Induction of nuclear anomalies in the gastrointestinal tract by polycyclic aromatic hydrocarbons

Cancer Letters, 56 (1991) 215-224

215

Elsevier Scientific Publishers Ireland Ltd.

Induction of nuclear anomalies in the gastrointestinal polycyclic aromatic hydrocarbons

tract by

T.V. Reddyb, J.A. Stoberb, G.R. Olsonc and F.B. Daniela “Biochemical and Molecular Toxicology Branch, Genetic Toxicology Division, bHealth Effects Research Laboratory, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive, Cincinnati, OH 43628 and %thology Associates Inc., 6217 Centre Park Drive, West Chester, OH 45069 (U.S.A.)

(Received 3 October 1990) (Revision received 31 December (Accepted 31 December 1990)

1990)

Summary A selective list of polycyclic aromatic hydrocarbons (PAl-l) with varied carcinogenic and mutagenic potencies, which are identified as common contaminants at industrial sites and which often contaminate the neighboring ground water, are investigated for their ability to induce nuclear anomalies (NA) in the mouse gastrointestinal (G.I.) tract. These studies examined the hypothesis that a relationship between NA induction and carcinogenic potency of these PAH exists. Among the PAH tested, 7,12dimethylbenzanthrene (DMBA) was most effectiue inducer of NA in all G.I. tract tissues examined, with the relative potency in Correspondence Molecular Health

to:

F.B.

Toxicology

Effects Research

tection Agency, OH 43628, Disclaimer:

Daniel,

Branch,

Ph.D.,

Genetic

Laboratory,

Biochemical

Toxicology

U.S.

and

Division,

Environmental

Pro-

26 West Martin Luther King Drive, Cincinnati,

duodenum of DMBA > > > benzo[a]pyrene (B[a]P) > > benzo[b]fluoranthene (B[b]F). The induction of NA by benzo[a]anthracene (B[a]A), pyrene (PY) and benzo[e]pyrene (B[e]P) was not different from that elicited by vehicle controls. MNU, a known potent inducer of NA in the mouse G.I. tract, yielded a high leuel of NA in duodenum and proximal colon but was less effective than DMBA in the forestomach. The data suggest that induction of NA by DMBA and B[a]P PAH are in approximate accordance with their relatiue carcinogenic potency in the gastrointestinal tract. When binary mixtures of some PAH were administered the yield of NA was less than that expected by simple additiuity and closer to that expected by averaging the actiuities of the two PAH comprising the mixture. Thus, this shortterm in vivo assay may be useful as a predictor of the genotoxic or carcinogenic strength of individual PAH and/or mixtures of these compounds.

U.S.A. This document

with U.S. Environmental ed for publication.

has been reviewed

Mention of trade names or commercial

ducts does not constitute endorsement use.

0304-3835/91/$03.50

in accordance

Protection Agency policy and approv-

0

1991

Published and Printed in Ireland

or recommendation

profor

Keywords: polycyclic aromatic hydrocarbons; gastrointestinal tract nuclear anomalies; polycyclic aromatic hydrocarbon mixtures

Elsevier Scientific Publishers Ireland Ltd

216

introduction PAH are widespread environmental contaminants [3-6,8,10,16,20] and these compounds have a wide range of carcinogenic and/or mutagenic potential. PAH exhibit genotoxic activity after being metabolically oxidized into reactive, electrophilic species [l,Z,ll-14,17,20,25,27,28,30-39,41441 and considerable efforts have been focused on the development of reliable biological markers to monitor the occupational exposure to PAH mixtures at industrial sites and in urban areas. In addition, mixtures of PAH have also been identified as contaminants at hazardous waste sites particularly in the neighboring ground water [3,16]. Many PAH were shown to be carcinogenic in various mouse strains typically producing forestomach tumors when administered orally [20], whereas topical application often results in skin cancer [11,36,37]. Recent work by Heddle and his colleagues [19] have demonstrated the feasibility of using the G.I. NA assay as an in vivo, tissue-specific indicator of the carcinogenic potential for a wide variety of chemicals including some PAH. Other investigators have reported similar induction of NA in forestomach, duodenum and proximal colon of both C57BL/6J and B6C3Fl mouse strains as well as in the proximal colon of Fischer 344 rats by several classes of tissue specific carcinogens [7,9,26,29,40]. In the present study we investigated the potential of some PAH and of binary mixtures of PAH on the formation of NA in forestomach, duodenum and proximal colon in B6C3Fl mice. The results of these studies are the subject of this manuscript. Materials

and Methods

Eight-week-old male B6C3Fl mice (from a cross between male C3Heb/FeJ and female C57B1/6Jc) were obtained from Charles River Laboratories (Portage, MI). After a 2-week quarantine, the mice were brought to the designated rooms where temperature

(20-22OC), humidity (40-60%) and a regular 12-h light cycle was maintained. These mice were kept in plastic cages (5 mice/cage) with hard wood chip bedding and were given free access to food and water. When the mice reached approximately 2 months of age they were randomly divided into treatment groups for experimentation. All aspects of these studies were conducted within guidelines recommended by the American Association for Accreditation of Laboratory Animal Care. Test chemicals B[a]P, B[b]F, B[a]A, DMBA, B[e]P and PY, were purchased (as highest purity available, typically 99%) from Aldrich Chemical Company, Milwaukee, WI. Nmethyl-N-nitrosourea (MNU), dimethylsulfoxide (DMSO) were obtained from Sigma Chemical Company, St. Louis, MO. Dosing

solutions

All test chemicals and corresponding vehicle solutions were administered by oral gavage. DMSO was used as a vehicle for all PAH while MNU was given in acetate-buffered (pH 5) saline. Mice were treated with test chemicals between 0800 h and 1100 h. The concentrations of the dosing solutions were prepared by weighing the individual chemical on an analytical balance and the concentration was confirmed using the electronic spectra of diluted solutions in conjunction with their appropriate molar absorbtivities. Experimental protocol: two separate experiments were performed Experiment 1. One hundred male mice were randomly divided into 10 groups (10 mice/group) and were individually weighed the day before the administration of test chemicals in the appropriate vehicle. Just administration, solutions of test before chemicals were prepared at a concentration such that the required dose was delivered in 10 ml/kg body weight. Groups l-10 were administered with one of the following test chemicals at the indicated dosage (mmol/kg body wt.): B[a]P, 0.76, 0.38, 0.19; B[b]F

217

0.76; B[a]A 0.76; PY 0.76; DMBA 0.38; B[e]P 0.75; MNU (positive control) 0.38 and DMSO (vehicle control, 10 ml/kg). The actual dose administered was determined by weighing the syringe and tubing before and after the administration using the actual weight of the solution administered divided by specific gravity of DMSO (1.48 g/ml) or water (1.0 g/ml). After dosing with appropriate test chemicals, all mice were returned to their respective cages and were given free access to food and water prior to sacrifice 24 h f 15 min later. Experiment 2. In the second experiment, 90 male mice were divided into 9 groups (10 mice/group). Groups l-2 were gavaged with B[a]P (0.76 mmol/kg body wt.) and Groups 3 and 4 with DMBA (0.38 mmol/kg body wt.). Groups 1 and 3 were killed 24 h after dosing and Groups 2 and 4, 48 h after dosing. Group 5 was given a mixture of B[a]P and DMBA (0.76 mmol/kg and 0.38 mmol/kg). Group 6 was given a mixture of B[a]P and B[e]P (0.76 and 0.76 mmol/kg) . Group 7 was dosed with B[a]P and PY (0.76 and 0.76 mmol/kg). Group 8 was given MNU (0.38 mmol/kg) and Group 9 gavaged with DMSO. Group 5 through 9 were killed 24 h f 15 min after treatment. All Groups (l-9) in experiment 2 were fasted for 16 h prior to the administration of the test chemicals. Mice were weighed at the time of dosing and other experimental conditions were the same as described for Experiment 1. Necropsy

At the designated intervals (24 or 48 h f 15 min) after dosing the mice were killed by carbon dioxide asphyxiation. The entire G.I. tract was removed, flushed with phosphate buffered saline, infused with 10% buffered formalin at intervals of 4-5 cm, and after careful examination, was stored in 10% neutral-buffered formalin. Histology

Sections from forestomach, duodenum and proximal colon were processed and embedded in paraffin and slides were prepared and stain-

ed by the Feulgen method and with a fast green counterstain. Slides were prepared in triplicate for each tissue. Slide evaluation

The scoring of the stained sections was restricted to those crypts in which a single continuous row of epithelial cells could be discerned from the proximal end of the crypt adjacent to the muscle layers to the distal end of the crypt at the mucosal surface. The NA scores consisted of micronuclei, pyknotic nuclei and karyorrhectic nuclei. The scoring criteria were those described by Blakely et al. [7] as modified by Daniel et al. [9]: (1) micronucleussame internal structure, shape and staining intensity as normal nucleus but l/3-1/4 smaller in diameter and is clearly disengaged from other nuclear fragments; (2) pyknotic nucleus - no discernible internal structure, darkly stained, usually smaller than a normal nucleus and frequently engulfed in a vacuole; (3) karyorrhectic nucleus - fragmented small nuclear bodies usually arranged in clusters, darkly stained with no internal structure and sometimes vacuolated. The term nuclear anomalies collectively includes all three lesions. Ten crypts per slide were scored in the duodenum and proximal colon (approx. 700-1000 cells) while the forestomach was evaluated on a sample population of 200 cells. Statistical

analysis

For each tissue site, the number of NA per animal was used as the response measure to test for a treatment related effect. The distribution of NA counts within any treatment group was highly skewed and for such data, the typical summary statistics and parametric analysis are not necessarily appropriate. Thus, the log rank test, a nonparametric procedure based on a rank transformation of the data, was used to compare individual treatment groups and, where appropriate, to test for a dose response trend [38]. Therefore, the mean and/or standard deviation alone should not be used in interpreting the results of the assay. Effects were deemed significant at the 0.05 alpha (a) level. The percentage of

218

animals with one or more NA was examined descriptively. To examine the data for an interaction effect between B[a]P and DMBA several different contrast procedures were used. In each case the general form of the contrast was as follows: Contrast

(C) = BD - BAP,,, + DMSO

- DMBA,,,,

where BD is the group dosed simultaneously with both 0.76 mmol/kg B[a]P (BAP,,,) and 0.38 mmol/kg DMBA (DMBA,,,,). If the combined effect of B[a]P and DMBA is simply the sum of their individual effects, the contrast should be zero. Note that the value background effect is contained in each dosed group (i.e. it is subtracted twice: once for BAP,,, and once for DMBA,,,,), thus it is adjusted by adding the NA of DMSO (the control group). Due to the nature of the data, two non-parametric methods were used to estimate the contrast; the first based on median differences (Hodges-Lehmann; [ 151)) the second based on ranks [38]. For the latter procedure it is also possible to obtain confidence intervals for the contrast. Results Mice exposed to B[a]P for 24 h (single oral dose at 0.19, 0.38 or 0.76 mmol/kg body wt.) exhibited significantly higher numbers of NA in duodenum and in proximal colon compared to the vehicle control, DMSO (Table I). In addition, the induction of nuclear anomalies exhibited dose-dependence in the duodenum (P < 0.0001) and proximal colon (P < O.Ol), but not in the forestomach (P < 0.43) as assessed by the log rank trend test. Induction of NA in G .I. tract by single doses of a variety of PAH are also shown in Table I. Among the PAH examined, DMBA (0.38 mmol/kg) was the most potent compound, inducing NA in all three tissues. B[a]P and B[b]F (0.76 mmol/kg), also induced NA in duodenum and proximal colon but did not consistently show activity in the forestomach.

In contrast, MNU, an established carcinogen to the rodent G.I. tract, is an effective inducer of NA in all three tissues with the duodenum being the most sensitive (Table I). Those PAH generally assumed to be noncarcinogenic, i.e., PY, B[e]P, B[a]A, did not produce a significant elevation of NA in any G.I. tract tissues examined. The effect of the time between gavage and sacrifice and the effect of fasting the animals before gavage on the induction of NA in G .I. tract by B[a]P, DMBA and MNU are presented in Table II. Fasting had an effect on the yield of an NA in proximal colon induced by B[a]P (an increase from 1.7 to 4.1, P < 0.001; Table I vs. II), but an influence of fasting was apparent for the forestomach or not duodenum (P > 0.10; Table I with Table II). In direct contrast, for DMBA, an effect of fasting was found in these same two tissues, (i.e., forestomach and duodenum; P < 0.01) and not the proximal colon. Fasting did not potentiate the numbers of NA observed in mice treated with MNU, in fact, there were significantly fewer NA in the forestomach of the fasted animals. When the time period between PAH administration and necropsy is increased from 24 to 48 h, the level of B[a]P induced NA in all three tissues decreased significantly (Table II). For DMBA, the number of NA observed in forestomach and proximal colon was also decreased at 48 h as compared to 24 h (from 7.22 to 2.7 and 3.3 to 1.5, respectively, Table II), however the yield in the duodenum was unaffected (16.7 vs. 17.1). For B[a]P the incidence of NA decreased significantly in all tissues when going from 24 to 48 h. Table III compares the results of PAH given both individually and in combination i.e., as binary mixtures on the induction of G.I. tract NA. This comparison demonstrates that if B[a]P is given in combination with an equimolar dose of B[e]P or PY (as a mixture) the level of NA induced is not significantly increased from B[a]P alone. Statistical analysis of the NA incidence from the co-administration of DMBA (0.38 mmol/kg) with B[a]P (0.76

219

Table I. Induction hydrocarbons”,b,c. Chemical

of G.I. tract nuclear

Dose (mmol/kg)

anomalies

Number

in mice 24 h after exposure

BIelP

0.76 0.76

0.10 (10) 0

WW=

0.76

WA

0.76

(0) 0.20 (20) 0

0.76

(0) 0.30

0.38

(30) 0.20

BIalP

0.19 DMBA MNU DMSOd

0.38

(20) 0.40 (40) 3.30

zt 0.32

Duodenum

Proximal

0.60

+ 0.70

0.20

* 0.70

(20) 0.3 zt 0.48

(50) 0.60 zt 0.42

(50) 3.40 f (100) 0.70 f

1.07”” 0.67

f

(30) 1.20 f (70) 0.20 f

colon 0.42

1.23” 0.42

zt 0.48

(60) 6.00

zt 0.42

(100) 3.20 f

zt 0.52

(100) 2.70 zt 1.34””

(60) 1.30 +z 1.42”’

f

1.49”’

(100) 8.90 f

(80) 2.50

0.9”’

(100) 37.30

0.38

(100) 1.10 f

-

(70) 0.20

ztz 0.42

(20) “Data from experiment 15 min after dosing.

aromatic

of NA (W Mice with NA)

Forestomach PY

to polynuclear

(100) 0.5 f (50)

zt 2.79”” 1.87””

4.58”” zt 6.65”” 0.53

1: mice were not fasted prior to dosing with the chemical.

bMean l S.D. is for 10 mice. Units of NA for various ‘Statistical analysis: ‘0.03 I P I 0.05; l*O.Ol 5 P values derived from pairwise, one-tailed log rank test “DMSO given at 10 ml/kg body weight. The MNU was studies have shown that this solvent does not induce

(20) 1.70 ztz 1.16”” (90) 0.80

zt 0.79’

zt 1.78””

(90) 3.50 zt 1.78”” (100) 0.30 + 0.48 (30)

Analysis for NA was made 24 h f

tissues described in Materials and Methods. I 0.03; “‘0.001 5 P 5 0.01; l***P 5 0.0001. The Pof dose groups versus control. delivered using acetate buffered saline as the vehicle. Previous nuclear anomalies in the mouse [9].

mmol/kg) by contrast analysis yielded contrast values less than zero at all three sites. This suggests a less than additive effect, but as the respective confidence intervals contain zero (i.e., forestomach (-31.3+9.95); duodenum (-26.2% + 14.98); proximal colon (-40.13 + 1.13) there was insufficient evidence to state that the effect is less than (or greater than) additive, i.e. the test for an interaction effect was non-significant. Potency measures based on the number of NA induced per crypt per unit dose (mmol/kg

body wt.) were calculated using the mean numbers of NA per animal given in Tables I and II (Table IV). The results of these calculations show that, on a per cell at risk basis, DMBA is the most potent PAH by a considerable margin when normalized for dose. Discussion The goal of this study was to examine the mouse G.I. tract NA test as a short-term in vivo bioassay for evaluating mixtures of en-

220 Table II.

Induction

Chemical

BfalP

of G.I. tract nuclear

Dose (mmol/kg)

Exposure time (h)

MNU* DMSOd

in mice exposed

Number

to different carcinogen@‘.

of NA (W Mice with NA)

Forestomach

Duodenum

Proximal

24

0.80

48

zrz 0.48’

7.00 f 1.49 (100) 2.4 zt 1.07’ *

4.10

(60) 0.30

0.38

24

(30) 7.22

zt 2.11

(100) 16.67

zt 3.94

(100) 3.33 + 1.32

0.38

48

(100) 2.70 f

(100) 17.10

zt 3.67

(100) 1.50 f

24

(100) 0.30 zt 0.48

(100) 34.90

zt 11.27

(90) 3.80

zt 1.87

(30) 0.10 (10)

(100) 0.80 zt 0.79 (60)

(90) 0.30

zt 0.48

0.76 0.76

DMBA

anomalies

0.38 -

24

zt 0.79

f

1.16””

0.32

l

l

colon 1.37

(100) 1.70 zt 0.67””

l

0.85’

l

*

(30)

“Data from experiment 2: mice were fasted 16 h prior to treatment. bMean f SD. for 10 mice (except 24 h DMBA group where IV = 9). Units of NA for various tissues described in Materials and Methods. 5 P 5 0.03; ** lP 5 0.0001. The P* lO.OOl 5 P 5 0.01; ‘Statistical analysis: l0.03 5 P 5 0.05; “0.01 values derived from pairwise, two-tailed log rank test of the 24 h group vs. the 48-h group given the same PAH. dMNLJ and DMSO groups were only analyzed at 24 h but may be compared with the appropriate non-fasted group in Table I. l

l

vironmental chemicals. In these experiments, our primary focus was on PAH, ubiquitous compounds that frequently exist as mixtures at many industrial and hazardous waste sites [16]. We were also interested in obtaining information concerning the ability of this

Table 111. Effect of PAH mixtures Chemical

Dose

on the induction Number

bioassay to predict relative genotoxic potency in this class of compounds both alone and in combination (i.e., mixtures of PAH). Three points are clear from our study. First, the carcinogenic PAH, such as DMBA and B[a]P, induced significant increases in the level

of G.I. tract nuclear anomalies

in mice 24 h after exposurea.b.

of NA

(mmol/kg)

BfalP DMBA

0.76 0.38

B[a]P + DMBA B[a]P + B[e]P B[a]P + PY

0.76 0.76 0.76

+ 0.38 + 0.76 + 0.76

Forestomach

Duodenum

Proximal

0.80 7.22

zt 0.79 zt 2.11

7.00 16.67

4.00 0.50 0.70

+ 1.76 0.53 f 0.82

18.10 6.50 5.00

4.10 3.33 3.50 2.40 2.80

l

f 1.49 zt 3.94 zt 5.86 + 2.92 zt 3.66

f zt f zt ztz

colon 1.37 1.32 1.43 1.65 1.03

“Data from experiment 2: mice were fasted 16 h before administration of the hydrocarbon. Mice were killed 24 h f min after hydrocarbon treatment. bMean f S.D. for 10 mice. Units of NA for various tissues described in Materials and Methods.

15

221 Table IV. Comparison of specific potency PAH in the mouse G.I. tract NA assay”. Test compound

of different

Nuclear anomalies/crypt (mmol/kg body wt.) Duodenum

Proximal colon

BW BloIA

0.23 0.4@ 0.08 0.09b 0.05 0.009

BIelP

0.008

PY

0.008

0.07 0.09b 0.02 0.05b 0.02 0.003 0.004 0.003

DMBA B[elP

“The corresponding numbers for MNU in the duodenum and the proximal colon were 9.8 and 0.92 for nonfasted mice (experiment 1) and 7.0 and 0.8 for the fasted animals (experiment 2). bData obtained from experiment 2: mice fasted for 16 h before chemical administration. cThe specific potency is calculated by dividing the total number of NA obtained for a given treatment group by the total number of crypts examined. The result is then divided by the dose in mmol/kg body weight.

of NA, whereas the non-carcinogens such as B[e]P and PY, did not. Secondly, it is also apparent from these data that DMBA is a more potent inducer of NA in G.I. tract than B[a]P and B[b]F, compounds that are also generally weaker carcinogens to rodents (Tables I, II and IV). And thirdly, the activities exhibited by binary mixtures of PAH do not appear to be greater than that predicted by simple additivity. The induction of NA appears to depend on several factors including the dose, time between gavage and sacrifice, the type and potency of the chemical, and the region of the G.I. tract. For example, B[a]P induces a dosedependent increase in NA in the duodenum and proximal colon (Table I) at 24 h. However, when the time between dosing and killing was increased from 24 to 48 h a significantly weaker induction of NA is observ-

ed. For the most part, all tissue sites demonstrated a greater response when evaluated at 24 h relative to 48 h (an exception was DMBA in the duodenum). Previous investigators have proposed that the formation of NA in the G.I. tract may be a useful tool for making judgments on the potency of specific chemicals or on the potential for radiation induce cancer exposures to [19,26,29,40]. The propensity of chemical and radiation to induce NA in G.I. tract correlates with both their site specificity for tumor induction along the G.I. tract and also with carcinogenicity their mutagenicity and [19,26,29,36,37,40] at other organ sites such as the skin [11,42]. We were also interested in evaluating mixtures of PAH for their potential to induce G.I. tract NA. Since the administration of B[a]P and DMBA, individually, were active in G.I. tract (Tables 1, I1 and III), it was expected that the co-administration of B[a]P and DMBA (as a mixture) would result in an increased induction of NA over that observed in the animals/groups receiving individual compounds. While this was observed, in comparison to the B[a]P group it was not for DMBA. These differences are probably due in part to the higher NA induction by DMBA relative to B[a]P. Additionally, the potency of the mixture (i.e., DMBA + B[a]P) differed in the various segments of the G.I. tract. The potency of the binary mixture of DMBA and B[a]P could be examined using at least two models: (1) assuming the potency of the mixture would be equivalent to that obtained by directly adding the results from comparable doses of the two PAH individually or by (2) assuming that the absorption and metabolism rates of the individual PAH were not independent and that they are competitive inhibitors for oxidation. In the latter case, one might expect that the number of NA induced in different regions of the gastrointestinal tract might be some precentage less than that which is induced by B[a]P and DMBA, individually. If the two PAH have equal access to oxidative enzymes and metabolic capability of the animal is

222

saturated the 24 h induction of NA by B[a]P + DMBA in forestomach might be approximately equal to the average of the individual activities. Our analysis of the former case indicated potency of a less than simple additivity, but this departure from expectation was not statistically significant. The level of NA induced by the mixture was approximately equal to the average of the B[a]P and DMBA individual results and might indicate that B[a]P and DMBA are, in fact, in competition for absorption and/or metabolism in the G.I. tract or that the capacity of either or both of these processes is exceeded. When the non-carcinogenogenic PAH, PY, and B[e]P was administered as a mixture with B[a]P, there was a reduction in the yield of NA in all three segments of G.I. tract compared to that seen with B[a]P alone (Table III). Our results suggest the response observed in the G .I. tract N.A. assay is in concert with the relative carcinogenic potencies of B[a]P and DMBA both as skin tumor initiators [ll], and as forestomach carcinogens in mice. For example, reported oral TD,, dose for DMBA and B[a]P are 0.29 mg/kg per day and 0.86 mg/kg per day, respectively, for the induction of G.I. tract cancer in the mouse [18]. Likewise, non-carcinogenic PAH (e.g., B[e]P and PY) do not increase the incidence of NA in any G .I. tract region examined. On the other hand the results with PAH mixtures would indicate that the effects are less than predicted by direct additivity. However, additional experiments using various total PAH doses and varying the PAH ratios would be required by for the observations reported here could be generalized. Hence, quantitation of NA in G.I. tract may prove useful as an index to predict the relative carcinogenic and/or mutagenic potency of environmental chemicals and use as a short-term bioassay for the evaluation of environmental chemicals and mixtures of chemicals.

for statistical analysis and Melda Hirth and Catherine Carr for typing the manuscript. References 1

Alfred, L.J. and Dipaolo, J.A. (1968) Reversible inhibition of DNA synthesis in hamster embryo cells in culture: Action 1.2~benzanthracene of and 7,12-dimethylbenz[a]anthracene. Cancer Res., 28, 60-75.

2

Atlas, S.A., Boobis, A.R., Felton, J.S., Thorgiersson, S.S and Nebert, D.W. (1977) Ontogenetic expression of polycyclic aromatic compound-inducible monooxygenase activities and forms of cytochrome P-450 in the rabbit.

3

4

5

6

7

8

drinking water samples for polynuclear aromatic hydrocarbons. EPA P.O. No. CA-7-2999-A and CA-8-2275B. Exposure Evaluation Branch, Health Effects Research Laboratorty, Cincinnati, OH. Bjorseth, A. and Lunde, G. (1977) Analysis of the polycyclic aromatic hydrocarbon content of airborne particulate pollution in a Soderberg paste plant. J. Am. Ind. Hyg. Assoc., 38, 224-227. Bjorseth, A., Bjorseth, 0. and Fjeldstad, E. (1978) Polycyclic aromatic hydrocarbons (PAH) in working atmospheres I. Analysis of the PAH-content in an aluminum reduction plant. Stand. J. Work Environ. Health, 4, 212-223. Bjorseth, A., Bjorseth, 0. and Fjeldstad, E. (1978) Polycyclic aromatic hydrocarbons (PAH) in working atmospheres II. Determination of the PAH-content in a coke plant. Stand. J. Blakey. D.H., Goldberg, M.T., Detection of NA

Work Environ. Health, 4, 224-236. Duncan, A.M.V., Wargovich, M.J., Bruce, W.R. and Heddle, J.A. (1985) in the colonic epithelium of the mouse.

Cancer Res., 45, 242-249. Colucci, J.M. and Begeman. C.R. (1971) Carcinogenic air pollutants in relation to automotive traffic in New York. Environ. Sci. Technol., 5, 145-150.

9

Daniel, F.B.. Olson, G.R. and Stober, J.A. (1990) The induction of gastrointestinal tract anomalies in B6C3Fl mice by 3-chloro-4-dichloro-methyl&hydroxy-2[5H] furanone and 3.4-dichloro-5-hydroxy-2[5H] furanone, mutagenic by-products of chlorine disinfection. Environ. Mol. Mutag., 17.

10

deMaio. L. and Corn, M. (1966) Polynuclear aromatic hydrocarbons associated with particulates in Pittsburgh air. J. Air. Poll. Control Assoc., 16, 67-71. Digiovanni. J., Slaga, T.J. and Boutwell. R.K. (1980) Comparison of tumor-initiating activity of

11

7-12.dimethylbenz(a)anthracene female SENCAR and CD-1 381-389.

Acknowledgements 12

The authors thank Deborah Schoborg and Karen Swallow for animal care, Huiling Feng

Evidence of temporal control and organ specificity of two genetic regulatory systems. J. Biol. Chem., 252, 4712-4721. Basu, D.K. and Saxena, J. (1977) Analysis of raw and

and mice.

benzo(a)pyrene Carcinogenesis

in 1,

DippIe, A. and Nebzydoskik, J.A. (1978) Evidence for the involvement of a diol epoxide in the binding of 7,12-dimethylbenzo[a]-anthracene culture. Chem. Biol. Interact., 20.

to

DNA

17-26.

in cells in

223

13

14

15 16

17

18

19

20

21

22

23

24

25

Dipple, A. (1985) In: Polycyclic Hydrocarbons and Carcinogenesis, pp. l-6. ACS Symposium Series No. 283. Editor: R.G. Harvey. American Chemical Society, Washington, DC. Eisenstadt, E. (1985) In: Polycyclic Hydrocarbons and Carcinogenesis, pp. 327-40. ACS Symposium Series No. 283. Editor: R.G. Harvey, American Chemical Society, Washington. Flenberg, S.A. (1977) The analysis of cross classified categorical data. The MIT Press, Cambridge, London. Garman, J.R., Freund, T. and Lawless, E.W. (1987) Testing for ground water contamination at hazardous waste sites. J. Chromat. Science, 25, 328-337. Glatt, H., Seidel, A., Bochnitschek, W., Marquardt, H.. Marquardt, H., Hodgson, R.M., Grover, L. and Oesch, F. (1986) Mutagenic and cell transforming activities of trio1 expoxides as compared to other chrysene metabolites. Cancer Res., 46, 4556-4565. Gold, L.S., Sawyer, C.B., Magaw, R., de Veciana, M., Levinson. R., Hooper, W.R., Bernstein, L., Peto, R., Pike, C. (1984) A carcinogenic potency date-base

Backman, G.M.. N.K., Havender, and Ames, B.N. of the standardiz-

26

27

28

29

30

ed results of animal bioassays. Environ. Health Perspectives, 38, 9-319. Heddle, J.A., Blakey, D.H., Duncan, A.M. ., Goldberg, M.T., Newmark, H., Wargovich, M.J. and Bruce, W.R. (1982) Micronuclei and related NA as a short-term assay for colon carcinogens. In: Indicators of Genotoxic Exposure, pp. 367-377. Editors: B.A. Bridges, B.C. Butterworth and I.B. Weinstein, Banbury Report 13.

31

International Agency for Research on Cancer (1973). Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 3, IARC, Lyon, pp. 45-81, 91-136, 178-196.

33

Kapitulnik, J., Wislocki. P.G., Levin, W.. Yagi, H., Jerina, D.M. and Conney, A.H. (1978) Tumorigenicity studies with diol epoxides of benzo [a]-pyrene which indicate that f -trans-7$3,cr-dihydroxy-9a, lOa-epoxy-7,8, 9.10~tetrahydrobenzo[a]pyrene is an ultimate carcinogen in newborn mice. Cancer Res.. 38, 354-358. Kouri. R.E. and Nebert. D.W. (1977) In: Originsof human cancer, pp. 811-835. Editors: H.H. Hiatt, J.D. Watson and J.A. Winston, New York, Cold Spring Harbor Laboratory, Vol. 4. LaVoie. E.J.. Amin, S.. Hecht, S.S., Furuya, K. and Hoffmann, D. (1982) Tumor initiating activity of dihydrodiols of benzo(b)fluoranthene, benzo-(j)fluoranthene and benzo(k)fluoranthene. Carcinogenesis. 3. 49-52. Levin, W.. Thakker, D.R., Wood, A.W., Chang, R.L., Lehr, R.E., Jerina, D.M. and Conney, A.H. (1978) Evidence that benzo[a]anthracene 3.4-diol-1,2-epoxide is an ultimate carcinogen on mouse skin. Cancer Res., 38, 1705-1710. Levin, W., Wood, A.W.. Wislocki. P.G., Chang. R.L., Kapitulnik, J., Mah, H.D., Yagi. H., Jerina, D.M. and Conney, A.H. (1978) Mutagenicity and carcinogenicity of benzo[o]pyrene and benzo[o]pyrene derivatives. In:

32

34

35

36

Polycyclic Hydrocarbons and Cancer: Environment, Chemistry, and Metabolism, pp. 189-202. Editors: H.V. Gelboib and P.O.P. Ts’o, Academic Press, New York. Maskens, A.P. (1979) Significance of the karyorrhectic index in 1,2-dimethylhydrazine carcinogenesis. Cancer Len., 8, 77-86. Phillips, D.H., Glan, H.R., Seidel. A., Bochnitschek, W., Oesch, F. and Grover, P.L. (1986) Mutagenic potential of DNA adducts formed by diol-epoxides. triol-epoxides and the K-region epoxide of chrysine in mammalian cells. Carcinogenesis., 7, 1739- 1743. Prough, R.A., Patrizi, V.W., Okita, R.T., Masters, R.S.S. and Jakobsson, S.W. (1979) Characteristics of benzo(a)pyrene metabolism by kidney, liver and lung microsomal fractions from rodents and humans. Cancer Res., 39, 1119-1126. Ronen, A. and Heddle, J.A. (1984) Site-specific induction of NA (apoptotic bodies and micronuclei) by carcinogens in mice. Cancer Res., 44, 1536-1540. Searle, C.E.. Ed. (1984) Chemical Carcinogenesis, 2nd Ed. ACS Monograph 182, American Chemical Society, Washington, D.C. Selkirk, J.K., Croy, R.G., Wiebel, F.J. and Gelboin, H.V. (1976) Differences in benzo(a)pyrene metabolism between rodent liver microsomes and embryonic cells. Cancer Res.. 36, 4476-4479. Shabad. L.M., Cohan, Y.L., Yl’nitskii, A.P., Khesina. A.Y.. Shcherbak, N.P. and Smirnov, G.A. (1971) The Carcinogenic benzo(a)pyrene Inst.. 47, 1179-1192. Sims, P. (1970) Studies 7-methylbenz[a]anthracene

38

39

on and

the metabolism of 7.12-dimethylbenz-

[alanthracene and its hydroxymethyl derivatives in rat liver and adrenal homogenates. Biochem. Pharmacol.. 19, 2261-2275. Sims, P., Grover, P.L., Swaisland, A., Pal, K.. and Hewer, A. (1974) Metabolic activation of benzo[a]pyrene proceeeds by a diol-epoxide. Nature, 252, 326-328. Slaga, T.J.. Viaje. A., Berry, D.L.. Bracken, W.. Buty, S.C. and Scribner, J.D. (1976) Skin tumor initiating ability of benzo[a]pyrene 4,5-, 7,8- and 7.8-diol-9.10-epoxides and 7,8-dial. Cancer Len., 2, 115-122. Slaga. T.J., Huberman, E., Selkirk, J.K., Harvey, R.G. and Bracken, W.M. (1978) Carcinogenicity and mutagenicity of benz[a]anthracene diols and diol epoxides. Cancer

87

in the soil. J. Natl. Cancer

Res., 38, 1699-1704.

Slaga, T. J., Gleason, G.L., Mills, G., Ewald, L., Fu. P.P. Lee, H.M.. and Harvey, R.G. (1980). Comparison of the skin tumor-initiating activities of dihydrodiols and diol epoxides of various polycyclic aromatic hydrocarbons. Cancer Res., 40. 1981-84. Maracuilo, L.A. and McSweeney. M. (1977) Nonparametric and distribution-free methods for the social sciences. Brooks/Cole Publishing Co., Monterey. CA pp. 310-312. Thakker, D.R., Yagi, H., Lehr. R.E., Levin, W., Buening, M.. Lu, A.Y.H., Chang, R.L., Wood, A.W.. Conney, D.M. (1978) Metabolism of A.H. and Jerina,

224

40

41

trons-9,10-dihydroxy-9.10-dihydrobenzo[a]pyrene occurs primarily by aryl-hydroxylation rather than formation of a diol epoxide. Mol. Pharmacol., 14, 502-513. Wargovich, M.J., Goldberg, M.T., Newmark, H.L. and Bruce, W.R. (1983) Nuclear aberrations as a short-term test for genotoxicity to the colon: evaluation of nineteen agents in mice. J. Natl. Cancer Inst.. 71, 133-137. Weinstein, I.B., Jeffrey, A.M., Jennette, K.W., Blobstein, S.H.. Harvey, R.G., Harris, C., Autrup, H., Kasai, H. and Nakanishi, N. (1976) Benzo[a]pyrene diol epoxides as intermediates in nuclei acid binding in vitro and in vivo. Science, 193, 592-595.

42

Wood, A.W., Levin, W., Chang, R.L., Lehr, R.E., Schaefer-Ridder, M., Karle, J.M., Jerina, D.M. and Conney, A.H. (1977) Tumorigenicity of five dihydrodiols of

benzo[a]anthracene

43

44

on mouse skin: Exceptional

activity of

benzo[a]anthracene 3.4.dihydrodiol. Proc. Nat. Acad. Sci., U.S.A., 74, 3176-3179. Yang, S.K. and Dower, W.V. (1975) Metabolic pathways of 7,12-dimethylbenz[a]anthracene in hepatic microsomes. Proc. Nat. Acad. Sci., U.S.A., 72, 2601-2605. Yang, S.K., McCourt, D.W., Roller, P.P. and Gelboin, H.V. (1976) Enzymatic conversion of benzo[a]pyrene leading predominantly to the dial-epoxide r-7,t-8-dihydroxy-t-9 , lo-oxy-7, 8, 9 , lo-tetrahydrobenzo[ a]-pyrene through a single enantiomer of r-7,t-8-dihydroxy-7,8dihydrobenzo[a]pyrene. Proc. Natl. Acad. Sci., U.S.A., 73, 2594-2598.