The mutagenicity of benzo[a]pyrene in mouse small intestine

Roger A. Brooks, Nigel J. Gooderham1, Robert J. Edwards1, Alan R. Boobis1 and Douglas J. Winton2

Cancer Research Campaign Human Cancer Genetics Research Group, Cambridge Institute for Medical Research, Box 139, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY and
1 Department of Clinical Pharmacology, Royal Postgraduate Medical School, DuCane Road, London W12 0NN, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have investigated the mutagenicity of benzo[a]pyrene (B[a]P) in small intestine using the Dlb-1 locus assay in the mouse. Administration of B[a]P by the oral and i.p. routes had markedly different effects on the number of Dlb-1 mutations and the pattern of induction of cytochrome P-4501A1 (CYP1A1). In Ahr-responsive animals i.p. injection resulted in marked induction in crypt cells along the length of the small intestine, with some induction in the villus cells. In contrast, after oral administration, CYP1A1 induction was evident only in the villus cells, and this declined distally. The intensity and speed of induction in Ahr-responsive animals was such that the genotoxic effect of a single injection of B[a]P could not be augmented by prior treatment with non-genotoxic inducers such as ß-napthoflavone and TCDD. Oral B[a]P treatment resulted in a decrease in the number of mutations when compared with the i.p. route. Studies in congenic Ahr-non-responsive versus Ahr-responsive mice indicated that induction of CYP1A1 was associated with increased numbers of Dlb-1 mutations. Mutation induction in Ahr-non-responsive mice in the absence of detectable CYP1A1 in either liver or small intestine indicates that an appreciable portion of B[a]P activation to a genotoxin must be by other than a CYP1A1 mediated route. These data show that B[a]P is a potent small intestinal mutagen at the Dlb-1 locus.

Abbreviations: B[a]P, benzo[a]pyrene; ß-NF, ß-naphthoflavone; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The small intestine is one of the sites most exposed to environmental chemicals, either via ingestion in the diet or swallowed following mucociliary clearance of inhaled material. However, it is very resistant to the tumorigenic effects of such compounds, despite the fact that many are carcinogenic at other sites (1). Indirectly acting genotoxic chemicals require activation and the enzymes responsible are widely distributed in cells and tissues, including the small intestine, in particular after exposure to inducing agents (2). Hence, it is important to define not only the extent to which epithelial stem cells are at risk from environmental genotoxins but also what factors might ameliorate that risk and whether such factors may bear on the resistance of the small intestine to carcinogenesis.

One important class of environmental genotoxins are the polycyclic aromatic hydrocarbons, the most extensively studied of which is benzo[a]pyrene (B[a]P). This compound is ubiquitous in the environment, is mutagenic in both prokaryotic and eukaryotic test systems and is an animal carcinogen (36). B[a]P is also classified as Group 2A (probably carcinogenic to humans) (7) and has recently been shown to bind to p53 at the sites most often mutated in bronchogenic carcinoma, implicating it as an etiological agent in cigarette smoke (8). B[a]P itself is unreactive, but it is readily converted to a highly reactive electrophile by enzymes of drug metabolism. The routes of metabolism of B[a]P are complex, but the ultimate genotoxic metabolite produced by the cytochrome P-450 system has been identified as the anti isomer of B[a]P 7,8-diol-9,10-epoxide (9,10). Three enzymatic reactions are required for its formation (11): initial epoxidation to yield the 7,8-epoxide, hydrolysis of this epoxide to yield the (–)-trans-7,8-diol and finally a second epoxidation of the diol to produce B[a]P-7,8-diol-9,10-epoxide (anti isomer).

The epoxidations involved in the activation of B[a]P to a genotoxin are catalysed primarily by members of the cytochrome P-450 family of enzymes, the cytochrome P (CYP)1A subfamily being particularly implicated, both in rodents and humans (1214). CYP1A1 is not expressed constitutively in either human (15) or rodent (16) tissues. However, this enzyme is readily induced by compounds such as polycyclic aromatic hydrocarbons, both in rodents and humans (15,16). Hence, differences in the inducibility of CYP1A1 could contribute to differences in susceptibility to the carcinogenic effects of polycyclic aromatic hydrocarbons, and there is evidence that this may be so (reviewed in ref. 17).

The Dlb-1 locus assay of somatic mutation in the mouse small intestine (18) remains one of only a very small number of assays able to measure mutation of an endogenous gene in vivo. In the assay, clones of cells arising from mutated stem cells are detected. It is thus ideally suited to determining the susceptibility of the stem cell population to mutation. The assay has been well characterized for its response to other genotoxic agents (1822). We have therefore investigated the mutagenicity of B[a]P in the Dlb-1 assay.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
B[a]P and ß-naphthoflavone (ß-NF) were obtained from Sigma (Poole, UK). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Dr S.S.Thorgeirsson (National Cancer Institute, Bethesda, MD).

Animals
C57BL/6J Dlb-1a congenic mice, homozygous for Dlb-1a were derived as previously described (23) and males were crossed with C57BL/6J females. The resulting F1 mice, heterozygous at Dlb-1, were between 6 and 12 weeks of age at the start of each experiment. C57BL/6J Ahrd congenic mice homozygous for the low affinity Ah receptor were obtained from Professor W.W.Weber (Department of Pharmacology, University of Michigan, MI). These mice which are Dlb-1b homozygotes were crossed with C57BL/6J Dlb-1a congenic mice which are homozygous for the high affinity Ah receptor (i.e. Ahrd). The resulting F1 mice are heterozygous at both Dlb-1 and Ahr and were backcrossed to the C57BL/6J Ahrd parents. The resulting mice were phenotyped following two i.p. injections of ß-NF (80 mg/kg) separated by 48 h. The distal 2 mm of the tail was removed 24 h after the second injection, embedded and prepared for immunocytochemistry as previously described (24). Five micrometre sections were immunostained for induced CYP1A1 as described below with cells in the sebaceous glands showing staining in Ahr-responsive mice. Further sections were stained with Dolichos biflorus agglutinin-peroxidase conjugate (DBA-Px) to determine Dlb-1 phenotype as previously described (23). After washing in 18 mM phosphate buffer (pH 7.4) containing 0.84% (w/v) NaCl (PBS) sections were incubated with a 1:250 dilution of the conjugate for 1 h at room temperature and developed with 0.025% w/v 3,3'-diaminobenzidine. The Ah receptor phenotype was retrospectively confirmed by injecting ß-NF (80 mg/kg) i.p. on two occasions separated by 48 h, the second injection being 24 h before the animals were killed for Dlb-1 mutation measurement. A piece of liver was taken from each animal, processed for immunocytochemistry and sections stained for CYP1A1. All mice were maintained on a 12 h day/night cycle and given water and No.1 modified expanded diet (Special Diet Services, Essex, UK) ad libitum.

Treatment of animals
Age matched mice were allocated to treatment groups without regard to sex. In all experiments B[a]P and ß-NF were administered in corn oil at 40 mg/kg and 80 mg/kg respectively. TCDD (10 µg/kg) was administered by i.p. injection in dimethyl sulfoxide.

The effect of B[a]P on Dlb-1 locus mutations was measured following the administration of 2, 4 or 6 doses of B[a]P given i.p., the doses being given at 96 h intervals. Control animals were treated with vehicle only. For all other mutation experiments B[a]P was administered as a single dose, i.p. or p.o., either alone or following cytochrome P-450 induction. Cytochrome P-450 was induced by either three i.p. injections of ß-NF each separated by 48 h with B[a]P administered 42 h before the last injection or by a single injection of TCDD given 72 h before B[a]P. Animals were killed 2 weeks following the final treatment. Two weeks is the minimum time for clones of non-staining cells to expand and become measurable on the villi (18).

For immunocytochemistry B[a]P was administered as a single dose given i.p. or p.o. and mice were killed 0, 9, 24, 48, 72, 96 and 182 h afterwards. Control animals received vehicle alone. Tissues were prepared as previously described (24).

Preparation of tissues for the Dlb-1 assay
Intestinal wholemounts were prepared as described previously (18). Animals were killed by cervical dislocation and the small intestine was removed. The intestinal lumen was flushed with ice cold PBS, briefly fixed with 5% (w/v) formal saline, cut along the mesenteric line and pinned onto wax dishes luminal side upwards. Following fixation in 10% (w/v) formal saline for 30 min the wholemounts were treated with 23 mM dithiothreitol and mucin was removed by gentle pipetting. The tissues were stained with DBA-Px and developed with 0.05% (w/v) 3,3'-diaminobenzidine.

Antibodies
Polyclonal antibodies were raised in rabbits against the C-terminus of rat CYP1A1 (25). The C-terminus of mouse CYP1A1 is identical to the rat form and the antibody binds specifically to CYP1A1 in mouse hepatic microsomal fractions (data not shown).

Immunocytochemistry
Immunocytochemistry was carried out as previously described (24). Briefly, 5 µM sections were rehydrated, blocked with a 1/20 dilution of normal goat serum in PBS and incubated with primary antibody overnight at 4°C. The antibody dilution determined by titration was 1/3200. Control sections were incubated with normal rabbit serum (NRS) at the same dilutions. After washing, sections were incubated with a 1/150 dilution of goat anti-rabbit peroxidase conjugate for 2 h at room temperature, developed with 3,3'-diaminobenzidine and counterstained with Carazzi's haematoxylin.

Dlb-1 locus assay
The Dlb-1 assay is a specific locus assay in which somatic mutations affecting one allele (Dlb-1b) of the polymorphic Dlb-1 locus on mouse chromosome 11 are detected in Dlb-1b/a heterozygotes by the visualization of epithelial clones which have lost their ability to bind the lectin Dolichos biflorus agglutinin. The detection of somatic clones and the fact that these form after a latent period (usually 2 weeks) which is long compared with the turnover time of the intestinal epithelium means that mutation is assayed in the epithelial stem cell compartment.

Quantification of Dlb-1 locus mutations
Scoring for Dlb-1 mutations involved determining the frequency of epithelial clones which remain unstained in intestinal wholemounts stained with a DBA-Px conjugate. These scores can be expressed as the mutation frequency per 104 villi (by an estimate of the local number of villi scored in the 50 microscopic fields viewed R). Alternatively, to allow comparison between the number of mutations detected at different intestinal sites data were converted to mutant clones per 105 crypts by correcting for the crypt:villus ratio determined as previously described (24).

Statistics
Within group variance was determined using one-way analysis of variance (ANOVA) and individual means compared by unpaired t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutagenicity of B[a]P
A clear relationship was found between the number of doses of B[a]P administered i.p. and the yield of Dlb-1 mutant clones induced (Table IGo). Animals receiving corn oil alone showed no increase in Dlb-1 mutation frequency above that seen in historical controls.


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Table I. Effect of multiple administration of B[a]P given i.p. on the number of Dlb-1 locus mutations
 
There was no evidence of a sex difference in the response to a single dose of B[a]P given either p.o. or i.p. Values are for p.o., 43.5 ± 9.2 versus 32.1 ± 10.7, n = 3, P = 0.234; and for i.p., 78.6 ± 25.2 versus 88.7 ± 19.2, n = 9, P = 0.364 (mean ± SD mutant clones per 105 crypts, female versus male; unpaired Student's t-test).

Immunocytochemistry
In uninduced mice there was no staining for CYP1A1 in any of the tissues examined (Figure 1aGo). In C57BL/6J (Ahrb) oral or i.p. treatment with B[a]P induced CYP1A1 in the liver. CYP1A1 was induced in the intestinal epithelium following a single dose of B[a]P with a pattern of immunoreactivity that was dependent on the route of administration.




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Fig. 1. Distribution of CYP1A1 in liver sections from either untreated mice (a) or mice injected i.p. with 40 mg/kg B[a]P (b). Bar = 45µm.

 
CYP1A1 staining 24 h after i.p. administration was predominantly in crypt epithelium although cells on the villus were also induced (Figure 2a and bGo). The extent and intensity of staining did not vary from proximal to distal small intestine. Capillary endothelial cells beneath the crypts and in the lamina propria were strongly stained (Figure 2cGo). Induction was rapid and the staining pattern described above could be seen by 9 h, which was the earliest time point analysed. By 48 h immunoreactive CYP1A1 in the epithelium had declined substantially and by 72 h had disappeared. Induction in hepatocytes occurred more slowly than in the intestinal cells and maximum induction was not reached until 48 h, thereafter it declined and was not visible at 182 h. The distribution was centrilobular (Figure 1bGo) with no staining periportally.








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Fig. 2. Localization of CYP1A1 in transverse sections of small intestine given 40 mg/kg B[a]P i.p. (a–c) or p.o. (d–f). (a) NRS control. (bf) anti-CYP1A1 staining showing immunoreactivity in the: (b) crypt epithelium; (c) capillary endothelium; (d–f) villus epithelium, (d) proximal, (e) mid and (f) distal small intestine. Bar = 45 µm.

 
Oral administration of B[a]P produced strong induction of CYP1A1 in the villus epithelium by 9 h. The extent of staining was greatest in the proximal intestine, decreased in the mid intestine and was only seen on the villus tips in the distal intestine (Figure 2d–fGo). No staining was seen in the crypts. By 24 h immunoreactivity had decreased in mid intestine and had disappeared distally. All staining was lost by 72 h. No induction of CYP1A1 was seen in capillary endothelial cells. In the liver a centrilobular distribution of immunoreactivity could be seen at 24 h but staining intensity was less than following B[a]P given by the i.p. route and immunoreactivity had disappeared by 72 h (results not shown). No immunoreactivity for CYP1A1 was observed in liver and intestine in samples taken from Ahrd/d mice treated with B[a]P (40 mg/kg i.p.) 9 or 24 h prior to killing or with ß-NF (80 mg/kg i.p.) given as two injections 72 and 24 h prior to killing (Figure 3Go).




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Fig. 3. Lack of CYP1A1 immunoreactivity in Ahrd/d mice treated with B[a]P 40 mg/kg i.p. (a) liver, (b) proximal small intestine. Bar = 45 µm.

 
Effect of CYP1A induction on mutagenicity
A single dose of B[a]P produced a significant increase in the number of Dlb-1 mutations (Table IIGo). Prior treatment with either ß-NF or TCDD failed to produce any increase in mutation frequencies.


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Table II. The effect of treatment with CYP1A inducing chemicals on Dlb-1 locus mutations produced by a single dose of B[a]P
 
Effect of route of B[a]P administration
The numbers of Dlb-1 mutations in the proximal small intestine seen following oral administration of B[a]P were 38.5% of those seen when B[a]P was given i.p. (Table IIIGo). There was no significant difference between the number of mutations in proximal and distal small intestine when B[a]P was given by either route.


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Table III. The effect of route of administration of B[a]P on Dlb-1 locus mutations in proximala and distalb small intestine
 
Mutation induction in Ahrd/d mice
Dlb-1 heterozygote mice which were either CYP1A1 inducible (Ahrb/b) or non-inducible (Ahrd/d) were treated with B[a]P. A significantly lower yield of Dlb-1 mutations was observed in the latter (Table IVGo).


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Table IV. The effect of Ahr-genotype on Dlb-1 locus mutations in response to B[a]Pa
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have characterized the mutagenic effects of B[a]P and investigated some of the factors determining intestinal stem cell mutation. The assay relies on mutation of an endogenous gene in stem cells for its read out. As a consequence, it is ideally suited to the analysis of both the mutability of constitutively expressed genes and the involvement of toxicokinetic factors.

A single i.p. dose of B[a]P is highly mutagenic to intestinal stem cells and after multiple doses its potency matches that of direct acting alkylating agents which are the most potent compounds tested in this assay so far (18,21). B[a]P has also been shown to be the most mutagenic of the compounds tested at the lacI locus in the spleen of transgenic C57BL/6 mice (26).

The mutagenicity of B[a]P will depend on the interplay between the enzymes of activation and detoxication (11) and in particular their cellular distribution and regulation. The major enzyme involved in the metabolic activation of B[a]P is CYP1A1 (1214). This enzyme is not constitutively expressed in mouse tissues but is inducible by polycyclic aromatic hydrocarbons and related compounds (27,28). In untreated mice CYP1A1 was not detected in either liver or small intestine using immunocytochemistry, which is the most sensitive method for detecting expression of specific apoprotein (29). Hence, it might be expected that prior induction of CYP1A1 would be required to obtain a maximal mutagenic response with B[a]P. This is not the case, and the mutagenicity of a single dose of B[a]P is as great alone as after prior induction with either ß-NF or TCDD. Congenic mice, non-responsive to hydrocarbon inducers, give a reduced mutagenic response to a single dose of B[a]P as compared with responsive mice. These results implicate CYP1A1 in the activation of B[a]P and indicate that, at the dose used, B[a]P is maximally auto-inducing in responsive animals. A corollary of this is that although the number of mutations in these animals increases with the number of doses of B[a]P administered, this is a consequence of accumulation of mutations rather than because of increasing induction of CYP1A1. Previous data on the kinetics of the accumulation of mutations in this model support this conclusion (22,24).

Consideration of the mutation data with the CYP1A1 distribution demonstrate that appreciable mutation also occurs in the absence of any detectable CYP1A1 either systemically (in Ahr-non-responsive mice) or locally (in the crypt epithelium of orally treated Ahr-responsive mice). Although CYP1A1 is the enzyme most strongly implicated in the mutagenic activation of B[a]P, other enzymes are clearly capable of fulfilling this role. There is evidence in humans that the second epoxidation can also be supported by CYP3A4 (13) and that the initial formation of B[a]P-7,8-epoxide can be supported by CYP2C11 in the rat (30). Peroxy radicals generated by a number of enzymes, including some of those involved in prostaglandin synthesis, can participate in the second epoxidation of B[a]P 7,8-diol to the genotoxic diol epoxide, at least in vitro (3134). Comparison of the mutagenic response in responsive versus non-responsive mice suggests that as much as 45% of the response in the former could be due to a genotoxic metabolite that is the product of enzymes whose induction is not through the Ahr locus.

The reduced number of stem cell mutations in oral as compared with i.p. treated animals demonstrates the protective effect of first pass metabolism. This is associated with strong induction of CYP1A1 on intestinal villi (and not within the crypt) and a decreased bioavailability of B[a]P as indicated by the reduced intensity of CYP1A1 induction in the liver. [It has been demonstrated previously (35) that the intensity of CYP1A1 induction by hydrocarbons depends on the dose, whereas the duration depends on the persistence of the exposure]. These observations show at least in respect of B[a]P that the crypt stem cells should be regarded as part of the systemic compartment and not of that compartment directly accessible to compounds present in the intestinal lumen.

The results of the present study have a number of important implications. They provide further evidence for the importance of factors other than mutation in determining tissue susceptibility to carcinogenicity. B[a]P is a potent mutagen in the small intestine but does not normally cause tumours in this tissue (4). Ingestion of hydrocarbons causes a very rapid and profound auto-induction of intestinal CYP1A1, but this is confined to villus epithelial cells. This appears to be a consequence of the kinetics of intestinal absorption, and serves as a protective mechanism against some dietary genotoxins. In contrast, systemic delivery of B[a]P to the crypt cells results in induction of CYP1A1 in these cells. The difference in induction pattern after p.o. and i.p. administration of B[a]P has important implications for the toxicokinetics of this compound. Following inhalation, a proportion of the dose is swallowed depending on the particle size (36). That proportion of a dose that is swallowed will have effects similar to those of an oral dose. However, that fraction of the dose absorbed across the lungs, like that absorbed through the skin, will have effects more resembling those of an i.p. dose. The extent to which these findings apply to humans is not known. Nevertheless, they do suggest that both the route of exposure and the inducibility of CYP1A1 can have profound impact on the mutagenicity of B[a]P, and presumably related hydrocarbons. In this context, it is of considerable interest that whilst the vast majority (>97%) of the human body burden of B[a]P is from the diet (37), there is no epidemiological evidence for a significant correlation between exposure to dietary B[a]P and the incidence of cancer (38).


    Notes
 
2 To whom correspondence should be addressed Email: djw{at}mole.bio.cam.ac.uk Back


    Acknowledgments
 
This work was supported by grants from the Cancer Research Campaign [CRC] and the EEC (EV5V-CT93-0246).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Potten,C.S., Li,Y.Q., O'Connor,P.J. and Winton,D.J. (1992) A possible explanation for the differential cancer incidence in the intestine, based on distribution of the cytotoxic effects of carcinogens in the murine large bowel. Carcinogenesis, 13, 2305–2312.[Abstract]
  2. Zhang,Q.Y., Wikoff,J., Dunbar,D., Fasco,M. and Kaminsky,L. (1997) Regulation of cytochrome P4501A1 expression in rat small intestine. Drug Metab. Dispos., 25, 21–26.[Abstract/Free Full Text]
  3. IARC (1973) Certain polycyclic aromatic hydrocarbons and heterocyclic compounds. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, vol. 3. IARC, Lyon.
  4. IARC (1983) Polynuclear aromatic compounds, part 1: chemical, environmental and experimental data. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Human, vol. 32. IARC, Lyon.
  5. Huberman,E. and Sachs,L. (1974) Cell-mediated mutagenesis of mammalian cells with chemical carcinogens. Int. J. Cancer, 13, 326–333.[ISI][Medline]
  6. McCann,J., Choi,E., Yamasaki,E. and Ames,B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl Acad. Sci. USA, 72, 5135–5139.[Abstract]
  7. IARC (1987) Overall evaluations of carcinogenicity: an updating of IARC monographs Volumes 1–42. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans (suppl. 7). IARC, Lyon.
  8. Denissenko,M.F., Pao,A., Tang,M. and Pfeifer,G.P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science, 274, 430–432.[Abstract/Free Full Text]
  9. Sims,P., Grover,P.L., Swaisland,A., Pal,K. and Hewer,A. (1974) Metabolic activation of benzo[a]pyrene proceeds by a diol-epoxide. Nature, 252, 326–328.[ISI][Medline]
  10. Sims,P. (1975) Epoxides as reactive intermediates in aromatic hydrocarbon metabolism. Biochem. Soc. Trans., 3, 59–62.[ISI][Medline]
  11. Yang,S.K., McCourt,D.W., Leutz,J.C. and Gelboin,H.V. (1977) Benzo[a]pyrene diol-epoxides: mechanisms of enzymatic formation and optically active intermediates. Science, 196, 1199–1201.[ISI][Medline]
  12. Eberhart,J., Coffing,S.L., Anderson,J.N., Marcus,C., Kalogeris,T.J., Baird,W.M., Park,S.S. and Gelboin,H.V. (1992) The time-dependent increase in the binding of benzo[a]pyrene to DNA through (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide in primary rat hepatocyte cultures results from induction of cytochrome P450IA1 by benzo[a]pyrene treatment. Carcinogenesis, 13, 297–301.[Abstract]
  13. Shimada,T., Martin,M.V., Pruess-Schwartz,D., Marnett,L.J. and Guengerich,F.P. (1989) Roles of individual human cytochrome P-450 enzymes in the bioactivation of benzo[a]pyrene, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons. Cancer Res., 49, 6304–6312.[Abstract]
  14. Vahakangas,K., Raunio,H., Pasanen,M., Sivonen,P., Park,S.S. and Gelboin,H.V. (1989) Comparison of the formation of benzo[a]pyrene diolepoxide–DNA adducts in vitro by rat and human microsomes: evidence for the involvement of P-450IA1 and P-450IA2. J. Biochem. Toxicol., 4, 79–86.[ISI][Medline]
  15. Sesardic,D., Pasanen,M., Pelkonen,O. and Boobis,A.R. (1990) Differential expression and regulation of members of the cytochrome P450IA gene subfamily in human tissues. Carcinogenesis, 11, 1183–1188.[Abstract]
  16. Sesardic,D., Cole,K.J., Edwards,R.J., Davies,D.S., Thomas,P.E., Levin, W. and Boobis,A.R. (1990) The inducibility and catalytic activity of cytochromes P450c (P450IA1) and P450d (P450IA2) in rat tissues. Biochem. Pharmacol., 39, 499–506.[ISI][Medline]
  17. Bartsch,H. and Hietanen,E. (1996) The role of individual susceptibility in cancer burden related to environmental exposure. Environ. Health Perspect., 104 (suppl. 3), 569–577.
  18. Winton,D.J., Blount,M.A. and Ponder,B.A.J. (1988) A clonal marker induced by mutation in mouse intestinal epithelium. Nature, 333, 463–466.[ISI][Medline]
  19. Winton,D.J., Gooderham,N.J., Boobis,A.R., Davies,D.S. and Ponder,B.A.J. (1990) Mutagenesis of mouse intestine in vivo using the Dlb-1 specific locus test: studies with 1,2-dimethylhydrazine, dimethylnitrosamine and the dietary mutagen 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline. Cancer Res., 50, 7992–7996.[Abstract]
  20. Tao,K., Urlando,C. and Heddle,J.A. (1993) Comparison of somatic mutation in a transgenic versus host locus. Proc. Natl Acad. Sci. USA, 90, 10681–10685.[Abstract/Free Full Text]
  21. Tao,K.S. and Heddle,J.A. (1994) The accumulation and persistence of somatic mutations in vivo. Mutagenesis, 9, 187–191.[Abstract]
  22. Winton,D.J., Peacock,J.H. and Ponder,B.A.J. (1989) Effect of gamma radiation at high- and low-dose rate on a novel in vivo mutation assay in mouse intestine. Mutagenesis, 4, 404–406.[Abstract]
  23. Winton,D.J., Gwynne,B. and Ponder,B.A.J. (1992) Derivation of a Dlb-1a homozygous mouse congenic to C57/BL6J. Mouse Genome, 90, 690–692.
  24. Brooks,R.A., Gooderham,N.J., Zhao,K., Edwards,R.J., Howard,L.A., Boobis,A.R. and Winton,D.J. (1994) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a potent mutagen in the mouse small intestine. Cancer Res., 54, 1665–1671.[Abstract]
  25. Edwards,R.J., Singleton,A.M., Murray,B.P., Davies,D.S. and Boobis,A.R. (1995) Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes. Biochem. Pharmacol., 49, 39–47.[ISI][Medline]
  26. Kohler,S.W., Scott Provst,G., Fieck,A., Kretz,P.L., Bullock,W.O., Sorge,J.A., Putman,D.L. and Short,M. (1991) Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc. Natl Acad. Sci. USA, 88, 7958–7962.[Abstract/Free Full Text]
  27. Forkert,P.G., Mirehouse-Brown,P., Park,S.S. and Gelboin,H.V. (1988) Distribution and induction sites of phenobarbital and 3 methylcholanthrene-inducible cytochromes P450 in murine liver: immunohistochemical localization with monoclonal antibodies. Mol. Pharmacol., 34, 736–743.[Abstract]
  28. Kimura,S., Gonzalez,F.J. and Nebert,D.W. (1986) Tissue-specific expression in the mouse dioxin-inducible P1450 and P3450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues. Mol. Cell. Biol., 6, 1471–1477.[ISI][Medline]
  29. Rich,K.J., Foster,J.R., Edwards,R.J., Davies,D.S. and Boobis,A.R. (1993) Ontogenetic development of the distribution of constitutive and 3-methylcholanthrene-induced CYP1A1 and CYP1A2 in rabbit liver. J. Histochem. Cytochem., 41, 915–925.[Abstract/Free Full Text]
  30. Todorovic,R., Devanesan,P.D., Cavalieri,E.L., Rogan,E.G., Park,S.S. and Gelboin,H.V. (1991) A monoclonal antibody to rat liver cytochrome P450 IIC11 strongly and regiospecifically inhibits constitutive benzo[a]pyrene metabolism and DNA binding. Mol. Carcinogen., 4, 308–314.[ISI][Medline]
  31. Byczkowski,J.Z. and Kulkarni,A.P. (1989) Lipoxygenase-catalysed epoxidation of benzo[a]pyrene-7,8-dihydrodiol. Biochem. Biophys. Res. Commun., 159, 1199–1205.[ISI][Medline]
  32. Dix,T.A. and Marnett,L.J. (1981) Free radical epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by hematin and polyunsaturated fatty acid hydroperoxides. J. Am. Chem. Soc., 103, 6744–6746.[ISI]
  33. Reed,G.A. and Marnett,L.J. (1982) Metabolism and activation of 7,8-dihydrobenzo[a]pyrene during prostoglandin biosynthesis: intermediacy of a bay-region epoxide. J. Biol. Chem., 257, 11368–11376.[Abstract/Free Full Text]
  34. Sivarajah,K., Lasker,J.M. and Eling,T.E. (1981) Prostaglandin synthetase-dependent cooxidation of (+)-benzo[a]pyrene-7,8-dihydrodiol by human lung and other mammalian tissues. Cancer Res., 41, 1834–1839.[ISI][Medline]
  35. Boobis,A.R., Nebert,D.W. and Felton,J.S. (1977) Comparison of beta-naphthoflavone and 3-methylcholanthrene as inducers of hepatic cytochrome(s) P-448 and aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. Mol. Pharmacol., 13, 259–268.[Abstract]
  36. Lippmann,M., Yeates,D.B. and Albert,R.A. (1980) Deposition, retention, and clearance of inhaled particles. Br. J. Ind. Med., 37, 337–362.[ISI][Medline]
  37. Hattemer-Frey,H.A. and Travis,C.C. (1991) Benzo[a]pyrene: environmental partitioning and human exposure. Toxicol. Ind. Health, 7, 141–157.[ISI][Medline]
  38. Stavric,B, and Klassen,R. (1994) Dietary effects on the uptake of benzo[a]pyrene. Food Chem. Toxicol., 32, 727–734.[ISI][Medline]
Received June 17, 1998; revised September 4, 1998; accepted September 4, 1998.