Alternative toxicological methods

ALTERNATIVE TOXICOLOGICAL METHODS ALTERNATIVE TOXICOLOGICAL METHODS Edited by Harry Salem Sidney A. Katz CRC PR E ...

Author: Harry Salem | Sidney A. Katz

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ALTERNATIVE TOXICOLOGICAL METHODS


Edited by

Harry Salem Sidney A. Katz

CRC PR E S S Boca Raton London New York Washington, D.C.

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.”

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CHAPTER

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Integrated In Vitro Approaches for Assessing Systemic Toxicity $QQD)RUVE\DQG%DV%ODDXERHU

CONTENTS ,QWURGXFWLRQ 7KH6WHSZLVH$SSURDFKIRU,QWHJUDWHG7HVWLQJ 7KH(&,7763URMHFW &RPSRXQGVDQG7HVW%DWWHU\ 5HVXOWV &RQFOXVLRQVDQG)XWXUH3HUVSHFWLYHV 5HÀQHPHQWRIWKH1HXURWR[LFLW\7HVW%DWWHU\ 'LIIHUHQW([SRVXUH7LPHV $FNQRZOHGJPHQWV 5HIHUHQFHV

INTRODUCTION &ODVVLFDOWR[LFRORJLFDOULVNDVVHVVPHQWLVEDVHGRQTXDQWLÀ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

43

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THE ECITTS PROJECT ,Q WKH (XURSHDQ 5HVHDUFK *URXS IRU $OWHUQDWLYHV LQ 7R[LFLW\ 7HVWLQJ (5*$77 LQLWLDWHGDQLQWHUODERUDWRU\SURMHFWLQDVVRFLDWLRQZLWKWKH6ZHGLVK%RDUG IRU /DERUDWRU\ $QLPDOV &HQWUDOD )|UV|NVGMXUVQl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

INTEGRATED IN VITRO APPROACHES FOR ASSESSING SYSTEMIC TOXICITY

1. Experimental

2. Modeling

3. Validation (literature data)

in vitro data on kinetics

kinetic modeling

kinetics in vivo

in vitro data on dynamics

prediction of target tissue

(e.g., CNC, EC20, EC50)

45

concentrations (e.g., NOEL, LOEL) prediction of dynamics

in vivo systemic

prediction of systemic

Figure 5.1

toxic doses

toxic doses

(e.g., NOED, LOED,

(e.g., NOED, LOED,

LD50)

LD50)

Building blocks of the ECITTS scheme.

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THE OCULAR SURFACE: BARRIER AND INJURY

93

Table 9.3 Grading of Eye Burns I

II

III

IV

Immediate signs Erosion Hyperemia

Large erosion Ischemia 1/3 Chemosis

Surface defect Ischemia >1/2 Rose chemosis Corneal opacity

Epithelia destroyed Deep ischemia >3/4 Dense corneal opacity Conjunctival necroses Sclera porcelain white Discoloration and atrophy of iris Fibrin exudate

Later signs Regeneration

Recirculation Regeneration

Persistent erosion Ulceration Vascularization Scars

Proliferation Large ulcerations Melting of cataract Glaucoma Scarification

Note: Grading of eye burns according to clinical signs. The upper part lists immediate damage visible, the lower one later secondary events. The classification of signs was developed from various authors and by clinical experiences (Hughes, 1946; Roper-Hall, 1965; Thoft, 1978; Reim and Kuckelkorn, 1995)

Figure 9.1

Eye with a mild alkali burn stage I (Table 9.3). The corneal epithelium was completely lost, but the stroma remained undamaged and clear. The conjunctiva showed hyperemia, but no swelling or ischemia. The damage healed within a few days.

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94

Figure 9.2


Lime burn stage II (Table 9.3). The whole corneal and some conjunctival epithelium was destroyed. The corneal stroma exhibited little superficial turbidities. The lower conjunctiva is demonstrated by upgaze. It was swollen (chemosis). Superficially in the conjunctiva, ischemia is recognized by the interrupted blood columns. With the lit lamp microscope, bloodstream could not be detected. Underneath the otherwise pale conjunctiva, intact sclera appeared with a faint rose background.

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Figure 9.3

Clinical appearance of a severe chemical injury grade IV. The inner margin of the upper lid showed a white line of necrosis. The conjunctiva appeared flat and white, also from necrosis, which presumably included visible parts of the sclera. In the upper left region, some hemorrhages were deposited in necrotic conjunctiva. Ischemia was evident. The cornea was completely turbid. The outlines of iris and pupil could be hardly identified.


Figure 9.4

95

Melting of the anterior eye segment, nine days after most severe burn from liquid metal. There were extended necroses of all conjunctival, subconjunctival, and scleral tissues, appearing homogeneously white and slippery. Only in the right upper region, some hemorrhages in necrotic tissues showed red color. The cornea was opaque in the upper marginal parts. The lower and central cornea was melted away and the iris and lens exposed. Since at that time (1977) corneal donor material was not available, the eye was lost and had to be removed. In the melting tissues of the anterior eye segment, high activities of N-acetylglucose aminidase (NAcGA, E.C.3.2.1.50) and cathepsin-D (E.C.3.4.23.5) were found (Reim, 1982a).

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96


Cornea and conjunctiva Severe lesion, severe response PGE2α, Interleukins, LT 4, Subst-P, VIP, CGRP Mild lesion weak response

IL-1, IL-6 IL-8, TNF

PMNs

PMNs, macrophages

T-lymphocytes B-lymphocytes Plasma cells

_

+

O2 OH -radicals lysosomal enzymes

Cellular and humoral antibodies Restitution

Inflammation Figure 9.5

Scars

Ulceration

Flow diagram of inflammatory cascade following chemical and thermal injuries of the eye. The inflammatory response is a quantitative process produced by the affected tissues and the leucocytes involved (Ghattacherjee et al., 1979; Reim et al., 1980, 1993, 1997; Reim, 1982a; Rochels et al., 1982; Kulkarni and Srinivasan, 1983; Becker et al., 1991, 1995; Reim and Leber, 1992; Reim and Becker, 1995).

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97

Table 9.4 Cytokines in Tears EGF p regeneration of epithelium TGFbeta 2 p inhibits proliferation TNFalpha, in inflammation Many others, but presumably released from damaged surface epithelia Note: Cytokines and growth factors in tears influencing the corneal epithelium (Mishima et al., 1991; Kruse and Tseng, 1994; Sotozono and Kinshita, 1998).

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100

10

1 Keratitis Inflammat.Cone Ulceration

Figure 9.6

Decompensation Dystrophy, Scars Keratoconus

Interleukin-1F (IL-1) and Interleukin-6 (IL-6) in human corneal buttons from keratoplasty. Total number of cases: 127. The logarithmic ordinate shows the concentrations found in pg/mg extractable protein. The symbols represent the median, squares stand for IL-1, rhombs for IL-6. The error bars demonstrate the 75% percentiles. In the abscissa, the diagnoses of the cases were indicated corresponding to the position of the symbols (Becker et al., 1995). Inflamed corneas revealed very high levels of IL-1 and IL-6. The levels in the uninflamed, quiete corneas were lower by an order of magnitude.

98


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99

N-Acetylglucose aminidase in human tears wmol/min/ml 10

Atopic conjunctivitis

1

Range of 8 samples from human burns Normal 0.24 s 0.09 (9)

0.1 Figure 9.7

Activity of N-acetylglucose aminidase (NAcGA, E.C.3.2.1.50) in human tears collected from nine human cases with eye burns stage I and II and an atopic patient. Please note that the ordinate is in logarithmic scale! The enzyme activity (QMol/min/ml) increased considerably in surface diseases.

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100


Table 9.5 Stroma of Rabbit Cornea, QMol/g H2O, mean ± SD

EDXA Na Cl S P Ca

Normal (n = 20) 125 123 105 12 3

± ± ± ± ±

Alkali Burn, Denuded, Rinsed for 16 Days, 4v daily with 0.9% NaCl (n = 8)

42 25 21 4 3

90 65 24 22 1

± ± ± ± ±

11 15 4 22 3

Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burn. The denuded stroma was rinsed with saline, four times daily for 16 days. Na, Cl, and especially S were decreased, P increased (Fischern et al., 1998).

Calcification and Contamination 0RUHRYHU ZKHQ H\HV GHQXGHG IURP HSLWKHOLXP ZHUH ULQVHG ZLWK SKRVSKDWH EXIIHURULIWKH\ZHUHWUHDWHGZLWKH\HGURSVFRQWDLQLQJSKRVSKDWHEXIIHUDVVROYHQW RUSKRVSKDWHVDOWVRIGUXJVVXFKDVSUHGQLVRORQHSKRVSKDWHUDSLGFRUQHDOFDOFLÀ FDWLRQZDVREVHUYHGLQFOLQLFDOSDWLHQWVDQGLQPRGHOH[SHULPHQWV7DEOH)LJXUHV ²5HLPHWDO6FKUDJHHWDO ,QDGGLWLRQKXPDQFRUQHDOEXWWRQV REWDLQHGIURPEXUQWH\HVRQNHUDWRSODVW\DVZHOODVFRQMXQFWLYDOVDPSOHVUHYHDOHG D UHPDUNDEO\ KLJK FRQWDPLQDWLRQ E\ YDULRXV PHWDOV DQG PLQHUDOV 6FKUDJH DW HO 6FKLUQHU HW DO 7KHVH FRQWDPLQDWLRQV ZHUH H[SODLQHG RQ RQH KDQGE\LPSXULWLHVLQWKHWHVWPDWHULDOVDQGRQWKHRWKHUKDQG E\VHFRQGDU\LQWUR GXFWLRQRISDUWLFOHVZLWKRSKWKDOPLFVROXWLRQVXVHGLQWRSLFDOWKHUDS\RQWKHGHQXGHG VWURPDVXUIDFH5HLPHWDO Scarring ,QPDQ\FDVHVWKHXOFHUDWLRQVGLGQRWSHUIRUDWH7KHQSUROLIHUDWLRQWLVVXHFRY HUHG WKH ZKROH DQWHULRU H\H VHJPHQW DQG WUDQVIRUPHG LW LQWR VKULQNLQJ VFDUV7KLV LQGXFHGV\PEOHSKDUDWKDWWKHPRWLOLW\RIWKHJOREHZDVUHVWULFWHG5HLPDQG6DULF 5HLPD RUWKHJOREHZDVWRWDOO\LPPRELOL]HG+HDOLQJRIWKHVHFDVHV Table 9.6 Stroma of Rabbit Cornea, QMol/g H2O, Mean ± SD

EDXA Na Cl S P Ca

Normal (n = 20) 125 123 105 12 3

± ± ± ± ±

42 25 21 4 3

Alkali Burn, Denuded, Rinsed for 16 Days, 4v Daily with Phosphate Buffer (n = 8) 105 88 28 623 435

± ± ± ± ±

22 33 4 307 198

Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burn and rinsing with isotonic phosphate buffer, four times daily for 16 days. Na, Cl, and especially S were decreased, but P and Ca were largely increased. Clinically, calcification of the cornea was observed (Schrage, 1997; Fischern et al., 1998; Haller, 2001).


Figure 9.8

101

Left eye of a 16-year-old boy six months after a most severe chemical injury. In this accident, a highly alkaline etching fluid used to work on electronic parts spilled into both eyes of the patient. In this case, a severe inflammatory response had developed and remained for years. The conjunctiva-like proliferation tissue surrounding the cornea was swollen and very hyperemic. The cornea was devoid of epithelium. It showed extended ulceration especially in its marginal parts and was generally thinned. The upper right cornea showed white calcification. To save the eye from melting, a keratoplasty was performed. The excised cornea was examined with electron dispersive x-ray analysis method (EDXA) (see Figures 9.9 and 9.10).

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Figure 9.9

Scanning electron microscopy (SEM) on a cross section of the cornea seen in Figure 9.8. Magnification v200. The upper part shows calcification, the lower one parallel corneal lamellae (Schrage et al., 1988, 1993, 1996).

102


Figure 9.10 Electron dispersive x-ray analysis (EDXA) of the calcified cornea as demonstrated in Figures 9.8 and 9.9. The spectra of the x-rays backscattered at scanning electron microscopy (SEM) showed as expected high peaks for calcium (Ca) and phosphorus (P). But the most prominent peak from this sample was emitted from silicon (Si). Thus, EDXA revealed an unexpected high contamination of the cornea by silicone, which might have explained the severe and longstanding inflammatory response in this case (Schrage et al., 1988, 1993, 1996).

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103

Figure 9.11 Eye of a 42-year-old male 2 years after severe lime burn. Heavy scar formation could not be prohibited. The cornea was covered with thick highly vascularized proliferation tissue. The conjunctiva developed strong scars between the globe and the lids, reducing eye motility. The conjunctival scars also deformed the lid margins. The hyperemic, red scar tissue showed that the inflammatory response had not subsided after 2 years. The eye was practically blind and had bad prognoses for surgical rehabilitation.

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Figure 11.1 Biotin surface labeling to visualize epithelial barrier. Cultured bovine cornea was incubated with sulfo-NHS-LC-biotin for 30 min and then embedded in OCT, snapfrozen, and sectioned (6 Qm). Cryostat sections (8 Qm) were (A) stained directly with hematoxylin to reveal corneal morphology (B) or incubated with rhodamineavidin D to visualize the bound biotin. The rhodamine staining represents biotinylation of accessible surface of normal bovine cornea and linear staining at the corneal surface indicates functional epithelial TJ barrier in cultured corneas. Ep, epithelium; BM, basement membrane; St, stroma that consists of fibroblasts. A and B are mirror orientations of the same corneal sections.

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Figure 11.2 Tight junction permeability assay of cultured bovine corneas in response to challenge of three hair care products. Corneas in culture were treated with 100%, 50% (not shown), and 25% chemicals, and TJ permeability of corneal epithelium was assessed by surface biotinylation as described in Figure 11.1. Inserts: corneal sections stained directly with hematoxylin to reveal corneal morphology. Note: extended biotinylation of the corneal surface caused by GA and GB exposure in a concentration dependent manner. However, no disruption of TJ was observed in GC treated cornea.

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Figure 11.3 EMSA analysis of NF-OB DNA-binding activity in bovine corneal epithelial cells in response to consumer product challenge. Panels showed cultured corneas were treated with different concentrations of three hair care products for 5 min, untreated cells were used as control (C). The corneas were then cultured for 10 min without the presence of the chemicals. Cell extracts from corneal epithelial cells treatment were probed with 32P-labeled AP-1 (upper panel) or NF-OB (lower panel) consensus oligonucleotide. EMSA experiments were repeated two times, and gels presented in the figure are from a representative set.

CORNEAL ORGAN CULTURE FOR OCULAR TOXICITY TEST

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b

stearalkonium chloride ceteth-24 dimethyl stearamine glyceryl stearate cocoamphodiacetate sodium nonoxynol-6phosphate quaternium-26 PEG-120-methyl glucose dioleate sodium lauryl sulfate disodium laureth sulfosuccinate butylene gylcol lauramide DEA ammonium lauryl sulfate lauramide DEA ethoxydiglycol hydroxypropyl methylcellulose

sodium laureth sulfate cocamidopropyl betaine

Surfactant Ingredients

5.0 0.5 12.0 2.0 0.4 0.15

25.0 15.0

1.14 1.0 0.67 0.44 15.0 6.0 1.5 1.5

25.0 5.0

Percent Formula (w/w)

anionic nonionic — —

anionic anionic nonionic nonionic

cationic nonionic cationic nonionic amphoteric anionic cationic nonionic

anionic amphoteric

Surfactant Class

MAS 57.4 CS 38.0 COS 2.0

MAS 37.8 CS 20.0 COS 1.0

MAS 22.0 CS 10.0 COS 0.5

MAS 4.8 CS 0.0 COS 0.0 MAS 14.2 CS 0.0 COS 0.0

Draize Scoresb

Concentration used in Draize test, and initial concentration for dilution in TEP assay. MAS is the maximum average score; CS is the corneal score; COS is the corneal opacity score. Federal Hazardous Substances Act (FHSA) classifications: –, negative; +, positive; –/+ repeat test (FHSA, 1979).

100%

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a

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Product Type

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Table 15.2 Prevalidation Study Test Materials and Draize Data

+

+

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–

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FHSA Classc

THE HUMAN CORNEAL HCE-T TEP ASSAY 177

178


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Draize MAS

THE HUMAN CORNEAL HCE-T TEP ASSAY

70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 –0.8

179

A

–0.4

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

Draize MAS

LogFR85 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 –0.8

B

–0.4

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

LogFR85 Figure 15.8 Overlay of the prevalidation study test results for five test materials in the MAS Prediction Model (PM). The solid line represents the nonlinear regression curve of the MAS PM, and the dashed lines are the 95% confidence intervals. (A) Overlay of the 60 log FR85 values in the MAS PM. There are 12 values for each of the five test materials on the plot, but, due to overlap in the data, all 12 points may not be distinct. (B) Fit of the average log FR85 value for each of the five test materials in the MAS PM. The result for each test material is the average of 12 assays.

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180


Table 15.3 The Nonirritant/Irritant Classification of the Five Test Materials as Determined by the Draize Test and by the HCE-T TEP Assay Test Material

Draize Score Classificationa

TEP Assay MAS PMb

TEP Assay CS PMb

TEP Assay COS PMb

Bubble bath Hair conditioner Cleansing gel Shampoo 1 Shampoo 2

1 1 2 2 2

1 1 2 2 2

1 1 2 2 2

1 1 1 2 2

a

Draize classification was the same across the MAS, CS, and COS scores, except for the cleansing gel which was an irritant by the MAS and CS, but a nonirritant by the COS. b Draize scores for the classifications: nonirritant MAS e 15; nonirritant CS < 5; nonirritant COS < 0.667. Note: Draize maximum average score, MAS; corneal score, CS; corneal opacity score, COS. Table 15.4 Average HCE-T TEP Assay Results for Five Test Materials in Three Laboratories; The Predicted MAS and Class from the TEP assay Are Compared to the Draize MAS and Class for Each Test Result Test Material Bubble bath Hair conditionerc Cleansing gel Shampoo 1 Shampoo 2 a b

c

Draize MASa

Draize Classb

Predicted MASa

Predicted Classb

4.8 14.2 22.0 37.8 57.4

1 1 2 3 4

4.20 1.82 16.15 34.62 41.18

1 1 2 3 3

MAS, maximum average score. The four classification cutoffs for MAS are based on the scheme proposed by Kay and Calandra (1962): MAS 0–15, minimal (class 1); MAS 15.1–25, mild (class 2); MAS 25.1–55, moderate (class 3); MAS > 55, severe (class 4). Hair conditioner was less water soluble than other test materials, which may account for its underprediction when it was diluted.

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181

Table 15.5 Variability in the Draize MAS Compared to the Predicted MAS for the HCE-T TEP Assay Prevalidation Study Data Test Material

Intralab CV (%) for Draize MASa

Bubble bath

22.82

Hair conditionerd

19.67

Cleansing gel

53.24

Shampoo 1

10.31

Shampoo 2

8.32

Intralab CV (%) for Predicted MASb 9.94 10.06 4.94 33.19 15.80 54.09 17.50 3.01 25.90 12.54 21.21 20.42 14.60 14.62 29.49

Interlab CV (%) for Predicted MASc 6.03

15.59

15.10

12.49

2.83

a

Evaluated using five to six animals from one lab. Evaluated using four replicates per lab for three labs. c Results from three testing labs. d Hair conditioner was less water soluble, which may account for its greater variability when it was diluted. Notes: Intralab is the within lab variability (repeatability); interlab is the between lab variability (reproducibility). Coefficient of variation, CV; maximum average score, MAS. b

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RESULTS OF THE STUDIES 7KHVWXG\UHSRUWVZHUHUHYLHZHGE\WKHÀ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

34.1 32.3 34.9 31.1 22.1 15.1

212.0 204.9 216.9 218.5 190.4

2571.9 2175.7 2322.7 2260.1 2261.4 1788.9

0.5 1 2 4 10 24

0.5 1 2 4 10

0.5 1 2 4 10 24

285.9 405.4 355.2 502.7 435.5 627.2

24.2 21.7 23.7 21.0 28.2

3.0 3.2 4.1 4.9 5.6 8.1

0.2 0.2 0.2 0.2 0.3 0.4

Cover

— — — — — —

— — — — —

— — — — — —

0.1 0.1 0.2 0.3 0.4 0.7

Carbon Filter

74.2 95.2 98.9 93.3 88.3 86.0

9.2 8.7 13.3 8.7 10.8

1.4 1.3 1.2 1.2 1.2 0.9

0.1 0.2 0.2 0.2 0.3 0.4

0.3 0.3 0.1 0.1 0.2 47.9

0.01 0.01 0.01 0.01 0.1

270 Qg/cm2 3.40 0.01 3.21 0.02 4.91 0.02 3.21 0.7 3.99 3.5 2934 Qg/cm2 2.53 0.1 3.24 0.2 3.37 0.5 2.18 2.3 3.01 15.2 2.92 97.1

0.001 0.001 0.005 0.003 0.3 2.1

0.001 0.001 0.001 0.001 0.01 0.2

Feces

42 Qg/cm2 3.18 0.004 2.99 0.02 2.78 0.02 2.81 0.2 2.66 2.1 1.83 5.5

0.01 0.01 0.01 0.02 0.2 0.5

Urine

3 Qg/cm2 4.00 6.00 7.17 7.93 9.63 12.53

Skin Qg/cm2 %

Absorbed is sum of urine, feces, cage wash, and carcass. Source: Lythgoe, 1990a.

a

0.22 2.2 2.0 1.8 1.2 0.9

Wash

0.5 1 2 4 10 24

Exposure (hr)

0.4 0.2 0.6 1.1 1.6 8.4

0.04 0.05 0.05 0.06 0.56

0.01 0.01 0.3 0.01 0.4 0.6

0.001 0.001 0.001 0.01 0.01 0.02

Cage Wash

40.3 150.9 98.5 74.8 114.3 216.7

12.9 15.1 8.1 12.3 21.1

1.7 2.5 1.8 3.6 7.0 4.4

0.1 0.1 0.2 0.3 0.4 0.2

Carcass

40.9 153.04 99.6 78.3 131.3 369.9

12.9 15.2 8.2 13.0 25.2

1.7 2.5 1.9 3.9 9.8 12.6

0.1 0.1 0.2 0.3 0.6 0.9

1.39 5.22 3.39 2.67 4.47 12.61

4.79 5.63 3.03 4.83 17.52

3.95 5.98 4.38 9.24 23.06 29.64

3.79 4.28 7.32 9.93 19.55 31.37

Absorbed Qg/cm2 %

Table 18.1 Acetochlor In Vivo (Mean dose distribution as Qg equivalents of acetochlor per cm2. Mean of four male rats.)

214 ALTERNATIVE TOXICOLOGICAL METHODS


215

Table 18.2 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in rat skin. Mean of four to seven skin samples. Results presented as Qg/cm2.) Exposure (hr)

Absorbed Qg/cm2 %

Wash

Skin Qg/cm2 %

Donor

Loop

3.60 3.77 3.84 2.98 2.52 2.75

0.08 0.04 0.03 0.05 0.01 0.02

0.55 0.44 0.50 0.43 0.59 0.41

2.30 2.47 3.09 2.28 2.11 1.73

1.41 0.97 0.57 0.58 0.78 0.14

1.03 1.02 1.25 1.38 0.98 1.21

4.06 5.22 8.84 5.09 7.11 4.53

10.5 14.3 19.4 4.41 5.59 5.52

13.4 16.7 8.25 11.6 11.6 13.3

Total Recovered

3.02 Qg/cm2 0.05 1 2 4 10 24

0.41 0.82 1.14 1.37 2.15 2.12

13.68 27.12 37.78 45.30 71.19 70.13

1.73 1.32 0.95 0.80 0.17 0.13

0.11 0.11 0.12 0.09 0.08 0.08

2.89 2.73 2.73 2.75 2.99 2.77

47.3 Qg/cm2 0.5 1 2 4 10 24

2.02 3.90 10.3 16.0 20.7 28.7

4.27 8.25 21.78 33.83 43.76 60.68

25.6 28.2 18.7 13.6 7.1 2.9

1.09 1.17 1.46 1.08 1.00 0.79

31.1 35.3 32.2 32.7 30.6 33.7

318 Qg/cm2 0.5 1 2 4 10 24

6.58 21.4 33.4 80.9 182 246

2.07 6.73 10.50 25.44 57.23 77.36

283 383 261 262 158 67

12.9 16.6 28.1 16.2 22.6 14.4

325 452 350 375 379 347

3095 Qg/cm2 1 2 4 10 24

17 104 137 264 757

0.55 3.36 4.43 8.53 24.46

2411 3146 2103 2344 1598

143 187 111 196 202

4.62 6.04 3.59 6.33 6.53

311 313 316 294 182

118 138 161 156 127

2999 3858 2828 3253 2866

Note: The 0.5-hr exposure was not performed at the high dose. Source: Clowes and Scott, 1990a.

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216


Table 18.3 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in human skin. Mean of six skin samples. Results expressed as Qg/cm2.) Exposure (hr)

Absorbed Qg/cm2 %

Wash

Skin Qg/cm2 %

Donor

Loop

Total Recovered

3.02 Qg/cm2 2 10 24

0.015 0.074 0.261

0.486 2.44 8.63

2.36 2.61 1.54

0.273 0.271 0.092

9.04 9.98 3.03

0.035 0.136 0.098

0.359 0.194 0.244

3.12 3.86 2.80

2.61 2.31 3.09

3.04 3.28 2.24

47.3 Qg/cm2 2 10 24

0.140 1.70 3.63

0.296 3.59 7.68

34.2 30.4 26.2

1.89 4.43 1.63

3.99 9.37 3.45

41.9 42.7 37.4

318 Qg/cm2 2 10 24

0.816 5.69 43.6

0.256 1.79 13.7

239 221 245

18.9 12.1 18.2

5.94 3.82 5.72

70.4 91.8 62.3

22.4 15.8 15.8

352 347 384

3095 Qg/cm2 2 10 24

4.57 22.4 32.5

0.148 0.722 1.05

2796 2057 1669

51.5 80.5 38.4

1.67 2.60 1.24

772 896 671

260 259 217

3884 3314 2628

Source: Clowes and Scott, 1990b.

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217

Table 18.4 Comparison of the Percent Absorbed In Vitro and In Vivo in Rat Skin; Data Are from Tables 18.1 and 18.2 Dose Qg/cm2) (Q

0.5

1.0

2.0

4.0

10.0

24.0

Exposure Duration (hr) 3.0 in vivo 3.02 in vitro 42.5 in vivo 47.3 in vitro 270 in vivo 318 in vitro 2934 in vivo 3095 in vitro

3.8 13.7 4.0 4.3 4.8 2.1 1.4 —

4.3 27.1 6.0 8.2 5.6 6.7 5.2 0.6

7.3 37.8 4.4 21.8 3.0 10.5 3.4 3.4

9.9 45.3 9.2 33.8 4.8 25.4 2.7 4.4

19.5 71.2 23.1 43.8 9.3 57.2 4.5 8.5

31.4 70.1 29.6 60.7 17.5 77.4 12.6 24.5

4.6 3.7 5.3 1.6

3.7 1.9 6.2 1.9

2.2 2.1 4.4 1.9

Ratio in Vitro/in Vivo 3.02/3.0 47.3/42.4 318/270 3095/2934

3.6 1.1 0.4 —

6.3 1.4 1.2 0.1

Dose 1 Dose 2

5.2 5.0 3.5 1.0

Dose 3 Dose 4

7 6

Ratio vitro/vivo

5 4 3 2 1 0 0

5

10

15

20

25

Exposure (h)

Figure 18.1 The ratio of the in vitro absorption to the in vivo absorption with exposure duration in the rat. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high dose ratio. Data are from Table 18.4.

DV\VWHPDWLFHUURULQWKHLQYLWURSURFHGXUH7KLVLQGLFDWHVWKDWWKHSURFHGXUHPD\ QRWEHFRUUHFWDEOH7KHGDWDVWURQJO\VKRZWKDWWKHSURFHGXUHLVQRWUHDG\WRHQWHU WKHWHVWYDOLGDWLRQVWHS :HKDYHQRKXPDQLQYLYRGDWDWRFRPSDUHZLWKWKHKXPDQ LQYLWURGDWDDQGVR FDQQRW GLUHFWO\ DVVHVV LWV YDOLGLW\ +RZHYHU WKH SRLQW KDV EHHQ UDLVHG WKDW HYHQ LI LQ YLWURGDWDGRQRWPDWFKLQYLYRRQHPD\FRPSDUHUDWDQGKXPDQLQYLWURGDWDWRREWDLQ

218


Table 18.5 Comparison of the Percent Absorbed In Vitro in Rat and Human Epidermal Membrane Preparations; Data Are from Tables 18.2 and 18.3 Dose Qg/cm2) (Q

2.0

10.0

24.0

Exposure Duration (hr) 3.03 rat human 47.3 rat human 318 rat human 3095 rat human

37.8 0.49 21.8 0.30 10.5 0.26 3.4 0.15

3.03 47.3 318 3095

77.1 72.7 40.4 22.7

71.2 2.44 43.8 3.59 57.2 1.79 8.5 0.72

70.1 8.63 60.7 7.68 77.4 13.7 24.5 1.05

Ratio Rat/Human 29.2 12.2 32.0 11.8

8.1 7.9 5.6 23.3

D UDW WR KXPDQ DEVRUSWLRQ FRQYHUVLRQ IDFWRU ,W LV JHQHUDOO\ DFFHSWHG WKDW UDW VNLQ LV PRUH SHUPHDEOH WR FKHPLFDOV WKDQ KXPDQ LQ WKH RUGHU RI WKUHH WR ÀYH WLPHV PRUH SHUPHDEOH$FRQYHUVLRQIDFWRUZRXOGHQDEOHRQHWRFRQYHUWUDWGHUPDOWR[LFLW\GDWDWR KXPDQGHUPDOWR[LFLW\7KHSHUFHQWRIGRVHDEVRUEHGLQYLWURLQWKHUDWDQGLQWKHKXPDQ HSLGHUPDOPHPEUDQHSUHSDUDWLRQDQGWKHLUUDWLRDUHSUHVHQWHGLQ7DEOH7KHUDWLRV RILQYLWURUDWWRLQYLWURKXPDQIRUHDFKGRVHDUHSORWWHGLQ)LJXUHDVUDWLRDJDLQVW vitro R/H 47.3 wg/cm2 vitro R/H 318 wg/cm2 vitro R/H 3095 wg/cm2

80 70

Ratio rat/human

60 50 40 30 20 10 0 0

5

10

15

20

25

Duration of Exposure (h)

Figure 18.2 The ratio of the rat in vitro absorption to the human in vitro absorption with exposure duration. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high dose ratio. Data are from Table 18.5.


219

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19

A Molecular Diagnostic Approach to Irritant or Allergic Patch Testing Using the DermPatch 0HOLVVD0)LW]JHUDOG9HUD%0RUKHQQ6WDFH\%+XPSKUH\ 1LUPDOD-D\DNXPDUDQG/DZUHQFH$5KHLQV

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Table 19.1 Skin Cytokins (adapted from Gerberick et al., 1998) Cytokines IL-1E IL-1F IL-3 IL-6 IL-7 IL-8 IL-10 IL-12 IL-15 G-CSF M-CSF GM-CSF TGF-E TGF-F TNF-E MIP-1E MIP-1F IP-10

Constitutive or inducible expression in Langerhans cells Keratinocytes Fibroblasts – + – + – – – –

+ – + + + + +

– – – – + – + + –

+ + + + + + – + +

+ + + – + – + + + – + – + – – – +

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IRRITANT OR ALLERGIC PATCH TESTING USING THE DERMPATCH

223

Table 19.2 mRNA Cytokine Profiles from Human Skin Biopsy or Human Cell Samples

TNF-E IFN-K IL-2 GM-CSF IL-1E (human cells) IL-1F (human cells) IL-4 IL-6 IL-10 IL-12 p35 (human cells) IL-12 p40 (human cells)

ACD

ICD

increased increased increased increased dependent on allergen increased increased increased increased no change increased

increased increased increased increased increased increased not determined not determined no change no change no change

Source: Adapted from Wakem and Gaspari, 2000.

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Allergic

Irritant

low molecular weight, lipid soluble less critical ++++ necessary interaction of antigen with primed T cells ++++ early ++++ ++ increased ++

acids, alkalies, surfactants, solvents, oxidants, enzymes more critical ++ not necessary damage to keratinocytes

Source: Adapted from Marks and DeLeo.

+++ later ++++ ++ decreased ++

224


Table 19.4 Clinical and Histological Aspects of Contact Dermatitis Feature

Allergic

Irritant

Itch Pain, burning Erythema Vesicles Pustules Hyperkeratosis Fissuring Sharp demarcation Reaction delay after contact Spongiosis Dermal edema Necrotic keratinocytes Ballooning degeneration Lymphocytic infiltrate Neurotrophilic infiltrate

++++ (early) ++ ++++ ++++ + ++ ++ yes days ++++ ++++ ++++ + ++++ +

+++ (late) ++++ (early) ++++ + +++ +++ ++++ yes minutes to hours ++++ ++++ ++++ +++ ++++ +++

Source: Adapted from Marks and DeLeo.

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Table 20.3 EpiDerm-200 Triton X-100 ET50 Database Summary Year 2000 1999 1998 1997 1996 1995 a

a

EPI-200 Triton ET-50 6.76 6.75 7.24 6.78 6.74 6.65

EPI-200 Triton C.V.

Lots

Avg. C.V.

16.4 18.2 17.9 15.9 14.6 77.8

89 146 175 228 184 112

6.2 5.7 9.2 9.9 9.6 4.9

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241

Figure 20.10 Changes in cancer-related gene expression following UVB-irradiation of EpiDerm tissue. AtlasTM human cancer cDNA expression array analysis of gene expression changes induced in EpiDerm tissues 6 hr after irradiation with 175 mJ/cm2 UVB.

Table 20.4 UVB-Irradiation of EpiDerm Tissue WAF-1 (+) MAP Kinase p38 (+) Growth-arrest-specific protein (+) c-Myc binding protein MM-1 (+) TRAF-interacting protein (+) Caspases (+) Death-associated protein kinase (DAP kinase 1, +) p53-induced protein (+) GADD45 (+) DNA excision repair protein ERCC1 (+) DNA-repair protein XRCC1 (+) Placenta growth factors 1+2 (+) TIMP-1 (+) T-plasminogen activator (+) Rho GDP dissociation inhibitor 1 (+) Endothelin 2 (–) IL-6 (–) Leukocyte interferon-inducible peptide (–) 60S ribosomal protein (–)

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242


– +

– +

– +

– +

Markers

273 bp

20

25

30

35

Cycles Figure 20.11 Induction of PlGF expression following UVB-irradiation of EpiDerm tissue. Agarose gel electrophoresis of products obtained by RT-PCR of total RNA isolated from EpiDerm tissue 20 hours following UVB-irradiation. (–) no irradiation. (+) UVB-irradiated. The expected PlGF PCR product is 273 bp.

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Figure 20.12 EpiDerm tissues cultured in 24 well (EpiDerm-224) or 96 well (EpiDerm-296) high throughput ALI formats. Each tissue has its own individual media reservoir to avoid cross-contamination of samples.


243

Figure 20.13 Total RNA-96 isolation kit designed for high throughput total RNA isolation from EpiDerm-296.

Figure 20.14 Agarose gel electrophoresis of total RNA isolated from EpiDerm-296 with the Total RNA-96 isolation kit. Average yield is approximately 10 Qg DNA-free total RNA/EpiDerm-296 tissue.

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Procedure

Date

Sample

Purity Level of DNA (260/280 Ratio)

Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells Purification of lymphocyte DNA using Boehringer Mannheim Kit for mammalian blood Phenol/chloroform extraction of HEK DNA

8/4/98 8/18/98 8/24/98 9/2/98 9/8/98 9/29/98 10/1/98 10/7/98 10/13/98 10/19/98 10/27/98 11/5/98

Control Control Control Control Control Control Control Control Control Control Control Control

1.35 1.65 1.35 1.82 1.81 1.93 1.22 1.35 1.47 1.90 1.86 1.86

Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells (modified procedure)

DNA Yield (mg/ml) 60 45 25 189 185 194 60 33 67 94 93 98

264


Table 22.2 Purification of HEK DNA using Boehringer Mannheim Kit for Mammalian Cells Exposed to Sulfur Mustard (HD) Date 11/19/98

11/26/98

12/8/98

Sample Control 50 mM HD 100 mM HD 300 mM HD Control 50 mM HD 100 mM HD 300 mM HD Control 50 mM HD 100 mM HD 300 mM HD

Purity Level of DNA (260/280 Ratio)

DNA Yield

1.81 2.10 1.89 1.82 1.85 1.99 1.86 1.81 1.98 1.96 1.91 1.86

122 163 150 199 194 213 198 172 183 179 176 199

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265

HeLa PARP enzyme activity

120

% of maximum response

100

80

60

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HEK PARP enzyme activity

140

% of maximum response

120 100 80 60 40 20 0 1

2

4

6

24

Time post-HD (h) x-axis vs. 100 µM HD x-axis vs. 10 µM HD

Figure 22.1 Time-dependent response of PARP activity following HD exposure of (top) HeLa cells and (bottom) HEK cells. Two concentrations of HD were used, 10 and 100 QM, and PARP activities are presented as percent maximal response.

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2.6

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2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 0

200

400

600

800

1000

1200

1400

Time (s) Figure 22.2 Increase in intracellular calcium in HEK (passage 2) exposed to 300 QM HD as measured by 340:380 nm ratio of Fura-2 AM. Table 22.3 Binding in HD-Exposed HEKa QM) Dose HD (Q 100 200

Hrs post HD C1q

0

8 16 24

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– ++ +++

CD32

0

50

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a

300

+ = weak; ++ = moderate; +++ = intense; NT = not tested. Grading of staining was judged visually by fluorescence microscope and HEK controls not exposed to HD were negative for fluorescence.

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4000 3500

pg/ml IL-8

3000 2500 2000 1500 1000 500 0 -50

0

50

100

150

200

250

300

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HD Concentration HD-exposed HEK

Figure 22.3 Concentration-dependent increase in the secretion of IL-8 from HEK exposed to HD.

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CHAPTER

23

Human Keratinocyte Inflammatory Transcript Gene Activity Following Sulfur Mustard* -RKQ-6FKODJHU.DVKLI$OL+LOPD5%HQMDPLQ&ODLUH)/HYLQH 'RXJODV3$YHU\DQG-DPHV+&ODUN

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HUMAN KERATINOCYTE SM INFLAMMATORY TRANSCRIPT GENE ACTIVITY

271

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272


Table 23.1 HEK Housekeeping Gene Transcripts at 16 Hr Following Sulfur Mustard Gene Ratioa Gene Transcript 14-3-3 zeta protein 23 kDa Highly Basic Protein E-Tubulin F-Actin Glyceraldehyde 3-phosphate dehydrogenase HLA class I histocompatibility antigen C-4 alpha chain Hypoxanthine-guanine phosphoribosyltransferase Ribosomal Protein S9 Ubiquitin a

b

Control Intensityb

25 QM SM

200 QM SM

317 1689 307 236 612 487

0.7 2.0 0.57 1.2 0.44 0.94

0.44 2.3 1.4 1.2 1.1 1.0

59

0.9

3.7

978 2064

1.2 0.8

2.1 0.73

Induction ratio from phosphorimage densitometeric measurements of listed gene transcript normalized to the sum total of the detected transcript intensity/pixel density. Control intensity (grayscale pixel density) adjusted from background. Background was set at the median intensity of the blank space between the six array panels. The subtracted background intensity ranged from 19 to 43.

Table 23.2 HEK Inflammation-Associated Transcripts at 16 Hr Following Sulfur Mustard Gene Ratioa Gene Transcript CD40 Interleukin 1 E Interleukin 1 F Interleukin 2 receptor E subunit Interleukin 6 Interleukin 7 receptor E subunit Interleukin 8 Interleukin 13 Interleukin 15 Macrophage inflammatory protein 2E S19 ribosomal protein Tumor necrosis factor E a

b

c

Control Intensityb

25 QM SM

200 QM SM

86 67 101 148 494 131 16 349 215 11 886 50

4.9 0.1 0.6 0.3 0.7 0.4 —c 3.0 — — 0.9 —

0.3 1.4 3.5 0.4 0.6 0.7 20 0.7 0.03 13 2.1 1.4

Induction ratio from phosphorimage densitometeric measurements of listed gene transcript normalized to the sum total of the detected transcript density. Control intensity (grayscale pixel density) adjusted from background. Background was set at the median intensity of the blank space between the six array panels. The subtracted background intensity ranged from 19 to 43. Transcript not detected above background.


273

1 2

Figure 23.1 Atlas cDNA array nylon blot image. HEK were exposed to 200 QM sulfur mustard for 16 h and mRNA isolated and 32P labeled as cDNA. The array contains doubledot blots of cDNA from 588 transcriptionally regulated genes and 9 housekeeping genes at the bottom of the array (see 3 housekeeping genes in rectangular box). Boxes 1 and 2 show expression of macrophage inflammatory protein 2E and interleukin 8, respectively. These inflammatory transcripts were at very low expression levels in control HEK.

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Figure 27.1 Basal cell adhesion complex. A microvesicle (lower left panel) showing details of the dermal–epidermal separations characteristic of a sulfur mustard blister. Hemidesmosomes (arrowhead), at the roof of the blister, are well displaced from the basement membrane (bm) and lamina densa (ld). An expanded model of the intact adhesion complex (circumscribed area) includes the intracellular keratin filaments K5 and K14 and their facilitated attachments to the transmembrane E6F4 integrin receptors. The exodomains of E6F4 are shown linked by laminin 5 to the basement membrane zone.

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Figure 27.2 Keratin 5 images from control cultures of HEK recorded by multiphoton imaging (A), showed the elaborate cytoskeletal matrix and distribution of these filaments within the cell cytoplasm. Image intensity and K5 concentration were greatest around the nucleus of each cell, and a lacy network of delicate filaments projected out toward the cell extremities. Analysis of confocal images from K5 controls (B) and HD-exposed populations (C) showed a statistically significant (p < 0.01) 29.2% decrease in intensity at 1 hr postexposure to sulfur mustard.

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Figure 27.3 Multiphoton keratin 14 images from control HEK cultures (A) showed elaborate cytoskeletal distribution comparable to that of keratin 5. Image intensity and K14 concentration were greatest around the nucleus. Lacy networks of filaments projected out to the cell extremities where they interfaced closely with those of adjacent cells (arrow). At 1 hr postexposure to sulfur mustard, K14 images (B) indicated a disruption of organization, resulting in withdrawal of filaments from the plasma membrane margins, appearance of punctate nodules (arrows), and a substantial loss of cytoskeletal definition.

Figure 27.4 Analysis of K14 confocal images from replicate cultures of HEK sham-treated controls (A and C) and HD-exposed populations (B and D) showed a statistically significant (p < 0.01) 30.14% decrease in image intensity (K14 expression) at 1 hr postexposure and a nearly complete loss of expression (79% decrease in image intensity) at 2 hr.

308


Figure 27.5 A multiphoton montage showing the organization of E6 integrins on an explant of human epidermis. Serial slices (A and B) illustrate the receptor outline on successive cross sections through the epidermal rete pegs. The three-dimensional reconstruction (C) and the stereo image (D) are from the same Z-series of serial slices. Together, they show the topographic complexity of ventral epidermis plus the circular shape and extensive distribution of E6F4-integrin receptors.

Figure 27.6 A multiphoton image (slice) of human epidermis showing in cross section the distribution and circular shape of F4 integrins (arrows) on the basal cell surface.


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Figure 27.7 Multiphoton images of E6F4 receptors in human epidermis exposed to sulfur mustard indicated unraveling and loss of circular shape at 1 hr postexposure (A) and an almost total loss of E6F4 expression at the basal cell surface by 2 hr postexposure (B, C). At 2 hr postexposure, only a basolateral pattern of residual fluorescence remained to outline the constituent basal cells. Loss of E6 and F4 integrin also occurred spontaneously in epidermal tissues following dermal–epidermal separation; therefore, the effects may not be strictly related to sulfur mustard exposure.

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Figure 27.8 Analysis of confocal images from HEK in replicate control and HD-exposed cultures indicated a statistically significant (p < 0.01) decrease of 27.3% and 26.3% in image intensity of E6 and F4 integrins, respectively, at 1 hr postexposure. The decrease was characterized by a loss of fluorescence from the surface of attached basal cells, resulting in a honeycomb pattern of residual, basolateral fluorescence. Postexposure image patterns from cultures were very similar to those recorded from intact epidermal tissues (see Figures 27.7B,C).

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cDNA selection PCR amplification Microarray construction Biological model

Sample preparation Treated vs. untreated Protein sample

mRNA sample isolation

Iso electrofocusing (1st D)

cDNA labeling

SDS-PAGE (2nd D)

Hybridization to microarray Bioinformatic analysis

Protein expression and posttranslational modification Mass spectrometry Protein database search mRNA expressed

Protein identity

Research lead

Toxicological marker

Toxicant signature

Figure 34.1 Schematic of genomic and proteomic technologies: The first step in toxicogenomics (right) is the construction of a microarray that involves the amplification by PCR and the immobilization of known DNA sequences (either cDNA or oligonucleotides) on a solid support. The mRNA prepared from a biological model can be labeled and hybridized to the microarray and visualized using phosphorimager scanning. Subsequent bioinformatic analyses using appropriate software allows determination of the extent of hybridization of the labeled probes to the corresponding arrayed cDNA spots, and a comparison of control with test samples permits quantitative assessment of changes in gene expression associated with treatment. Total protein content from a biological model treated with a toxicant is separated on two-dimensional gel electrophoresis according to isoelectric point (first dimension) and molecular weight (second dimension), allowing bioinformatic analysis of differences in protein expression of treated versus untreated samples. Therefore, there is no preliminary work such as array construction for proteomic studies (left), but proteins with altered expression have to be identified subsequently. Individual proteins of interest are excised from two-dimensional-gels, digested with trypsin, and applied to a mass spectrometer. Identification of these proteins is obtained by searching protein databases with mass spectrometry data.


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Table 35.1 IARC Class I or 2A Human Carcinogen, or NTP Reasonably Anticipated Human Carcinogen Tg.AC Topical

TgrasH2

p53+/–

XPA–/–/p53+/–

+ + – + – – nd

+ nd + nd +/– + +

+ + + + + – nd

nd* + nd +** nd – nd

+ + +# + +

+/– + – nd nd

+ + +/– –

+ + + nd nd

Genotoxic Benzene Benzo(a)pyrene Cyclophosphamide 7,12-Dimethylbenzanthracene Melphalan Phenacetin Procarbazine Nongenotoxic Cyclosporin A Diethylstilbestrol 17-F-estradiol (or ethinyl estradiol#) Oxymetholone 2,3,7,8-TCDD

* nd: no adequate data available on the performance of the compound in that model. **Positive in 6 month XPA–/– and positive in 6 month p53+/–; not tested in XPA–/–/P53+/– bitransgenic. Table 35.2 Genotoxic Trans-Species Rodent Carcinogens

p-Cresidine 2,4-Diaminotoluene Diethylnitrosamine Dimethylnitrosamine N-Ethylnitrosourea Glycidol N-Methylnitrosourea Phenolphthalein Thiotepa Urethane 4-Vinyl-1-cyclohexene-diepoxide*

Tg.AC Topical

TgrasH2

P53+/–

XPA–/–/P53+/–

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+ nd + + + + + – + + +

+ – nd + + – + + nd + +

+ nd nd nd nd nd nd nd nd nd nd

* Applied dermally to each model tested.

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Table 35.3 Nongenotoxic Rodent Carcinogens and Human Carcinogenicity Unlikely or Uncertain Tg.AC Topical

TgrasH2

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XPA–/–/P53+/–

nd + nd – nd nd nd + – – – –

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Chlorpromazine Clofibrate Dieldrin Diethylhexylphthalate Haloperidol D-Limonene Metaproterenol Pentachlorophenol Phenobarbital Reserpine Sulfamethoxazole WY-14643

* Positive in 6 month XPA–/–, not tested in XPA–/–/p53+/– bitransgenic. Table 35.4 Rodent Noncarcinogens

Genotoxic p-Anisidine 2-Chloroethanol 1-Chloro-2-propanol 2,6-Diaminotoluene 8-Hydroxy-quinoline Nongenotoxic Ampicillin Benzethonium chloride D-Mannitol Oleic acid diethanolamine Phenol Resorcinol Rotenone Sulfisoxazole

Tg.AC Topical

TgrasH2

P53+/–

XPA–/–/P53+/–

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– nd – – –

nd nd nd nd nd

nd – nd – – + – –

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Alternative toxicological methods

Alternative Toxicological Methods

Alternative methods of regression

Alternative methods of regression

Alternative Methods of Regression

Toxicological profiles - Chlorfenvinphos

Toxicological profiles - Chloromethane

Toxicological profiles - Manganese

Toxicological profiles - Hexachlorobutadiene

Toxicological profiles - Mercury

Toxicological profiles - Formaldehyde

Toxicological profiles - Nitrophenols

Toxicological profiles - Methylenedianiline

Toxicological profiles - Radon

Toxicological profiles - Methyl parathion

Toxicological profiles - Methyl mercaptan

Toxicological profiles - Endosulfan

Toxicological profiles - Hydrogen sulfide

Toxicological profiles - Vinyl Acetate

Toxicological profiles - Nitrobenzene

Toxicological profiles - Thorium

Toxicological profiles - Ionizing radiation

Toxicological profiles - Iodine

Toxicological profiles - Chloroform

Toxicological profiles - Silver

Toxicological profiles - Tin

Toxicological profiles - Pyridine

Toxicological profiles - Copper

Toxicological profiles - Fuel oils

Toxicological Chemistry and Biochemistry

Toxicological profiles - Polychlorinated Biphenyls

Alternative toxicological methods