSAI assesses masculine and feminine play behavior scored on three subscales: Masculine, Feminine, and Composite. One hundred sixty PSAI questionnaires were returned (mean age þ SD: 7.5 years þ 0.4). Higher prenatal PCB levels were related with less masculinized play behavior in boys and with more masculinized play behavior in girls. Higher prenatal dioxin levels, available for BF children, were associated with more feminized play in boys as well as in girls, assessed by the Feminine scale. There was no evidence that lactational exposure to PCBs and dioxins was related to play behavior in the total BF group nor in boys and girls separately. The results are suggestive of steroid hormone involvement in the neurotoxic mechanism of action of prenatal exposure to environmental levels of PCBs and dioxins (Vreugdenhil et al., 2002b).

G.

Neuropsychological Functions

Half of the Rotterdam cohort, the lowest prenatally exposed (p25; n ¼ 26) and the highest prenatally exposed children (p75; n ¼ 26) of both feeding groups (total n ¼ 104) were invited to participate in neuropsychological and neurophysiological assessments at 9 years of age. From the invited children, 80% (n ¼ 83) were willing to participate in this follow‐up study (mean age þ SD: 9.2 þ 0.2). Exposure levels of the participating and nonparticipating children were comparable. The assessment of neuropsychological functions included the Rey Complex Figure task, the Auditory–Verbal Test, the Simple Reaction Time task, and the Tower of London (TOL). Prenatally high‐exposed children had, adjusted for confounding variables, longer reaction times and more variation in their reaction times, and lower scores on the TOL than did prenatally low‐exposed children. On the latter task, assessing predominantly executive or planning functions, in contrast to the other tasks, children that were BF for a long period (>16 weeks) scored significantly lower than did FF children. The results of this study are suggestive of multifocal or diVuse neurotoxic eVects of prenatal exposure to PCBs and related compounds. For lactational exposure, the negative eVect on the TOL scores may suggest that processes related to the prefrontal cortex are involved in the neurotoxic mechanism of exposure to PCBs and related compounds. This can be hypothesized since the frontal cortex shows a delayed maturation rate compared to other brain regions and developing brain structures are more

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vulnerable to exposure to PCBs and dioxins. A complex task such as the TOL may also serve as a sensitive outcome parameter to assess neurotoxic eVects of early exposure to PCBs and related compounds (Vreugdenhil et al., 2004a). H.

Neurophysiological Endpoints

The P‐300 is considered to be a cognitive component of event‐related brain potentials and occurs with a latency of about 300 milliseconds when a person is actively processing (‘‘attending to’’) incoming stimuli. Prenatally high‐exposed children had significantly longer P‐300 latencies than prenatally low‐exposed children, adjusted for confounding variables. The results gave no evidence of diVerences in P‐300 latencies related to lactational exposure to PCBs and dioxins. Instead, a longer duration of breast‐feeding (>16 weeks) was associated with shorter P‐300 latencies compared to children that were BF for 6 to 16 weeks and to FF children. No diVerences in P‐300 amplitudes were seen relative to prenatal or postnatal exposure to PCBs and dioxins or to the duration of breast‐feeding. These results suggest that prenatal exposure to PCBs and dioxins delays CNS mechanisms that evaluate and process relevant stimuli at school age, whereas breast‐feeding accelerates these mechanisms (Vreugdenhil et al., 2004b).

VI. A.

DISCUSSION

Neurotoxic Mechanisms

Prenatal exposure to PCBs and dioxins can be regarded as chronic exposure of the developing CNS and many processes of the CNS are likely to be sensitive to exposure to PCBs and dioxins, including neuronal and glial cells, neurotransmitters, and endocrine systems (Brouwer et al., 1995, 1999; Mariussen & Fonnum, 2001; Tilson & Kodavanti, 1998). Consequently, eVects of prenatal exposure to PCBs and dioxins are likely to be of multifocal or diVuse nature. The results of the Dutch PCB/dioxin study and other prospective human PCB studies suggest eVects of prenatal exposure to PCBs on several neurodevelopmental outcome variables, including general cognitive and motor development (Gladen et al., 1988; Jacobson & Jacobson, 1996; Koopman‐Esseboom et al., 1996; Patandin et al., 1999b; Rogan & Gladen, 1991; Vreugdenhil et al., 2002a; Walkowiak et al., 2001; Winneke et al., 1998), verbal comprehension skills (Jacobson & Jacobson, 1996; Patandin et al., 1999b), processing speed (Grandjean et al., 2001; Vreugdenhil et al., 2004a), attention and concentration (Jacobson & Jacobson, 1996;

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Patandin et al., 1999c; Vreugdenhil et al., 2004a), memory skills (Jacobson & Jacobson, 1996; Vreugdenhil et al., 2002a), planning or executive functions (Vreugdenhil et al., 2004a), and on a neurophysiological endpoint that assesses processing and evaluation of auditory stimuli (Vreugdenhil et al., 2004b). In the neuropsychological study, scores on some tests were not related to perinatal exposure to PCBs and dioxins. This may reflect diVerences in sensitivity of neuropsychological tests to measure subtle neurotoxic eVects of perinatal exposure to neurotoxic compounds. The diVerence in sensitivity in a relatively small cohort may aVect the power to detect eVects and may, therefore, result in missing eVects of perinatal exposure to PCBs and dioxins (increasing Type II errors). Postnatally, maturation of diVerent areas in the brain occurs at diVerent rates. The frontal cortex shows the slowest maturation rate. Since developing CNS structures are known to be especially vulnerable to adverse eVects of exposure to PCBs and dioxins, structure‐related eVects of lactational exposure can be hypothesized. Some evidence in support of this hypothesis can be found in the finding that performance on the TOL was the only outcome that was suggested to be related to lactational exposure to PCBs. In planning or executive functions, processes of the prefrontal cortex are especially involved, in which higher cortical functions from several areas of the brains are integrated. In monkeys that were only exposed to PCBs through lactation, PCB‐induced behavioral deficits were also suggestive of prefrontal cortex involvement (Rice, 1999). Moreover, brain dopaminergic systems have been shown to be aVected (Mariussen et al., 2001; Seegal et al., 1997) by exposure to PCBs and some major dopaminergic pathways are known to serve the prefrontal cortex (Saper et al., 2000). Another important aspect of neurotoxic eVects of prenatal exposure to PCBs and dioxins is that the neurodevelopmental consequences of neurotoxic actions of prenatal exposure to environmental levels of PCBs and dioxins may be influenced by parental and home environmental conditions. Global measurements of cognitive and motor abilities are relatively strongly related to parental and home environmental conditions. The results of the Dutch PCB/dioxin study are suggestive of compensation of negative eVects of perinatal exposure on cognitive and motor development in children raised in more favorable parental and home environmental conditions or of cumulative deficits in children raised in less favorable conditions. These results may be in line with animal studies which show a positive impact of an enriched environment on the eVects of brain lesions (Brenner et al., 1985; Diamond et al., 1975; Kolb, 1989) and with some human studies addressing eVects of perinatal exposure to lead and methyl mercury (Bellinger et al., 1988; Winneke & Kraemer, 1984) and the outcome in very low birth

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weight children (Landry et al., 1997; PfeiVer & Aylward, 1990; Weisglas‐ Kuperus et al., 1993). Neurotoxic eVects of perinatal exposure to PCBs and dioxins may be mediated by hormone‐disrupting properties of PCBs and dioxins, for example, in regard to steroid and thyroid hormone systems. The (sex‐specific) eVects of perinatal exposure to PCBs and dioxins on childhood play behavior suggest mediation of behavioral eVects of prenatal PCB and dioxin exposure by the sex‐steroid hormone system. However, evaluation of the relation between prenatal steroid hormone status and PCB and dioxin exposure is needed to further confirm these findings. In this cohort, maternal and infant thyroid hormone levels were related to maternal levels of PCBs and dioxins. Prenatal alterations in prenatal thyroid hormone levels may cause long‐lasting neurodevelopmental deficits (Porterfield, 1994; Rovet & Ehrlich, 1995). However, in the Dutch PCB dioxin study, maternal thyroid hormone status was not statistically related to the level of cognitive and motor development from 3 to 84 months of age. The presently used analyses, therefore, do not provide evidence that prenatal thyroid hormone status is one of the key mechanisms in the neurotoxic eVects of prenatal exposure to PCBs and dioxins on general cognitive and motor development. Animal studies show diVerences in the neurotoxic eVects of nonplanar PCBs and dioxins and dioxin-like PCBs (Fischer et al., 1998; Tilson & Kodavanti, 1997). Humans are exposed to complex mixtures of PCBs and dioxins and their related compounds, such as hydroxylated PCBs. Not finding associations between outcome variables and TEQs or total TEQs may suggest that neurotoxic eVects of PCBs and dioxins were not mediated by the Ah receptor, as is in line with animal studies that report more pronounced neurotoxic actions of nonortho‐substituted PCB congeners than of dioxins and dioxin-like PCBs. The studies presented here show more pronounced eVects on general cognitive and motor abilities of the four nonplanar PCBs (IUPAC nos. 118, 138, 153, and 180) as assessed in plasma compared to the dioxin TEQs and total TEQs assessed in breast milk. However, based on these results, we believe that we cannot diVerentiate eVects of diVerent types of congeners, since the levels of diVerent types of congeners were strongly related (Koopman‐Esseboom et al., 1994a). Moreover, dioxins as well as a more elaborate number of PCB congeners were assessed in breast milk that was available for only half of the cohort. Analyses in this subpopulation may have increased the risk of Type II errors, which may consequently increase the risk of missing associations. However, in regard to play behavior, some evidence of diVerent neurotoxic eVects of PCBs and dioxins can be hypothesized. Prenatal exposure to the sum of the four nonplanar PCBs was suggested to be related with opposite eVects in boys and girls on masculine play behavior, whereas higher levels of prenatal

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exposure to dioxins, expressed in TEQs, were related to more feminized play behavior in both boys and girls in a similar direction. In conclusion, the mechanisms of neurotoxic eVects of prenatal exposure to PCBs and dioxins may include multifocal, or diVuse, neurodevelopmental impairments. Due to diVerences in the maturation of the CNS, lactational exposure may be related to more focal eVects, in which processes related to the prefrontal cortex are suggested to be involved. Neurotoxic eVects on neurodevelopmental outcome that is more strongly related to parental and home environmental conditions may be modified by these conditions. Moreover, steroid hormones are suggested to be involved in the neurotoxic mechanism of eVects of prenatal exposure to PCBs on a sex‐specific nonreproductive behavior. B.

Is Breast‐Feeding Safe?

Although BF children are exposed to relatively large amounts of PCBs and dioxins, negative eVects of lactational exposure to PCB and dioxins are only suggested on the scores on the TOL. The results of this study, therefore, may indicate that eVects of prenatal exposure to PCBs and dioxins are more pronounced than eVects of exposure to PCBs and dioxins through lactation. This is in agreement with most of the human studies that address perinatal exposure to environmental levels of PCBs and dioxins (Gladen et al., 1988; Jacobson & Jacobson, 1996; Jacobson et al., 1990; Rogan & Gladen, 1991). Only two studies have described adverse eVects of lactational exposure on scores on these developmental tests. Lactational exposure to PCBs and dioxins was related to lower psychomotor abilities at 7 months of age (Koopman‐ Esseboom et al., 1996) in the Dutch study and to lower general cognitive abilities at 42 months of age in the German cohort (Walkowiak et al., 2001). There are some methodological aspects that should be considered in risk assessment studies that address neurodevelopmental eVects of lactational exposure to PCBs and dioxins, especially in Western societies. Based on the studies described in the previous paragraph, it can be hypothesized that negative eVects of lactational exposure may, similarly to eVects of prenatal exposure, be counteracted or masked by optimal parental and home environmental conditions. In The Netherlands, comparable to most Western societies, the parents’ choice for breast‐feeding their child generally also reflects diVerences in, for example, levels of parental and home environmental conditions. Studies that explore subtle adverse eVects of lactational exposure to PCBs and dioxins may therefore benefit from more advanced modeling techniques in which the interrelationships of these neurodevelopmental determinants and exposure can be more properly modeled. Moreover, more insight is needed into potential beneficial eVects of breast‐feeding, such as

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the eVects of brain‐stimulating substances that are provided by breast milk and not by formula milk. The design and aims of the studies described in this paragraph are not adequate to address this aspect of breast‐feeding. However, the results of the neurophysiological study presented in this paragraph may suggest positive eVects of a longer duration of breast‐feeding in which potentially brain‐stimulating eVects of substances in breast milk are involved. Animal studies show evidence of profound neurodevelopmental eVects in monkeys that were exposed only to low levels of PCBs and dioxins through lactation (Rice, 1997, 1999; Rice & Hayward, 1997, 1999). These results indicate the potential for neurodevelopmental eVects of lactational exposure to PCBs and dioxins in humans. Moreover, these behavioral deficits in animals were suggestive of prefrontal cortex involvement. Since structure‐ related eVects of lactational exposure can be hypothesized considering maturation diVerences of brain structures, risk assessment studies that address lactational exposure should include a more elaborate neuropsychological test battery and larger study populations in which children were breast‐fed for longer durations than in the Dutch cohort (median of breast‐feeding duration 16 weeks). This may increase the knowledge of neurotoxic eVects of lactational exposure as well as help to diVerentiate eVects of prenatal and lactational exposure to PCBs and dioxins. Although infants are exposed to relatively large amounts of PCBs and dioxins through lactation, neurodevelopmental eVects of prenatal exposure to environmental levels of PCBs and dioxins were generally more pronounced. Subtle eVects of postnatal exposure to PCBs and dioxins were detected on one of the neurodevelopmental outcome variables that were explored in the Dutch PCB/dioxin cohort. On the other hand, the neurophysiological data may suggest beneficial eVects of a longer duration of breast‐ feeding in which potentially brain‐stimulating eVects of substances in breast milk are involved. These results do not warrant restrictions on breast‐feeding or reductions of the period of breast‐feeding in the Western societies. Neurodevelopmental eVects of lactational exposure to PCBs and dioxins and eVects of breast milk brain‐stimulating substances should be studied more thoroughly, using advanced modeling techniques in addition to addressing specific cognitive domains in larger cohorts as well as animal research. C.

Magnitude of Estimated Neurodevelopmental Effects

The magnitude of neurodevelopmental eVects that were associated with PCBs and dioxin exposure is relatively small in the Dutch cohort, and is not likely to be clinically relevant to the individual child. The level of cognitive development from 3 to 84 months of age, for example, in children born to

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younger mothers, was approximately 3 points lower in high prenatally exposed children (75% equivalent) compared to their low-exposed counterparts (25% equivalent). Under less favorable parental and home environmental conditions, however, the magnitude of cognitive and motor decrements may be larger. The Rotterdam cohort consists of families willing to participate for at least 7 years in this study. Parental and home environmental characteristics of this group are therefore likely to be more advantaged than in the average Dutch population or in populations in which educational possibilities or potential for cognitive stimulation are limited. When considering these subtle eVects in a large population, a lower average IQ shifts the distribution and increases the number of individuals who can be classified as retarded (IQ < 85). Additionally, it decreases the number of gifted and exceptionally gifted individuals (IQ > 130). For example, if the average IQ is shifted by 5 points (in a normal distribution with a mean of 100 and a standard deviation of 15), the number of children that score below 70 increases by a factor of 2 (Rice, 1998b). Neurodevelopmental eVects of perinatal exposure to PCBs and dioxins are detectable in a cohort of normal children. The magnitude of the eVects is relatively small and not likely to be clinically relevant to the individual child. The magnitude of neurodevelopmental eVects may be somewhat larger in populations in which conditions for child development are less favorable. For the whole society, however, these subtle decrements may have long‐term consequences.

VII.

FUTURE PERSPECTIVES

The epidemiological PCB studies described in this chapter draw attention to a number of important aspects that should be considered in this type of prospective follow‐up risk assessment studies that address eVects of perinatal exposure to PCBs and dioxins on neurodevelopmental outcome. First, the results of these studies may illustrate the importance of nonrandom attrition of the subjects of the original cohort. Ten percent of the original cohort was lost to follow‐up at school age. Although exposure levels were not statistically diVerent between participating and nonparticipating children, the latter group was significantly more formula‐fed, breast‐fed for shorter periods, and maternal age, parental education level, and verbal IQ were significantly lower in this lost to follow‐up group. Since developmental eVects of prenatal exposure to PCBs and dioxins may be influenced by parental and home environmental conditions, this is an important change in the study population. At preschool age, adverse eVects of prenatal PCB

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exposure on cognitive development were seen in the total cohort, whereas at school age, significant adverse eVects were seen only when parental and home characteristics were less optimal. The higher mean levels of these background variables in the population at school age might explain that no eVect of prenatal PCB exposure was seen in the total cohort, adjusting for the mean population levels of the confounders. Therefore, changes in the distribution of these variables in a cohort are a point of great attention in prospective follow‐up studies that address neurodevelopmental risks of perinatal exposure to PCBs and dioxins, since it may cause missing neurodevelopmental eVects in older children. Second, the choice of neurodevelopmental outcomes to detect adverse eVects of prenatal and lactational exposure to PCBs and dioxins should be addressed with great care in risk assessment studies. For example, general cognitive abilities, as measured with developmental tests, may not be the most sensitive outcome to detect neurotoxic eVects of lactational exposure to PCBs and dioxins since this outcome is particularly sensitive to parental and home environmental conditions. The process of learning or more specific neuropsychological functions as well as motor development may be more sensitive outcomes in risk assessment studies addressing subtle eVects of lactational exposure to neurotoxicants. Third, due to the complex interrelationships of various neurodevelopmental determinants and maternal PCB and dioxin levels, risk assessment studies may benefit from using sophisticated statistical modeling techniques. Moreover, these analyses make it possible to address the developmental course of functions in addition to addressing the level of functions at a certain point during the development of those functions. Fourth, due to diVerences in eating habits or area diVerences in environmental PCB and dioxin mixtures, populations worldwide are exposed to dissimilar mixtures of PCBs and dioxins. For example, results from the Oswego Study indicated that the cord blood of women who consumed Lake Ontario fish contained a significantly higher proportion of the most heavily chlorinated PCBs relative to non‐fish eaters. Levels of PCBs of lighter chlorination as well as the total PCB levels were similar in these groups (Stewart et al., 1999). Moreover, the cord blood levels of the highly chlorinated PCBs correlated more strongly with breast milk PCB levels than lower chlorinated PCBs. The results of the neurodevelopmental analyses in this cohort (children from birth to 12 months of age) showed some evidence of more pronounced neurodevelopmental eVects of exposure to the higher chlorinated PCBs (Darvill et al., 2000). The initial findings of this study as well as the results of laboratory studies therefore suggest that risk assessment studies can benefit from addressing more thoroughly the eVects of diVerent types of congeners to which children are exposed.

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Fifth, environmental levels of PCBs and dioxins are generally declining, due to worldwide control of sources, regulations of disposal practices, elimination of production, and natural attenuation. In The Netherlands, eVorts to minimize dioxin emissions, as were performed from the late 1980s, clearly show decreasing levels of PCBs and dioxins in food in the past 10 years (Liem et al., 2000). In breast milk, dioxin levels even decreased up to 50% during the past decade (Liem et al., 2000; Van Leeuwen & Malisch, 2002) (see Fig. 2). Children, however, are perinatally exposed to a large number of other potentially neurotoxic-persistent environmental pollutants such as heavy metals, pesticides and insecticides, flame retardants, and cleansers. For example, although breast milk levels of PCBs and dioxins have decreased over the past years, an increase is seen in the levels of another group of persistent organic pollutants, polybrominated diphenyl ethers (PBDEs) (Meironyte et al., 1999). PBDEs are used as flame retardants and are presently applied throughout the world. The chemical structure of the PBDEs resembles the structure of PCBs and dioxins and their neurotoxic properties have been recognized (Darnerud et al., 2001). Furthermore, of the over 80,000 chemicals that are used in commerce and industry, only a small proportion has undergone testing for neurodevelopmental toxicity. PCBs and dioxins are among the few contaminants subjected to extensive research exploring their neurotoxic properties and neurodevelopmental consequences for humans exposed to environmental levels of these compounds. The subtle neurodevelopmental decrements detected in the prospective follow‐up studies in healthy‐born children who were perinatally exposed to relatively low levels, environmental levels of PCBs and dioxins may be illustrative for the

FIG. 2. Temporal trends of levels of PCDD/PCDF in human milk (Van Leeuwen & Malisch, 2002).

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Leeuwen, F. X., Liem, A. K., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M., Younes, M., Waern, F., & Zacharewski, T. (1998b). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife [see comments]. Environmemtal Health Perspectives, 106, 775–792. Van der Meulen, B. F., & Smirkovsky, M. (1985). Mos. 2 1/2 –8 1/2 McCarthy Ontwikkelingsschalen. The Netherlands: Swets & Zeitlinger B.V. Van Leeuwen, F. X. R., & Malisch, R. (2002). Results of the third round of the WHO‐ coordinated exposure study on the levels of PCBs, PCDDs, and PCDFs in human milk. Organohalogen Compounds, 56, 311–316. Vreugdenhil, H. J., Lanting, C. I., Mulder, P. G., Boersma, E. R., & Weisglas‐Kuperus, N. (2002a). EVects of prenatal PCB and dioxin background exposure on cognitive and motor abilities in Dutch children at school age. Journal of Pediatrics, 140, 48–56. Vreugdenhil, H. J., Slijper, F. M., Mulder, P. G., & Weisglas‐Kuperus, N. (2002b). EVects of perinatal exposure to PCBs and dioxins on play behavior in Dutch children at school age. Environmental Health Perspectives, 110, A593–A598. Vreugdenhil, H. J. I., Duivenvoorden, H. J., & Weisglas‐Kuperus, N. (2002c). Neurodevelopmental EVects of Perinatal Exposure to Environmental Levels of PCBs and Dioxins in Children at School Age. Rotterdam, the Netherlands: Erasmus University Thesis. Vreugdenhil, H. J. I., Mulder, P. G., Emmen, H. H., & Weisglas‐Kuperus, N. (2004a). EVects of perinatal exposure to PCBs on neuropsychological functions in the Rotterdam cohort at 9 years of age. Neuropsychology, 18, 185–193. Vreugdenhil, H. J. I., Van Zanten, G. A., Brocaar, M. P., Mulder, P. G. H., & Weisglas‐ Kuperus, N. (2004b). Prenatal PCB exposure and breast‐feeding aVect auditory P300 latencies in 9‐year‐old Dutch children. Developmental Medicine and Child Neurology, 46, 398–405. Walkowiak, J., Wiener, J. A., Fastabend, A., Heinzow, B., Kramer, U., Schmidt, E., Steingruber, H. J., Wundram, S., & Winneke, G. (2001). Environmental exposure to polychlorinated biphenyls and quality of the home environment: EVects on psychodevelopment in early childhood. Lancet, 358, 1602–1607. Weisglas‐Kuperus, N., Baerts, W., Smrkovsky, M., & Sauer, P. J. (1993). EVects of biological and social factors on the cognitive development of very low birth weight children. Pediatrics, 92, 658–665. Weisglas‐Kuperus, N., Patandin, S., Berbers, G. A., Sas, T. C., Mulder, P. G., Sauer, P. J., & Hooijkaas, H. (2000). Immunologic eVects of background exposure to polychlorinated biphenyls and dioxins in Dutch preschool children. Environmental Health Perspectives, 108, 1203–1207. WHO (1989). Levels of PCBs, PCDDs, and PCDFs in breast milk: Results of the WHO‐ coordinated interlaboratory quality control studies and analytical field studies. Environmental Health Series, 34. Winneke, G., Bucholski, A., Heinzow, B., Kramer, U., Schmidt, E., Walkowiak, J., Wiener, J. A., & Steingruber, H. J. (1998). Developmental neurotoxicity of polychlorinated biphenyls (PCBS): Cognitive and psychomotor functions in 7‐month‐old children. Toxicology Letters, 102–103, 423–428. Winneke, G., & Kraemer, U. (1984). Neuropsychological eVects of lead in children: Interactions with social background variables. Neuropsychobiology, 11, 195–202. WolV, J. R., Laskawi, R., Spatz, W. B., & Missler, M. (1995). Structural dynamics of synapses and synaptic components. Behavioural Brain Research, 66, 13–20.

Interactions of Lead Exposure and Stress: Implications for Cognitive Dysfunction DEBORAH A. CORY‐SLECHTA ENVIRONMENTAL AND OCCUPATIONAL HEALTH SCIENCES INSTITUTE, A JOINT INSTITUTE OF ROBERT WOOD JOHNSON MEDICAL SCHOOL, UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY, AND OF RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY, PISCATAWAY, NEW JERSEY

I.

HISTORY AND CURRENT UNDERSTANDING OF LEAD EFFECTS

Although the toxicity of lead (Pb) was recognized as far back as the time of the ancient Romans (Hernberg, 2000), the versatility of this metal virtually guaranteed its continued application. Throughout the years, Pb was utilized in cosmetics such as rouge and mascara, as a spermicide, broadly as a sugar and preservative for cooking and wines, and for pewter, coins, and ceramic glazes. Lead pipes served as the basis of the plumbing system that supplied the Roman Empire with water. The high levels of exposure and Pb body burdens at that time as a consequence of these uses has repeatedly been documented in analyses of bone lead levels of the ancient Romans. New uses of Pb emerged during the Industrial Revolution, with the manufacture of ammunition and glassware and the introduction of printing processes, among others. But by far the greatest increase in worldwide distribution of Pb, and consequently of human exposure, arose as a result of two new applications in the 1920s. The first was the incorporation of Pb into gasoline in the United States as a highly eYcacious anti‐knock agent. This occurred despite the presence of warnings of potential health hazards, such as those issued in the Surgeon General’s report of 1926 (Rosner & Markowitz, 1985). Leaded gasoline was critical to the development and success of the automotive industry. Lead was also incorporated at about the same time into paint as a pigment, again resulting in broad distribution in the environment through the application of such paints in homes, buildings, and other structures. INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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It was almost 50 years later that recognition of the adverse health eVects resulting from Pb exposure reached a level of concern suYcient to provoke a phase‐out of Pb from gasoline (1973) and paint (1978) in the United States and the implementation of public screening programs for elevated Pb exposure. These actions were prompted largely by deaths resulting from acute lead poisoning in children. Over the period 1951 to 1953, for example, 94 pediatric deaths due to lead poisoning were documented in New York City, Cincinnati, St. Louis, and Baltimore. Similar cases were reported in other countries as well, with such fatalities typically occurring at blood lead (PbB) levels of 80 mg/dL and above. Moreover, contrary to existing beliefs at the time, reports began to emerge indicating that children who survived acute lead encephalopathy, rather than fully recovering, were left with residual and permanent sequelae such as mental retardation, recurrent seizures, cerebral palsy, and optic atrophy (Byers & Lord, 1943; Perlstein & Attala, 1966). These events made two facets of Pb toxicity evident. First, they demonstrated that the central nervous system was a critical target organ for Pb. Second, they made clear that children were particularly vulnerable to these eVects. Based on these findings, consequent eVorts aimed at understanding the health hazards associated with Pb in children focused predominantly on the central nervous system and associated behavioral manifestations, with two specific hazards serving as the focus. The first was whether chronic low‐level Pb exposure was reliably associated with diminutions of cognitive function, and, a second was the blood Pb (PbB) levels associated with such eVects, i.e., the nature of the corresponding dose–eVect relationship. The initial studies in children designed to address these questions were largely cross‐sectional in design and focused on the validity of an association between elevated PbB and decrements in IQ, with IQ generally measured using standardized psychometric tests. Even when considered in light of their limitations, these studies suggested that PbBs as low as 30 mg/ dL could be associated with detrimental eVects, a significant departure from the 80 mg/dL level. Subsequently, prospective cohort studies were initiated in the United States as well as in various other countries, which enrolled pregnant women and longitudinally tracked Pb exposure levels and outcomes in oVspring at various intervals thereafter (D. Bellinger et al., 1987, 1989; D. C. Bellinger et al., 1992; Dietrich et al., 1991, 1993b, 2001; Fulton et al., 1987; McMichael et al., 1988; Wasserman et al., 1997). As these studies proceeded, their collective findings provided accumulating and compelling evidence for an inverse relationship between elevated PbBs and IQ score, indeed, with similar magnitudes of eVects detected across divergent populations. Based on the outcome of these longitudinal studies, with their greater

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power to detect Pb eVects and to control significant potential confounding variables, the PbB level of concern for IQ reductions in children was lowered to 10 mg/dL by the CDC in 1991, where it has since remained. The World Health Organization set a similar level of concern. More recent studies have been directed to questions of whether a threshold PbB level exists for an association between Pb exposure and cognitive deficits, and to determining what specific behavioral processes aVected by Pb contribute to the decrements in cognition (D. Bellinger et al., 1987, 1989; D. C. Bellinger et al., 1992; Dietrich et al., 1991, 1993a, 2001; Fulton et al., 1987; McMichael et al., 1988; Wasserman et al., 1997). These newer eVorts provide evidence for cognitive deficits at PbB levels lower than 10 mg/dL in children and also demonstrate deficits in working memory and behavioral flexibility as additional components of the behavioral toxicity (Canfield et al., 2003, 2004). Paralleling these prospective cohort studies have been an extensive number of experimental studies aimed at further evaluating the behavioral and neurotoxicity arising from Pb exposure, and determining the mechanisms by which they occur. Importantly, both the nature of the observed eVects of Pb and the PbB levels at which eVects occur in experimental studies are remarkably similar to those reported for human populations (Cory‐Slechta, 1984, 1988, 1995a, 2003; Rice, 1996). In addition, the toxicokinetics of Pb shows significant similarities across species. This further enhances the credibility of findings from the experimental models.

II.

Pb EXPOSURE IN THE CONTEXT OF ENVIRONMENTALLY REALISTIC CONDITIONS

Both the human and experimental studies of Pb were critical for establishing an association between elevated Pb exposure and reduced cognitive function. They have also been informative with respect to neurobiological and behavioral mechanisms of action. However, the environmental realities of Pb exposure can be quite diVerent from the conditions under which it has been examined to date. First, environmental chemical exposures do not occur in isolation: human populations are exposed to mixtures of chemicals, the nature and levels of which may change dynamically across time. The resultant mixtures could modify the eVects of Pb in ways that are currently unknown, by adding or potentially even reversing its eVects. Second, Pb exposures occur in a context of many other potential host or extrinsic risk‐modifying factors, including genetic background, age, gender, developmental period of exposure, nutritional status, lifestyle, stress levels,

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and underlying disease state, among others. As with mixtures, these other variables may also modify the eVects of Pb exposure, by increasing or decreasing eVects on Pb toxicity. At the present time, almost nothing is known about the ability of such factors to modify Pb risk. In fact, our current understanding of the eVects of Pb on cognitive function, and virtually all other outcomes, is based almost exclusively on studies of its eVects in isolation. While many of the prospective cohort studies have had the potential to examine risk modification, such variables are generally controlled statistically, and thus potential risk modification is not generally evaluated (Burns et al., 1999; Dietrich et al., 1993a,b; Gomaa et al., 2002; Tong & Lu, 2001; Wasserman et al., 2000). This is often attributed to sample sizes deemed insuYcient to produce reliable interaction evaluation. In experimental studies, Pb is almost invariably studied in isolation from other potential risk modifiers, and most often in healthy young rodents that are almost exclusively males. Thus, the extent to which our current understanding of Pb realistically reflects its toxicity or adverse eVects on cognitive functions may not be accurate, particularly if additive, synergistic, or potentiated interactions occur with other chemical exposures or risk factors. This chapter describes our observations on one such new interaction, that of environmental stress with Pb, as determined in experimental models. It presents the rationale for hypothesizing such an interaction, followed by highlights of some current studies based on this hypothesis. Notably, this interaction of Pb with environmental stress resulted in a profile of eVects that diverges notably from those produced by Pb exposure alone. These interactions are relevant to understanding the role of Pb as a contributor to human disease and dysfunction, to our understanding of mechanisms of neurotoxicity and other target organ toxicities associated with Pb, and, moreover, may be particularly pertinent to criteria for screening programs for the prevention of adverse eVects of Pb.

III. RISK MODIFIERS FOR Pb NEUROTOXICITY: ENVIRONMENTAL STRESS AS A CASE STUDY A.

Elevated Lead Exposure Preferentially Impacts Low Socioeconomic Status Populations

After the removal of Pb from gasoline in the 1970s, PbB levels of the U.S. population declined markedly, as has been documented across successive years in the National Health and Nutrition Examination Surveys, a national representative survey of the civilian U.S. population (Brody et al., 1994; MahaVey et al., 1982; Pirkle et al., 1998). Indeed, the prevalence of elevated PbBs (10 mg/dL) in children between 1 and 5 years of age has


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continued to decline from an estimated 88% as reported in the 1976–1980 NHANES II survey to values of 2.2% based on 1999–2000 estimates (Meyer et al., 2003). When considered in relation to various demographic factors, however, a diVerent story emerges, since levels of elevated PbB in children actually diVer markedly in relation to race/ethnicity, income level, and residence type. Based on NHANES III reports from 1994, the percentage of elevated PbB values in African‐American children, for example, at 22%, was five times higher than that of the general population, while the percentage of children with elevated PbB levels from low‐income families living in pre‐1946 homes, residences endemic to our inner cities, was 16%, almost four‐fold higher than in the overall population (Pirkle et al., 1998). Although the diVerences in percentages have since narrowed, the disparitites across racial/ethnic groups remain, with 2001 figures of 2% for white non‐Hispanic children, 9% for black non‐Hispanic (four‐fold higher), and 6% (almost three‐fold higher) for Hispanic children (Meyer et al., 2003). Figure 1 shows these diVerential levels of elevated PbBs in relation to race/ethnicity across various PbB clusterings based on 2001 United States data. Total numbers of black non‐ Hispanic and Hispanic children exceed those of white non‐Hispanics in every PbB category shown. As Fig. 1 shows, elevated Pb exposure has now become a demographically circumscribed health problem, one of particular concern for socioeconomically disadvantaged, medically underserved inner-city minority children who reside in old houses with Pb‐based paints. Indeed, PbB histories that include levels in excess of 40 mg/dL still occur in many low socioeconomic (SES)

FIG. 1. Numbers of children with elevated PbB levels (>10 mg/dL) in relation to racial and ethnic groups across various blood lead level (BLL) groupings based on 2001 U.S. data. Unknown category represents individuals who did not identify race/ethnicity. From Meyer et al. (2003).

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inner-city children. These are populations that lack the economic aZuence and social capital to successfully lobby for Pb abatement programs. Because of the vicious cycle of poverty, the parents and grandparents of these children often lived in these same neighborhoods; thus they too experienced the highest Pb exposures in the country, making the cycle of poverty coincide with the cycle of Pb exposure and maintaining Pb exposure as an intergenerational public health problem in these populations. Even as this problem endures, at least one study suggests that not only do Pb eVects in children occur even below 10 mg/dL and that there may be no threshold for deficits in IQ, but that these problems can be of even greater magnitude than eVects occurring at higher PbBs (Canfield et al., 2003). B.

Low SES Is Already a Known Risk Factor for Various Diseases and Behavioral Dysfunctions

What is particularly notable about the current demographics of elevated Pb exposure is that low SES is already a well‐documented risk factor for adverse health impacts and behavioral and neurological dysfunctions, even after other pertinent covariates have been statistically controlled. In adults, for example, the risk of mortality, the prevalence of numerous diseases, increased blood pressure, and the prevalence of schizophrenia (Dohrenwend, 1990) and depression (Hirschfeld & Cross, 1982) have all been shown to be inversely related to employment grade, occupational status, income, and years of education (Adelstein, 1980; Dyer et al., 1976; Marmot et al., 1984; Pappas et al., 1993; Pincus et al., 1987). In addition to Pb poisoning, links between low SES and greater health risk in children have been reported for vision problems, otitis media, hearing loss, cytomegalic inclusion disease, and iron deficiency anemia (Starfield, 1982). Lower SES children also have higher levels of mental retardation, learning disabilities, emotional and behavioral problems, and deficits in language, memory, and attentional capacities (Anderson & Armstead, 1995; Ardila & Rosselli, 1995). Low SES has been described as the underlying basis of academic risk in minority children (Arnold & DoctoroV, 2003). C.

Links Between Low SES, Stress, and Cortisol

1. ELEVATED STRESS IS POSTULATED TO UNDERLIE THE INCREASED INCIDENCE OF DISEASE AND DYSFUNCTION ASSOCIATED WITH LOW SES

One hypothesis that has been proposed to account for the association between lower SES and the increased prevalence of adverse health outcomes and behavioral dysfunctions is a greater exposure of these populations to


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environmental and psychosocial stressors (Lupien et al., 2001). Stress has been defined as life events that require adaptation (Selye, 1950) or a state in which the individual perceives that the demands of the environment exceed the ability to cope (Adler et al., 1994). Lower SES individuals report greater exposures to stressful life events as well as greater impacts of these stresses on their lives (Baum et al., 1999; Dohrenwend, 1973; Marmot & Wilkinson, 1999, 2001; Taylor & Seeman, 1999). The physical and social environments associated with low SES, characterized by deteriorating housing, high crime and violence rates, increased drug use, fewer two‐parent families, and chronic unemployment, no doubt contribute to this. Low SES populations are subjected to a greater frequency of threatening and uncontrollable events in life, higher levels of environmental hazards and violence, and lower levels of family stability (Bradley & Corwyn, 2002). The corresponding lower income levels may mean inability to consistently aVord required expenses, such as home mortgage or rent payment, food, day care, transportation, or health insurance, contributing to the ongoing cycle of poverty. There is a chronic strain associated with unstable employment situations and persistent economic hardships. The hypothesis that stress contributes to the greater prevalence of disease and dysfunction in low SES populations is based on numerous studies showing that, like low SES, higher levels of stress are associated with poorer health outcomes, including hypertension, cardiovascular disease and death, the extent of chronic illness, and altered immune function, among others (Brosschot et al., 1992; Calabrese et al., 1987; Kennedy et al., 1988; Rahe & Lind, 1971; Wyler et al., 1971). Increased stress adversely aVects mood and cognitive function (Lupien & McEwen, 1997) and correlates with measures of anxiety and depression (Vinokur & Selzer, 1975). Increased stress is also associated with poorer academic achievement (Harris, 1972) and impairments in cognitive functions, including attention and memory functions (Mueller, 1976). Stress may actually exert its most detrimental eVects in children and have eVects that are particularly long‐lived (Cohen et al., 1973; Tennes & Kreye, 1985). 2. STRESS STIMULI ACTIVATE THE HYPOTHALAMIC– PITUITARY–ADRENAL AXIS

Stressful stimuli are known to activate adrenal cortical glucocorticoids, an eVect considered an adaptive response to stress, as shown schematically in Fig. 2 (left side) (Vazquez, 1998). Physiological or psychological stressors cause release of corticotropin‐releasing hormone (CRH) and arginine vasopression (AVP) from the periventricular nucleus (PVN) of the hypothalamus. This stimulates release by the adrenal pituitary of adrenocorticotropin (ACTH), which then acts on adrenal cortex receptors to elevate plasma glucocorticoids that subsequently activate glucocorticoid receptors,

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FIG. 2. Schematic depicting the influence of low SES producing stress that then acts on the hypothalamic–pituitary–adrenal (HPA) axis (left side). Physiological or psychological stressors cause release of corticotropin‐releasing hormone (CRH) and arginine vasopression (AVP) from the periventricular nucleus (PVN) of the hypothalamus. This stimulates release by the adrenal pituitary of adrenocorticotropin (ACTH), which then acts on adrenal cortex receptors to elevate plasma glucocorticoids that subsequently activate glucocorticoid receptors, including those in brain. Feedback loops to pituitary, hypothalamus, and hippocampus regulate the secretion of corticosteroids. Hippocampal regulation of the HPA axis is particularly significant in relation to cognitive outcome. The HPA axis interacts extensively with the mesocorticolimbic dopamine system (right outset) of the brain. In this system, the nucleus accumbens receives dopaminergic input from neurons in the ventral tegmental area as well as glutamatergic projections, both NMDA and AMPA/kainite‐mediated, from the septo‐hippocampal system and from the prefrontal cortex. The prefrontal cortex also receives dopaminergic input from the ventral tegmental area and glutamatergically mediated information from the septo‐hippocampal system. Notably, both the hippocampus and prefrontal cortex have been reported to play an inhibitory role over HPA axis function (Diorio et al., 1993; Figueiredo et al., 2003; Jacobson & Sapolsky, 1991; Sullivan & Gratton, 1999). Increased stress resulting in elevated cortisol levels can impact cognitive function through interactions of the HPA axis with the mesocorticolimbic system. As indicated, Pb exposure also impacts the mesocorticolimbic systems of the brain as well as HPA axis function, as illustrated here.

including those in the brain. (Cortisol is the main glucocorticoid of human and nonhuman primates, whereas corticosterone is the main glucocorticoid of the rat.) Feedback loops to pituitary, hypothalamus, and hippocampus regulate the secretion of glucocorticoids. Hippocampal


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regulation of the HPA axis is particularly significant, given its critical role in numerous cognitive functions. In fact, hippocampectomy or transaction of the fornex is known to elevate basal HPA activity (Jacobson & Sapolsky, 1991). Glucocorticoids act via two types of receptors: Type I, or mineralocorticoid (MR) receptors, and Type II, or glucocorticoid (GR) receptors. In brain, MR receptors are located primarily in limbic regions, including the septo‐hippocampal system and amygdala. In contrast, GR receptors are distributed throughout brain. GR receptors in particular are activated by higher levels of corticosterone such as those associated with stress (Joels & de Kloet, 1994), while low basal corticosterone levels activate MR receptors. 3. LOW SES IS ASSOCIATED WITH INCREASED CORTISOL

The higher levels of stress in low SES populations are thought to produce chronic elevation of associated stress hormones such as glucocorticoid, a presumption supported by an increasing number of studies. Low SES children from 6 to 10 years of age living in Montreal had higher morning salivary cortisol levels than did children from more aZuent families (Lupien et al., 2001). In another study, elevated salivary cortisol levels in children were associated with lower SES as well as the mother’s extent of depressive symptomatology, eVects that emerged as early as 6 years of age (Lupien et al., 2000). In adults, job strain and the expression of anger were associated with elevation of free cortisol early in the working day (Steptoe et al., 2000). A 2002 study reported that family SES was inversely related to initial cortisol levels in a population of young adult African‐American males (Kapuku et al., 2002). 4. ELEVATED GLUCOCORTICOIDS ARE ASSOCIATED WITH INCREASED DISEASE AND DYSFUNCTION

Again, in concert with stress as the potential mediator of increased disease and dysfunction in low SES populations, increasing evidence also documents a relationship between chronic elevation of glucocorticoids and disease and dysfunction, with sustained elevations of cortisol reported to increase resistance to insulin, and to cause hypertension, hypercholesterolemia, arteriosclerosis, and immunosuppression (Munck et al., 1984). In a review, Kristenson and colleagues (Kristenson et al., 2004) describe the fact that in several studies, an SES gradient can be demonstrated for all causes of mortality and for diseases that include coronary heart disease, diabetes, gastrointestinal disease, respiratory diseases, arthritis, and adverse birth outcomes.

96

Deborah A. Cory-Slechta IV.

A.

BOTH Pb AND STRESS CAN PRODUCE COGNITIVE DEFICITS

Stress During Development, Sustained Cognitive Deficits, and Alterations in Structure and Function of the Brain and the HPA Axis

Particularly important in the current context is that circulating glucocorticoids can cross the blood–brain barrier to act on GR and MR receptor sites in the central nervous system. In studies in humans, various measures of stress/anxiety in mid‐pregnancy were inversely related to developmental indices in oVspring at 8 months of age as measured on the Bayley Scales. Moreover, morning cortisol levels of mothers were also inversely related to developmental outcomes at both 3 and 8 months of age (Buitelaar et al., 2003). O’Connor and colleagues (O’Connor et al., 2003) report an association between higher levels of self‐assessed maternal anxiety and depression and higher rates of behavioral and emotional problems (conduct problems, emotional problems, hyperactivity/inattention) in their children at 81 months of age after controlling for several potentially confounding variables. EVect levels were generally comparable in males and females, and similar eVects had been described in this cohort at 47 months of age, indicating their persistence. Using a naturally stressed cohort of mothers in Quebec who had been exposed to an ice storm, it was reported that the level of prenatal anxiety could account for 11 to 12% of the variance in Bayley Mental Development Index (MDI) scores and productive language abilities, and for 17% of the variance in receptive language abilities in 2‐year‐olds (Laplante et al., 2004). EVects from both nonhuman primate and rodent studies provide evidence that the nature of stress eVects on cognition depends upon the parameters of stress exposure, oVspring gender, and the behavioral paradigm used (i.e., stress eVects reflect risk modification), and that significant individual diVerences in responsiveness occur as well. Prenatal stress consisting of exposure of mothers to unpredictable noise during mid‐ to late gestation, for example, altered social behaviors of juvenile monkeys (18‐month‐old oVspring), elevated basal cortisol and ACTH hormone levels, and produced higher levels of stress‐induced ACTH (Clarke & Schneider, 1993; Clarke et al., 1994; Coe et al., 2003). OVspring of monkeys that were subjected to noise stressors during diVerent periods of gestation were reported to exhibit reduced attention span and neuromotor capabilities during the first month of life (Schneider et al., 2002). Aspects of both learning and memory were evaluated in these oVspring at 32 to 34 months of age using a nonmatching to sample memory task. In this case, monkeys of prenatally


97

stressed mothers required the same number of trials to criterion as their nonstressed counterparts, and exhibited no alterations in accuracy across diVerent delay values of up to 120 s, suggesting no residual deficits in either acquisition or memory (Schneider et al., 2001). However, even at 120 s delay values, these monkeys maintained accuracy levels ranging from approximately 83 to 86%, i.e., they exhibited minimal reductions in memory. It would be important to determine whether more challenging memory conditions, where delays suYcient to reduce accuracy levels to chance, for example, would reveal memory deficits. Nevertheless, this cohort of primates did sustain excess levels of metabolites of norepinephrine and dopamine in cerebrospinal fluid consistent with residual changes in brain catecholamine function. In addition, several studies have shown decreases in hippocampal volume of 10 to 12% and reductions in neurogenesis in male and female oVspring of monkeys that had been exposed to acoustic startle either in mid‐ or late gestation for 25% of the gestational period. Basal and stress‐induced cortisol levels were also elevated in stressed oVspring (Coe et al., 2003). Magnetic resonance imaging scans of corpus callosum revealed decreases in volume in male stressed oVspring and increases in females, relative to controls (Coe et al., 2002). Studies in rodents provide evidence for cognitive dysfunction, but also of improved performance, as well as individual diVerences in stress responsivity, again dependent upon experimental parameters. For example, female oVspring whose dams were exposed to corticosterone in drinking water throughout gestation and lactation exhibited improvements in learning in a water maze paradigm at 21, 30, and 90 days of age, as well as in active avoidance measured at 15 months of age (Catalani et al., 2002). In another study, gestational restraint stress had no eVect on object recognition memory and improved performance of female oVspring in a radial arm maze, eliminating the gender diVerences seen in controls (Bowman et al., 2004). In contrast to those findings, however, another study reported that prenatal stress retarded the acquisition of reversal learning (Weller et al., 1988) and of spatial learning in a water maze (Lemaire et al., 2000; Nishio et al., 2001; Szuran et al., 2000). One of the more complete studies (Vallee et al., 1999) demonstrated increased numbers of errors in a radial arm maze at 22 months of age in male oVspring from dams subjected to restraint stress during the final week of gestation. In addition, stressed oVspring showed an accelerated age‐related decline in the HPA axis response to stress and, at least at one time point of measurement, elevated basal corticosterone levels. Encompassing both improvements and adverse eVects, de Kloet and Oitzl (de Kloet & Oitzl, 2003) observed that maternal separation stress in rats significantly increased, relative to controls, the percentage of rats

98


exhibiting impaired performance on a water maze learning task, but also the percentage exhibiting no impairment, thus moving performers toward both extremes of the distribution. Both outcomes resulted from a decrease in the number of partially impaired performers. How this related specifically to gender was not described. Such findings underscore the widely reported individual diVerences in stress responsivity and recovery that, no doubt, also contribute to the inconsistencies across studies (Dellu et al., 1996; Garcia & Armario, 2001; Irwin et al., 1989; Kabbaj et al., 2000; Liu et al., 1997; Tomie et al., 1987). B.

Pb Exposure and Deficits in Cognitive Function

Like low SES, and in some cases stress, elevated PbB levels in children have been associated with adverse cognitive eVects in several prospective epidemiological studies based on psychometric measures of intelligence (Needleman, 1993; Schwartz, 1994; S. Tong et al., 1996), with reductions in IQ scores in response to increasing PbBs. Similar eVects have been reported in cohorts from several diVerent countries and thus diVerent environmental conditions, including Boston and Cleveland in the United States, Australia, Scotland, and Yugoslavia (D. Bellinger et al., 1987, 1989; D. C. Bellinger et al., 1992; Dietrich et al., 1991, 1993a, 2001; Fulton et al., 1987; McMichael et al., 1988; Wasserman et al., 1997), indicating the generality of the eVects, findings that ultimately served as the basis for the designation of a level of 10 mg/dL of Pb in blood as a level of concern. Later studies suggest not only that Pb‐induced reductions in IQ may occur at PbBs even below 10 mg/dL (Canfield et al., 2003), but that the nature of the dose–eVect curve is not linear, since the magnitude of the deficit at PbBs 564 ppb blood 16–598 ppm hair Unknown

Davis et al. (1996)

22

Paresthesias 4 mothers Paresthesias

Unknown

Harada (1968)

293 ppm

Saito (2004a)

United States Minamata, Japan Niigata, Japan

1

Engleson & Herner (1952) Amin Zaki et al. (1974) Marsh et al. (1978)

Number is based on children with a neurological score >4, but it is not clear if all had functional neurological impairment. a

poisoning, but the mother did not. The mother was pregnant when the grain was consumed. The infant appeared normal at birth and did not have elevated Hg in the urine. However, she had severe mental retardation. No follow‐up data were reported. In the winter of 1971–1972, an epidemic of poisoning from MeHg‐treated seed grain occurred in Iraq (Bakir et al., 1973). Similar Hg poisonings had already occurred in Iraq and physicians recognized the clinical picture early. Soon after the first cases were identified, studies of the exposure began. Prenatal exposure was determined by measuring the level of total Hg (THg) in maternal hair. Total Hg in hair is composed of over 80% organic Hg, mainly MeHg. The amount of THg deposited in the hair appears to be stable over time and has been shown to correspond well to the blood level. Hair grows at a known rate of about 1.1 cm a month and by cutting the hair sample into short segments for analysis, the timing and degree of exposure can be determined with reasonable accuracy (Phelps et al., 1980). Some investigators believe that mercury enters hair and brain by similar mechanisms and thus hair mercury measurements accurately reflect brain exposure. Hair is the only biological marker that has been directly correlated with Hg concentrations in the brain (Cernichiari et al., 1995a). Following the Iraq poisoning, Amin‐Zaki and colleagues (Amin‐Zaki et al., 1974, 1976) reported on a number of children exposed to MeHg in utero. Six of them manifested evidence of poisoning and ‘‘the lowest

DEVELOPMENTAL DISABILITIES & PRENATAL METHYL MERCURY EXPOSURE

147

measured blood level in infants associated with signs of poisoning was 564 ppb.’’ Subsequently, Amin‐Zaki and colleagues reported 5‐year follow‐up of 32 infants with prenatal MeHg exposure (Amin‐Zaki et al., 1979). All of the infants with symptoms or signs had maternal peak hair Hg concentrations above 100 ppm. At age 5 years, 5 of the children had cerebral palsy and 18 had speech delay. Marsh and colleagues reported 81 children from Iraq with prenatal exposure, of whom some had neurological impairment (Marsh et al., 1987). Four children having prenatal exposure of 405 ppm in maternal hair or greater were clearly neurologically impaired while an additional seven had neurological scores above 4 and might have been impaired. Snyder (Snyder, 1971) reported one case of prenatal MeHg exposure from the United States. A family in New Mexico fed MeHg‐ contaminated grain to their hog and subsequently consumed the pork. The mother was pregnant when the pork was consumed. The level of exposure was measured only in her urine, but other family members had hair mercury levels of 186, 329, 1398, and 2436 ppm (Davis et al., 1994). The infant was born with severe neurological problems including seizures and died at age 21 years. B.

Prenatal Poisoning from Consumption of Contaminated Seafood

In the 1950s, the consequences of a massive industrial pollution at Minamata, Japan, were first recognized. A local chemical factory had been dumping untreated waste directly into Minamata Bay for many years, but in 1954, a mysterious neurological disease was first recognized in the area. The disorder was initially named Minamata disease (MD) because it was presumed to be an infectious disorder. Following several years of investigation, it was determined that MD was secondary to pollution and resembled MeHg poisoning. The fish and shellfish from Minamata Bay were reported to have MeHg levels in their flesh as high as 47 ppm (Tsubaki & Irukayama, 1977). It was not clear why human illness did not occur earlier since the factory had been dumping waste since 1935. However, two possible reasons were finally determined. In the 1950s, the company changed the chemical catalyst for acetaldehyde production to one that resulted in a greater production of MeHg as a by‐product. In addition, the factory increased the total production of acetaldehyde steadily from the late 1940s to 1960 (Nomura, 1968). The neurological findings of MD were ascribed primarily to MeHg, but the actual exposure was to multiple toxins present in the factory discharge. In addition to mercury, the factory waste was known to contain large

148

Gary J. Myers et al.

amounts of silicon, iron, aluminum, calcium, magnesium, potassium, sodium, ammonia, copper, arsenic, manganese, chlorine, phosphorus, sulfur, and lead (Harada, 1972). Several of these toxins were originally considered to be the cause of MD. However, the patients’ symptoms were very similar to those of patients with known poisoning by MeHg and it was eventually ascribed to that cause alone. The contribution, if any, of the concomitant toxic exposures was never determined. In 1968, Harada reported 22 cases of fetal MD (Harada, 1968). The children were from six of the seven small fishing villages where cases of postnatal poisoning were found. All of the children reported had severe neurological deficits, including microcephaly, intellectual disability, cerebral palsy, and seizures. The children’s mothers appeared to be healthy, but five mothers reported having mild sensory symptoms. Among the 22 children described, 17 were from families whose occupation was fishing and seafood was a major component of their diet. In most cases, there were other family members with clinical MeHg poisoning. The children’s prenatal exposure levels were not determined because all of them were over 1 year of age when Harada first evaluated them. However, mercury levels were measured in the hair of the children and their mothers when they were evaluated. The children’s hair mercury levels ranged from 5 to 100 ppm and the mothers’ from 1 to 191 ppm. The hair mercury content of healthy infants under 1 year of age living in the Minamata district at that time ranged from 0 to 158 ppm. Following that report, there were other children who were said to have congenital MeHg poisoning, but the details of those cases have not been reported. In the mid‐1960s, a similar industrial pollution occurred near Niigata, Japan. Local health care workers recognized the cause of the poisoning early and MeHg levels were measured during pregnancy or shortly after delivery (Tsubaki & Irukayama, 1977). Health authorities oVered mothers with a hair Hg level of over 50 ppm the opportunity to terminate their pregnancies (Watanabe & Satoh, 1996). Only one infant at Niigata was formally diagnosed with congenital MeHg poisoning. Her hair THg level, measured shortly after birth, was 77 ppm. Her mother’s hair THg level measured at the same time was 293 ppm (Saito, 2004a). One other child was suspected of having prenatal poisoning, but this was not oYcially confirmed (Saito, 2004a). There were 12 mothers with hair Hg levels measured between 51 and 115 ppm who were pregnant and chose to deliver their infants. At delivery, all of those infants and their mothers appeared healthy. The children were examined by a pediatrician at age 5 years and reported to be normal (Moriyama & Takizawa, 2001). The nature of the evaluation was not specified. Adult follow‐up of those subjects was reported in 2004 (Saito et al., 2004b). Three of them completed middle school, six completed


149

high school, and one each completed nursing school, business school, and university. Although MD appears to be poisoning with MeHg, those aVected were actually exposed to a mixture of toxic chemicals that included MeHg. The last subject with clinical symptoms from consumption of fish or seafood reported was born in Japan in 1965 (Saito, 2004a). Although there have been media reports that similar industrial pollution has occurred elsewhere, such as China, no cases of prenatal MeHg poisoning from consuming fish or seafood apart from those in Japan have been documented. Whether MeHg exposure from fish consumption in the absence of other toxins might cause similar findings is diYcult to determine from the industrial poisonings in Japan.

III.

DEVELOPING A HYPOTHESIS ABOUT PRENATAL LOW‐LEVEL METHYL MERCURY EXPOSURE

The episodes of poisoning from contaminated seed grain and seafood previously outlined led to the hypothesis that levels of exposure which have little or no detectable eVect on the mother can seriously damage the developing brain. However, of interest is that among the reported cases from Japan and elsewhere, there were no children with mild or moderate disability. There was one epidemiological study carried out at Minamata that tried to determine if there was such a spectrum. The study compared schoolchildren from the Minamata area with those from schools where minimal exposure was believed to have occurred (Fujino et al., 1976). The authors looked at the prevalence of abnormal mental and motor functions among school students. Their findings raised the intriguing possibility that varying levels of disability might have followed prenatal MeHg exposure in Japan. However, the study was not able to link the findings exclusively to MeHg because the exposure was to a mixture of toxins over several years and was not directly measured. In addition, there were some limitations in the survey methodology which included not measuring exposure in the children studied and pollution later found in the control area. Even so, the study raised the possibility of a spectrum of disability related to MeHg exposure. The poisoning in Iraq provided an opportunity to determine whether this hypothesis was correct. The outbreak was an acute exposure that took place over 3 to 4 months. In the winter of 1971–1972, the Iraq government imported over 70,000 metric tons of seed grain treated with MeHg (Bakir et al., 1973). The grain arrived after the planting season and some of it was ground into flour and consumed. The exposure was only to MeHg

150


and the victims varied in the amount they consumed. An estimated 50,000 people were exposed, with over 6000 hospitalizations and 450 deaths documented (Bakir et al., 1973; Greenwood, 1985). The amount consumed varied within and among families and, consequently, it presented a unique opportunity to examine the eVects of prenatal exposure of varying degrees. Following the outbreak, a group of investigators from the University of Rochester undertook a study of children who were in utero during the exposure (Marsh et al., 1987). Mothers who were known to be pregnant during the exposure were interviewed in their homes. The children’s developmental milestones, such as age at which they first walked, were ascertained and the children examined neurologically. The children’s prenatal Hg exposure was determined by measuring the THg in the mother’s hair growing during pregnancy. The women in Iraq allow their hair to grow very long and, when the Hg in their hair was measured by segmental analysis, it provided an accurate picture of their exposure over the prior one or more years. When the association between the children’s prenatal exposure and their developmental milestones and neurological findings was examined, it showed a dose–response relationship (Cox et al., 1989). That relationship indicated that maternal hair levels as low as 10 ppm might be associated with adverse eVects on a child’s neurodevelopment. It was known that people who consume fish with no overt Hg contamination could have THg levels of 10 ppm or above in their hair (Airey, 1983; Matthews, 1983). Consequently, the studies in Iraq raised the question of whether these levels of exposure actually pose a risk to the child’s development. The Iraq studies were not adequate to answer this question because fish consumption was minimal and there were other study limitations (Marsh, 1994). Studies of people who actually consume fish were needed to confirm the hypothesis. Since millions of people around the world consume fish daily, it is possible to study this issue directly (Vannuccini, 2003).

IV. EPIDEMIOLOGICAL STUDIES OF FISH‐CONSUMING POPULATIONS A number of epidemiological studies were designed to test the hypothesis that the levels of exposure achieved by consuming fish that contain only background levels of contamination actually present a risk to the developing fetal brain. Investigators postulated that if low‐level exposure to MeHg did have adverse eVects on the developing brain that the manifestations


151

would be subtle. Consequently, the study would require detailed evaluations of a large cohort in which individual fish consumption varied (and, hence, MeHg exposure) and careful control for the many covariates already known to have a significant influence on child development. Although many people consume fish, finding a population with a suitable exposure that could be studied in such detail was a diYcult task. So far, epidemiological studies have been reported from Brazil, Canada, French Guiana, New Zealand, Peru, the Philippines, and the Faroe and Seychelles Islands (Cordier et al., 2002; Davidson et al., 1998; Grandjean et al., 1997; Kjellstrom et al., 1986, 1989; Marsh et al., 1995; McKeown‐Eyssen et al., 1983; Myers et al., 2003; Ramirez et al., 2000, 2003). These epidemiological studies present a number of challenges to investigators and all of the studies have limitations. After finding a population with a suitable exposure, the investigators must measure it accurately. Studies that use surrogate measures of prenatal exposure, such as maternal hair measured when the children are several years old, are diYcult to interpret (Murata et al., 1999). Next, the cohort must agree to the study and reside in a setting where a study can be carried out. Ideally, the population should be studied repeatedly over time. Other limitations can include logistical issues, limited numbers of subjects, exposure to multiple coexisting potential toxins, unmeasured covariates, and other design limitations (Davidson et al., 2004). Ensuring reliable data collection presents diYculties that can, to a great degree, be overcome by rigorous study design, but there remain the issues of how to categorize and analyze the data and interpret and communicate the findings. Table II summarizes the epidemiological studies that have been reported to date. The table includes the authors’ opinion about whether the study shows an adverse association between low‐level prenatal MeHg exposure and various endpoints or not. Some investigators report associations and others do not. Among those reporting an association, the endpoints studied and reported to show an adverse association with prenatal MeHg exposure have varied. Among these studies, there are three that looked at relatively large populations over time and had suYcient power to detect subtle adverse changes in endpoints if they were present. These three studies have received the most attention and they will be discussed in more detail. The other studies are of interest, but a variety of factors, such as smaller cohorts, limited control of covariates, or single evaluations, makes them more diYcult to interpret. These limitations also make it diYcult to be confident that any changes detected might be causally related to prenatal MeHg exposure.

152


TABLE II SUMMARY OF EPIDEMIOLOGY STUDIES EVALUATING PRENATAL (FETAL) METHYL MERCURY EXPOSURE Country/Cohort

Exposure source

N

Authors’ conclusion

Exposure

Reference Grandjean et al. (1999b) McKeown‐Eyssen et al. (1983) Cordier et al. (2002) Grandjean et al. (1997) Steuerwald et al. (2000) Kjellstrom et al. (1986) Kjellstrom et al. (1989) Marsh et al. (1995) Ramirez et al. (2000, 2003) Myers et al. (1995a) Davidson et al. (1998) Myers et al. (2003)

381

þ

Mean 11 ppm**

Canada

Gold mining Fish

234

þ

Mean 6 ppm**

French Guiana

Fish

378

þ

Faroes Main

Whale meat

1022

þ

Means 1.4 to 10.2 ppm** 1‐350 ppb*

Faroes PCB

Whale meat

182

þ

1‐102 ppb*

New Zealand 4y

Fish

79

þ

1‐89 ppm**

New Zealand 6y

Fish

350

þ

1‐89 ppm**

Peru

Fish

131

–

1‐30 ppm**

Philippines

78

þ

0‐130 ppb*

Seychelles Pilot

Gold mining Fish

781

–

1‐37 ppm**

Seychelles Main

Fish

789

–

1‐27 ppm**

Brazil

*, cord blood; **, maternal hair. Authors’ conclusion: þ indicates that the authors interpreted the study to show an adverse association between prenatal MeHg exposure and the endpoints studied.

A.

New Zealand

The first large study of prenatal low‐level MeHg exposure was reported from New Zealand (Kjellstrom et al., 1986, 1989). The cohort children were exposed to prenatal MeHg when their mothers consumed fish and chips. Fish and chips is widely consumed in New Zealand and is often made using shark meat. Some of the shark contained up to 4 ppm of Hg, a level not generally considered as background (Mitchell et al., 1982). The investigators reported adverse associations between the level of prenatal Hg exposure and several of their endpoints. The original study was published in a Swedish technical bulletin and it is not clear that it was peer reviewed (Marsh, 1994; U.S. EPA, 1997).


153

However, the data were later reanalyzed and published in the peer‐reviewed literature (Crump et al., 1998). The study was complicated by several design issues that have been discussed elsewhere in detail (Marsh, 1994). These included a small sample size, the inclusion of children from three diVerent ethnic groups, each with a distinct and diVerent culture (Maori, European, and Pacific Islanders), and testing of controls and subjects at diVerent ages. When the reanalysis was done, an adverse association was again found. However, it was present only when one child with a very high level of exposure (89 ppm) was excluded. The National Academy of Sciences (NRC) chose to include this study in its review (2000), but some investigators continue to be cautious about how to interpret it. There are two large ongoing studies that are generally recognized as having adequate study designs and subject numbers that they might be able to determine whether prenatal MeHg exposure from consumption of fish containing background levels of MeHg might adversely aVect child development. One study is in the Republic of Seychelles and the other in the Faeroe Islands. Both studies have longitudinal designs and have been published extensively in the peer‐reviewed literature. B.

Seychelles Islands

In 1989–1990, the University of Rochester team that carried out the Iraq study initiated a longitudinal study of a population in the Seychelles Islands, the Seychelles Child Development Study (SCDS). The SCDS enrolled a main cohort that consisted of 779 children with prenatal exposure to MeHg from maternal fish consumption. In Seychelles, the ocean fish contains background Hg levels that average 0.3 ppm and the population does not consume sea mammals (Cernichiari et al., 1995b). At enrollment, the mothers reported consuming fish on average with 12 meals each week. Prenatal exposure was estimated by determining total and inorganic mercury in maternal hair growing during pregnancy. The prenatal exposure averaged 6.9 ppm with a range of 1 to 27 ppm. There was no evidence of exposure to other toxins and measured levels of lead and polychlorinated biphenyls (PCBs) were within normal limits (Davidson et al., 1998). A variety of covariates were measured, including maternal intelligence and the family’s socioeconomic status. Each family was visited at home to assess the child’s home environment using the Caldwell‐Bradley Home Observation of Measurement of the Environment (HOME). Each family participated in six test batteries through the child’s first 10.5 years of life. The children were evaluated when they were 6, 19, 29, 66, 107, and 128 months of age. Testing started with global measures and became increasingly focused on specific cognitive processes as the children matured (Davidson et al., 1995,

154


1998; Myers et al., 1995b, 2003; Shamlaye et al., 1995). Through 2005, over 60 primary outcomes have been measured and the association of each with prenatal MeHg exposure examined using both linear and nonlinear models (Axtell et al., 1998, 2000; Huang et al., 2005). Evaluations over the first 9 years of the children’s lives found four statistically significant associations between prenatal MeHg exposure and specific outcomes. One association was adverse (the grooved pegboard using the nonpreferred hand at 9 years of age), two were beneficial (the Preschool Language Scale Total Language Score at 66 months and the Connors Teacher Rating Scale at 9 years of age), and one was diYcult to categorize (males had lower activity scores on the Bayley Scales of Infant Development–Infant Behavior Rating Scale at 29 months of age). The authors concluded that these associations were consistent with chance and that the study provided no support for an adverse association between child neurodevelopment and prenatal exposure to MeHg from maternal consumption of ocean fish at the levels being studied (Myers et al., 2003). The study was not able to rule out adverse eVects above 10 ppm in maternal hair because the cohort had a limited number of children with exposures in that range. The study also could not exclude the possibility that subtle adverse eVects might occur and be detected as the children mature. The study is ongoing and continues to be double blinded. Evaluations are planned for the cohort as they reach their teen years.

C.

Faeroe Islands

In 1986–1987, the Faeroe Islands research team enrolled a main cohort of 1022 children. The children’s prenatal exposure was from the maternal consumption of seafood during pregnancy. Fish in the Faeroe Islands are relatively low in Hg content, but the population also consumes whale meat and this was the primary source of Hg (Grandjean et al., 1997). Whale meat can contains up to 3 ppm of Hg, with equal parts MeHg and inorganic (Andersen et al., 1987). They measured prenatal exposure to Hg in both maternal hair and in cord blood taken at delivery. The geometric mean for the cord blood concentration of Hg was 22.9 ppb (interquartile range was 13.4–41.3 ppb with a maximum of 350 ppb) and for the maternal hair Hg 4.27 ppm (interquartile range 2.6–7.7 ppm with 15% over 10 ppm) (Grandjean et al., 1997). Whale blubber is also consumed in the Faeroes and it contains PCBs and other toxins. The investigators measured exposure to PCBs in umbilical cord tissue and reported elevated levels. The relationship of PCBs that are lipophilic measured in cord tissue that has virtually no fat to values present in other tissues is not clear.


155

A number of covariates, such as maternal intelligence and socioeconomic status, were measured. The children were examined at 7 and 14 years of age, using an extensive battery of neuropsychological and neurophysiological tests. Following the 7‐year evaluations, the investigators reported the association between prenatal MeHg exposure measured in cord blood and 20 neuropsychological endpoints (Grandjean et al., 1997). Eleven of these endpoints had a significant association on one of three analytical models using ‘‘. . .a one‐tailed statistical significance level of 0.05. . .’’ (p. 425). These included adverse associations ‘‘. . .in the domains of language, attention, memory, and to a lesser extent in visuospatial and motor functions’’ (p. 417). Associations with maternal hair mercury levels were said to be present, but not as strong, and were not reported in detail. The contribution of PCBs and other toxins to these findings was addressed statistically in that and subsequent papers (Grandjean, 2004; Grandjean & Budtz‐Jorgensen, 1999; Grandjean et al., 1997, 2001). The investigators concluded that the adverse findings on multiple endpoints were primarily related to the MeHg exposure and that these eVects could be detected at a few weeks of life (Grandjean et al., 1997, 1998; Steuerwald et al., 2000). After the results of the 7‐year evaluations were published, oYcials in the Faeroes directed the investigators to tell the subjects their Hg values. Subsequently, the study was no longer double blind. The subjects were evaluated a second time at age 14 years. Only neurophysiological endpoints have been reported so far after that evaluation (Grandjean et al., 2004; Murata et al., 2004). These reports describe finding an association between prenatal MeHg exposure and several parameters of brainstem auditory evoked responses (BAERs). The authors reported ‘‘latencies of peak III and V increased by about 0.012 ms when the cord blood mercury doubled.’’ They also reported an association between prenatal MeHg exposure and heart rate variability as measured by the R–R interval on the EKG. However, they did not find an association between prenatal MeHg exposure and blood pressure (BP) that was reported at age 7 years (Sorensen et al., 1999). D.

Differences Between the Seychelles and Faeroe Islands Studies

These two studies were both well designed and executed and reached diVerent conclusions about the risk to a child’s neurodevelopment following prenatal MeHg exposure. Both studies examined large populations with significant MeHg exposure, used extensive test batteries, and included

156


a large number of covariates. However, there are important diVerences between them. Perhaps most striking are the diets and exposure. In the Seychelles, the diet is fish, fruits, and vegetables while the Faeroes diet includes whale meat and blubber. Consuming whale blubber leads to exposure to other toxins such as PCBs, cadmium, pesticides, and other persistent organic pollutants. In addition, the Faeroe Islands are in the North Atlantic while the Seychelles are near the equator and essential micronutrients in fish, such as omega 3 fatty acids, are reported to diVer, depending upon the temperature of the sea. Fatty acids and micronutrients have been shown to have beneficial eVects on child development (Grantham‐McGregor & Ani, 1999; Koletzko et al., 2001). There are also genetic diVerences between the cohorts. The heritage of the Faeroes is Scandinavian while that of Seychelles is primarily African. In addition, there are a number of diVerences in the study designs, covariates measured, statistical analysis plan, and other aspects of the studies that may account for the diVerent findings.

V. A.

INTERPRETING THE AVAILABLE DATA

Governmental Interpretations

Using the data from the studies described, health and environmental authorities have tried to establish what they consider safe levels of MeHg exposure. In 1990, an expert panel organized by the World Health Organization concluded that ‘‘. . .a prudent interpretation of the Iraqi data implies that a 5% risk may be associated with a peak mercury level of 10–20 m{ts}/g [ppm] in the maternal hair’’ (WHO, 1990, p. 103). In 1997, the U.S. Environmental Protection Agency (EPA) reviewed the data (U.S.E.P.A., 1997) and determined that the reference dose (RfD) should be set at 0.1 mg/kg/day. This translates to a maternal hair level of approximately 1.2 ppm, or a blood level of 5.8 ppb. The EPA defines an RfD as ‘‘a safe dose to consume daily over a lifetime. . ..’’ To establish the RfD, the EPA determined what they felt was the lowest level of exposure associated with any adverse eVect (58 ppb measured in blood). They then took one‐tenth of that number so there would be a safety or uncertainty factor. They based their determination on the Iraq study initially, but when the Faeroe Islands study was published in 1997, they recalculated the RfD. Although the studies in Iraq and the Faeroe Islands diVered markedly, the RfD determination was identical. The FDA in 1979 established an ADI (acceptable daily intake) for Hg in adults of 0.4 ug/kg/day (Tollefson & Cordel, 1986). In 1999, the Agency for Toxic Substances and Disease Registry (ATSDR) set the permissible


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exposure level at 0.3 mg/kg/day (ATSDR, 1999), based on data from the Seychelles studies. At the international level, the Joint FAO/WHO Committee on Food Additives met in 2003 and determined that 1.5 ug of MeHg/kg bw/week or 0.21 ug/kg/day could be consumed daily over a lifetime. In Japan, the Ministry of Health, Labour, and Welfare established a PTWI (permissible tolerable weekly intake) in 1973 that translates to about 0.4 ppm of THg, or 0.3 ppm of MeHg. These determinations of a safe dose to consume by varying health authorities are all based on individual interpretations of the same data. Some authorities based their determination on one study. The EPA based their RfD on the Faroes study while ATSDR used the Seychelles study. Others, such as the Joint FAO/WHO Committee on Food Additives, considered both studies. Despite these diVerent approaches and the use of diVerent uncertainty factors, the determinations are fairly similar. It is not clear presently which determination is most appropriate to protect human health. B.

Benchmarks

When one determines the benchmark dose from the various studies, they too seem to fall within a narrow range. Nearly all of the outcomes from the various studies on which a benchmark dose has been determined indicate that an exposure level around 10–20 ppm in maternal hair may carry a risk to the infant. This is shown in Fig. 1 (Clarkson & Strain, 2004). Benchmark dose calculations from the Faeroe Islands fall in the lower part of this range, while those from the Seychelles fall in the upper part of the range (Crump et al., 2000). C.

Factors Contributing to the Differing Interpretations

Numerous factors probably contribute to these diVerences in interpretation. However, the following may be especially important. 1. EXPOSURE BIOMARKER

Toxins can be measured in many biological media and mercury is no exception. Selecting the biomarker that most accurately defines the exposure is critical. With MeHg, the critical organ is the brain, but there is no direct way to measure brain levels in living subjects. Hair measurements have been the standard for many years and in both the Seychelles and Faeroe Islands, exposure was measured in maternal hair growing during pregnancy. However, in the Faeroe Islands, exposure was also measured in cord blood. Cord blood recapitulates exposure that occurs during the last few weeks of pregnancy. A bolus exposure occurring earlier in pregnancy, such as from

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FIG. 1. Benchmark evaluations of epidemiological studies. NOAEL, no observed adverse eVect level. There are two calculations from New Zealand, depending on whether the single child with the highest exposure is included (1) or excluded (2). Reprinted with permission from Clarkson, T. W., & Strain, J. J. (2004). Methyl mercury: Loaves versus fishes. Seychelles Medical Dental Journal, 7(1), 64.

consumption of a whale meal, would not be detected by measuring cord blood. If hair is long enough and analyzed in segments, it can recapitulate exposure during the entire 9 months of pregnancy. Consequently, a larger exposure at any time during the pregnancy would appear in hair. Autopsy studies have confirmed that maternal hair Hg levels correlate highly with brain levels (Cernichiari et al., 1995a). The association of cord blood mercury and brain levels has not been reported. Presently, the choice of the optimal biomarker is controversial. 2. CONCOMITANT EXPOSURES

Most human exposures are to mixtures of toxins, such as occurred with the pollution from factories in Japan. Although the toxicity of one toxin may predominate, there may be contributions from the other toxins present. For example, there are reports that concomitant exposure to MeHg and PCBs may have synergistic toxicity (Risher et al., 2003; Stewart et al., 2003). Investigators with the Faeroes study report their findings are mainly associated with MeHg exposure and that they can statistically factor out the contribution of other toxins. Some authorities feel that statistical methods can accomplish this while others do not.


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3. OUTCOME MEASURES

The selection of the most sensitive outcome to measure presents a special problem. Studies in the Iraq poisoning showed a dose–response relationship between exposure levels and the attainment of developmental milestones and neurological findings on examination (Cox et al., 1989). However, with low‐ level exposure from fish consumption, investigators are looking for more subtle developmental diVerences. The Seychelles and Faeroe Island studies approached the outcome measures diVerently. In the Seychelles, subjects were evaluated first for mild diVuse cognitive changes, such as general intelligence, neurological findings, and milestones. As the children matured, testing focused on increasingly specific motor and cognitive functions and processes. In the Faeroe Islands, testing focused on domain‐specific functions and neurophysiological tests for vision, hearing, and central processing from the beginning (White et al., 1993). The problem selecting an outcome is highlighted by the National Research Council (NRC) review (2000). The committee was charged with reviewing the available data and asked to determine ‘‘. . . the appropriateness of the critical study, end points of toxicity, and uncertainty factors used by EPA in the derivation of the reference dose for MeHg’’ (p. xii). The endpoint that had the lowest Benchmark Determination (BMDL) turned out to be the McCarthy Scales of Children’s Abilities (MSCA) from the New Zealand study. However, the NRC committee rejected it, based on the limitations of that study (p. 285). The next lowest BMDL was on the Continuous Performance Test (CPT) from the Faeroe Islands study. The committee also rejected it because of technical diYculties in test administration (p. 286). The third lowest BMDL was on the Boston Naming Test (BNT) from the Faeroe Islands. The panel chose the BNT stating that it was ‘‘. . . the most sensitive, reliable end point’’ (p. 299). Using the BMDL from the BNT, the panel concluded that the EPA’s RfD was appropriate. However, selection of the BNT raises additional concerns. The BNT was developed to detect aphasia and brain damage in adults and is not a standard part of child neuropsychological testing. No biological reason has been proposed as to why the BNT should be particularly sensitive to prenatal MeHg exposure. The Faeroe Islands investigators themselves have reported concerns about the BNT. In 1997, they wrote, ‘‘. . . especially for the Boston Naming Test, the PCB concentration appeared to be an important predictor’’ (Grandjean et al., 1997, p. 425). They later stated ‘‘. . . the cord PCB concentration was associated with deficits on the Boston Naming Test. . .’’ and ‘‘. . . the association between cord PCB and cord‐blood mercury (r ¼ 0.42) suggested possible confounding’’ (Grandjean et al., 2001, p. 305). Selecting an appropriate outcome to focus upon presents a substantial

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challenge to investigators and those interpreting their studies to use in making public policy. 4. STATISTICAL ANALYSIS

There are many approaches that can be taken to analyze the data and investigators must choose. The Seychelles study established a priori a linear analysis plan that they considered the primary analysis. Subsequent nonlinear and other analyses were considered confirmatory or secondary. The Faeroe Islands study performed three diVerent analyses on the data from their 7‐year‐old evaluations and reported all three models (Grandjean et al., 1997) Among the models, there were 11 endpoints that showed statistically significant adverse associations with prenatal MeHg exposure. Interpreting these findings was challenging because the significant endpoints were not consistent among the diVerent models. In addition to the factors already highlighted, there are numerous others such as the selection and measurement of covariates that make epidemiological studies challenging to carry out and to interpret (Davidson et al., 2004).

VI.

WHAT CONSTITUTES A DEVELOPMENTAL DISABILITY?

The term ‘‘developmental disability’’ is a political one. The definition presented by the Center for Communicable Diseases (CDC) of the U. S. Department of Health and Human Services is ‘‘Developmental disabilities are a diverse group of severe chronic conditions that are due to mental and/ or physical impairments. People with developmental disabilities have problems with major life activities such as language, mobility, learning, self‐help, and independent living. Developmental disabilities begin anytime during development up to 22 years of age and usually last throughout a person’s lifetime.’’ (CDC, 2005). The subtle diVerences in cognitive and motor performance that have been reported to have an association with prenatal MeHg exposure are all far below the level of clinical significance (see this volume’s Introduction). As an example, the diVerence in finger‐tapping reported to be present between high‐ and low‐exposure groups in the Faeroe Islands consisted of four finger taps using both hands in 15 seconds. Tapping with either hand individually was nearly identical and there was substantial overlap in scores between the two groups (Grandjean et al., 1998). Finger‐tapping and the other reported findings from epidemiological studies are statistical associations that have no clinical relevance for an individual. Even so, it is important to know whether prenatal MeHg exposure can consistently be shown to be associated with a change in these endpoints. If so, it would strengthen the conclusion that


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there is a causal relationship between low‐level prenatal MeHg exposure and adverse eVects on the developing nervous system. However, the diVerences reported to date by epidemiological studies could not be considered developmental disabilities since they would have no impact on the child’s ability to function. VII.

DOES EXCEEDING THE EPA’S RFD PLACE A CHILD AT DEVELOPMENTAL RISK?

If we accept that the reported associations between prenatal MeHg exposure and child development are indeed true, although this is by no means a certainty, would they constitute a developmental disability? Several papers have equated exceeding the RfD as placing the child at developmental risk (MahaVey et al., 2004a; Trasande et al., 2005). To understand whether this is likely to be true, it is helpful to examine the definition of the RfD. As noted earlier, the RfD is the dose that can be consumed daily over a lifetime without a risk of adverse eVects. The RfD determined by the EPA for MeHg includes a safety or uncertainty factor of 10, that is, the EPA determined what they believed was the lowest level of exposure thought to be associated with adverse eVects and divided that number by 10. The lowest level thought to be associated with harm to the fetus is, of course, open to substantial controversy. It seems unlikely that exceeding the RfD, even on a regular basis, would place the child at significant risk of any clinical health problems. To illustrate the clinical impact of the reported changes, one of the strongest pieces of evidence for a detectable change among subjects of varying prenatal MeHg exposure from the Faeroe Islands study is on the BAERs at 14 years of age (Murata et al., 2004). Murata and colleagues reported that the latency of wave III and V increased by 0.012 ms for each doubling of the cord Hg exposure. There are no data to suggest that a change of 1/100,000th of a second would constitute a significant impairment in auditory processing and it would not be considered a developmental disability. Thus, there is no evidence that exceeding the RfD, even on a regular basis, would place the child at risk of having a developmental disability. VIII.

HOW DID THE NRC AND THE EPA DETERMINE THE RISK TO UNITED STATES CHILDREN?

The process by which the NRC determined that 60,000 U.S. children were at risk from MeHg exposure was explained in a letter from the committee chair to the U.S. Food and Drug Administration dated December 1, 2000, and later published (MahaVey et al., 2004a). The determination was made as follows:

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Start with the RfD determined by the EPA

0.1 ug/kg/d

Take the population of U.S. women of childbearing age (15–44 years). [Based on the U.S. Census Bureau data from 1989]

60,208,000

Take the percentage of women who reported consuming fish [based on the Continuing Surveys of Food Intake by Individuals conducted in 1989–1990, this is approximately 30.5%]

18,363,440

Take the number of those women who reported consuming over 100 grams of fish per day (approximately 5%)

918,172

Take the birth rate for U.S. women

65.6/1000

Multiply the number of women consuming over 100 grams of fish per day by the birth rate for U.S. women

60,232

This formula determines the number of U.S. children who might exceed the RfD established by the EPA. It does not determine the number of children who might have a developmental disability from MeHg exposure secondary to maternal consumption of fish. Indeed, the number was obtained without requiring that there be even one child with a developmental disability secondary to MeHg exposure from fish consumption. Subsequently, the number of U.S. children at risk was increased to 300,000, based on a recalculation of this sequence of assumptions using more recent data from the 2000 NHANES survey (MahaVey et al., 2004a; Rice, 2003). Not surprisingly, fish consumption was substantially higher in the NHANES survey since health authorities had encouraged it for its reported cardiac benefits. The number was later increased to over 600,000 U.S. children at risk based on the recognition that fetal hemoglobin binds more tightly to MeHg (Trasande et al., 2005). This had been known for many years (Amin‐Zaki et al., 1976), but was apparently not considered when the EPA originally determined its RfD.

IX.

CONCLUSIONS

Mercury is ubiquitous in our environment and the amount that we come in contact with is increasing. The organic form of MeHg is very toxic to the central nervous system. All fish contain small amounts of MeHg and everyone who consumes fish or seafood is exposed to it. Once inside the body, MeHg readily crosses the placenta and the blood–brain barrier and enters the central nervous system. Methyl mercury poisoning episodes have clearly demonstrated that the developing fetal brain is especially sensitive and can be seriously impaired, even when the mother has no symptoms.


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The events in Japan confirm that consuming fish that is heavily contaminated with MeHg can damage the developing central nervous system. However, interpreting the Japanese experience is complicated by the very high exposures (at Minamata, some fish contained up to 50 ppm of MeHg or 100 times the background level usually found) and the presence of other toxins in the factory waste. Even so, studies of poisoning in Iraq suggested that a level of prenatal exposure of around 10 ppm measured in maternal hair might adversely aVect the developing fetus. Individuals who consume fish with only background levels of MeHg can achieve a hair level of 10 ppm or greater. However, there are no reported cases of mild disability after prenatal MeHg exposure and no reports of poisoning from fish consumption outside of Japan. The evidence that low levels of MeHg exposure from fish consumption might adversely aVect a child’s neurodevelopment presently comes only from epidemiology studies in Iraq and elsewhere. As has been noted, these studies are diYcult to carry out and equally hard to interpret. To date, these studies have not been conclusive and the evidence that MeHg exposure at the levels achieved by consuming fish that is not overtly polluted adversely aVects the fetal brain is very controversial. The level of MeHg exposure at which adverse eVects to the developing brain first occur is not presently known. DiVerent health and environmental authorities interpret the epidemiological studies of MeHg exposure from maternal fish and seafood consumption diVerently and the risk of fetal neurological harm from this source requires further study. There is general agreement among health authorities that fish consumption has significant benefits. Fish is an important source of protein, long chain fatty acids, and micronutrients. The American Heart Association encourages the consumption of two fish meals a week to maintain a healthy heart (AHA, 2005). Consequently, it is important to maintain a balanced view of fish consumption. Although fish consumption carries theoretical risks from MeHg exposure, it also carries proven benefits. Many scientists are not prepared to make the multiple assumptions necessary to arrive at the conclusion that large numbers of U.S. children are presently at significant risk for a developmental disability from exposure to low‐dose MeHg. There is agreement, however, that the high‐quality study done in the Faeroe Islands shows conclusively that the consumption of whale meat and blubber can have adverse health eVects and health authorities there have appropriately discouraged its consumption by women of childbearing age (Weihe et al., 2005). However, the Faeroes study does not clarify which toxin present in whales causes the harm. Since millions of people around the world consume fish daily, it should be possible to study this issue directly without extrapolating from populations having unusual

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Environmental Agents and Autism: Once and Future Associations SUSAN L. HYMAN DEPARTMENT OF PEDIATRICS (GOLISANO CHILDREN’S HOSPITAL AT STRONG), UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

TARA L. ARNDT DEPARTMENT OF ENVIRONMENTAL MEDICINE, UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

PATRICIA M. RODIER DEPARTMENT OF OBSTETRICS AND GYNECOLOGY, UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

I.

INTRODUCTION

Autism is a neurodevelopmental disorder whose symptoms are manifest along a continuum, hence, the common use of the term, Autism Spectrum Disorders (ASD). The Diagnostic and Statistical Manual—IV (American Psychiatric Association, 1994) defines a general set of symptoms related to deficits in social reciprocity, communication, and repetitive behaviors that can be clustered into four specific diagnoses within the spectrum. However, the heterogeneity of clinical presentations even within those subgroups suggests that there is no single etiology or neurobiologic cause. Evidence for the genetic contribution to the etiology of autism was reported as early as 1977 (Folstein & Rutter, 1977). The genetics of autism has been an active area of epidemiologic and molecular genetic pursuit (Bailey et al., 1995; Stodgell et al. [rev.], 2001b). The support for a genetic component to the etiology of autism is based on the observation of recurrence in siblings of almost 4% (Chakrabarti and Fombonne, 2001), which is much greater than the risk for the disorder in the general population. The concordance for INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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autism is 60% in identical twins, with some symptoms of autism present in over 92% of the identical twin of an aVected individual (Bailey et al., 1995). Larger twin studies will be an important avenue to investigate the role of genetic and environmental factors in the etiology of autism. The role of environmental agents and the environment itself in the development of the symptoms of autism has been the source of speculation and hypothesis generation since Leo Kanner first described autism in 1943 (Kanner, 1943). Indeed, some of the hypotheses generated based on clinical observations in his original cases remain areas of study to this day, the genetic contribution being a prime example. Some of the early etiologic hypotheses have succumbed to advances in behavioral and medical science, however. This chapter summarizes the hypotheses related to role of the environment in autism. It will not only examine how environmental factors might be etiologically related to autism but also how the thinking around environmental influences shaped treatment for and beliefs about the disorder.

II.

THE FAMILY ENVIRONMENT

In 1943, Kanner first reported a case series of children who had social isolation, atypical language development, and intense and unusually ritualized behaviors (Kanner, 1943). He noticed that, among his first 11 patients with this symptom complex, there seemed to be a pattern of cold and obsessive parents, professional families, and atypical attachment of parent and child. He did note that he thought the disorder was of organic origin. The idea of a broader phenotype, where family members could share some lesser characteristics with the aVected family member and still be subthreshold for the diagnosis, was not yet conceptualized (Dawson et al., 2002). The prevailing explanations for behavioral disorders in the years after World War II lay in psychological theories related to the relationship of an individual with his/her parents. This was specifically applied to autism by Bruno Bettelheim, a self‐proclaimed expert in child psychology and development whose Ph.D. was in philosophy. He advanced the hypothesis that autism was the result of maternal rejection (Bettelheim, 1967). He advocated that aVected individuals could only be helped by removal from their families and application of treatments that involved both language‐based therapies and physical management to repair a dramatically abnormal bonding experience with a ‘‘refrigerator’’ mother. The Orthogenic School he ran in Chicago was the site of much of this treatment. Later review of the case histories of his patients suggests many would not have been diagnosed with

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autism as defined by Kanner. The belief that autism was based on poor parenting, however, was the prevailing theory explaining autism through the 1950s and into the 1960s. A generation of families suVered shame and guilt because they thought that they provided a socially toxic environment that caused their child’s autism. It is possible that the disorder was underdiagnosed because of the stigma of blame for the parents in addition to the lack of acceptable or eVective treatments. Insight‐oriented psychotherapy and other language‐based psychodynamic interventions, which were the treatments used by psychiatrists in the 1950s and 1960s, did not address the core symptoms of the disorder and were not successful in teaching people with autism how to interact socially.

III. A.

CONSIDERATION OF BIOLOGIC ETIOLOGIES

The Prenatal Environment

1. OBSTETRIC COMPLICATIONS

By the end of the 1950s, the biologic bases for many neurologic and psychiatric disorders were being investigated. For example, the role of asphyxia, metabolic instability, and placental compromise during gestation and delivery were increasingly linked with developmental disabilities. The National Institute for Child Health funded the largest study to date to identify the factors associated with birth injury and later neurologic handicap in the Collaborative Perinatal Project. Between 1959 and 1961, exhaustive data were collected on over 50,000 pregnancies. For seven years, targeted neurologic disorders manifesting in childhood were prospectively monitored in this sample. Autism was one of them (Torrey et al., 1975). At that time, the diagnostic criteria for autism were relatively vague and the distinction from childhood psychosis and mental retardation was not always clear. Only 14 children were reported to have had autism in this birth cohort. (The prevalence would be less than 3:10,000 if all children with autism were identified.) When the obstetrical data were analyzed for this subset of children, the presence of bleeding in pregnancy compared to controls was the only obstetric finding that almost reached statistical significance. It was noted that more fathers of children with autism were employed as chemists than would have been expected by chance. This finding was rediscovered in a modern sample examining the potential for cognitive profiles of technical professions such as engineers and computer scientists as opposed to social workers that might be associated with a genetic predisposition to autism (Wheelwright & Baron‐Cohen, 2001). This modern interpretation of the observation suggested that a genetic predisposition for features of autism

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in family members related to enhanced visual problem‐solving skills rather than an occupational exposure might be the association leading to autism in the oVspring. Subsequent to the Collaborative Perinatal Project, investigators probed the potential association of obstetric factors with autism using retrospective examination of birth records and birth histories of children later diagnosed with autism compared to cohort controls and unaVected siblings. These more recent studies had the benefit of more discrete diagnostic criteria for autism, so cases defined as having autism probably represented a more homogeneous group. The families who participated in an epidemiological study in Nova Scotia (Bryson et al., 1988) reported a higher incidence of neonatal respiratory distress in children with autism. However, no consistent findings related to gestational age, birth weight, asphyxia, or obstetric intervention has been identified across studies. It has been noted that increased obstetric risk characterizes the families of children with autism (cases and unaVected siblings compared to controls), suggesting that the obstetric findings are not causal for the autism but are somehow related to a genetic and/or environmental risk in the family (Table I). Although no consistent associations of individual events with autism have emerged when the obstetric and neonatal histories are examined, the neuroanatomical findings reported in brains of people with autism indicate that the abnormalities most often seen are of prenatal origin. Bauman and Kemper (1985) reported increased hippocampal cellular density and decreased cellularity of the cerebellum. Rodier et al. (1996) reported hypoplasia of brain stem nuclei. Bailey et al. (1998) identified heterotopias and atypical development of the inferior olive in some cases of autism. Both autopsy and imaging studies have identified the cerebellum as abnormal in some cases of autism. The most consistent pathologic finding is decreased number of Purkinje cells in the cerebellum (Bailey et al., 1998; Bauman & Kemper, 1985). Courchesne has reported hypoplasia of the cerebellar vermis on MRI, although this finding is variable (Courchesne et al., 1988). The neuroanatomic studies have not identified a specific brain region involved in all cases or pinpointed a time in prenatal development that the neurologic insult occurred. However, the reports all identify abnormalities that occurred between the first weeks and last trimester of gestation. The periventricular leukomalacia, cysts, and ventricular dilatation associated with asphyxic injury do not appear in the histology of idiopathic autism (Bailey et al., 1998). Prenatal events related to atypical brain development and subsequent atypical function are likely to play a role in the etiology of autism. How genetic factors, environmental embryologic events, and obstetric optimality may interact is an area deserving of further study.

TABLE I SEVERAL STUDIES IDENTIFY AN INCREASED RATE OF OBSTETRIC AND PERINATAL COMPLICATIONS IN BOTH CHILDREN WITH ASD AND SIBLINGS, SUGGESTING A FAMILIAL RISK Authors

Cases

Controls

Diagnosis

Glasson, Bower, Petterson et al. (2004)

465 ASD

481 Sibs 1313 population controls

Record review, DSM–IV criteria

Hultman, Sparen, Cnattangius et al. (2002)

408 ASD

Matched control 2040

Swedish Medical Birth Registry

Zwaigenbaum, Szatmari, Jones et al. (2002) Juul‐Dam, Townsend, & Courchesne (2001)

78 ASD

88 Sibs

ADI/ADOS diagnosis

61 Autism 13 PDD, NOS

Report of Final Natality Statistics, 1995

ADI/ADOS

110 Autism

50 Down syndrome

ADI, ADOS

Bolton, Murphy, MacDonald et al. (1997)

Findings Cases had older parents, were firstborns, fetal distress, Apgar

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International Review of Research in Mental Retardation, Volume 33: Developmental Epidemiology of Mental Retardation and Developmental Disabilities

International Review of Research in Mental Retardation, Volume 24 (International Review of Research in Mental Retardation)