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

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 markedlydifferent effects on the number of Dlb-1 mutations and the patternof induction of cytochrome P-4501A1 (CYP1A1). In Ahr-responsiveanimals i.p. injection resulted in marked induction in cryptcells along the length of the small intestine, with some inductionin the villus cells. In contrast, after oral administration,CYP1A1 induction was evident only in the villus cells, and thisdeclined distally. The intensity and speed of induction in Ahr-responsiveanimals was such that the genotoxic effect of a single injectionof B[a]P could not be augmented by prior treatment with non-genotoxicinducers such as ß-napthoflavone and TCDD. Oral B[a]Ptreatment resulted in a decrease in the number of mutationswhen compared with the i.p. route. Studies in congenic Ahr-non-responsiveversus Ahr-responsive mice indicated that induction of CYP1A1was associated with increased numbers of Dlb-1 mutations. Mutationinduction in Ahr-non-responsive mice in the absence of detectableCYP1A1 in either liver or small intestine indicates that anappreciable portion of B[a]P activation to a genotoxin mustbe by other than a CYP1A1 mediated route. These data show thatB[a]P is a potent small intestinal mutagen at the Dlb-1 locus.     

Cytotoxicity and genotoxicity of ({+/-})-benzo[a]pyrene-trans-7,8-dihydrodiol in CYP1A1-expressing human fibroblasts quantitatively correlate with CYP1A1 expression level

Cytochrome P450 1A1 (CYP1A1) activity is associated with increasedsusceptibility to lung cancer induced by polycyclic aromatichydrocarbons such as benzo[a]pyrene (BP). In non-hepatic humantissues, CYP1A1 is the principal enzyme responsible for themetabolic activation of the proximate BP mutagenic metabolite,(–)-benzo[a]pyrene-trans-7,8-dihydrodiol, to (+)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide,the ultimate BP mutagen. We have genetically engineered bothDNA repair-deficient (xeroderma pigmentosum group A) and DNArepair-proficient human skin fibroblasts to express human CYP1A1under control of the inducible mouse metallothionein-I promoter.CYP1A1 activity was induced by CdSO₄ and monitored by followingthe O-deethylation of ethoxy fluorescein ethyl ester or of 7-ethoxyresorufin.Induced CYP1A1 activities were similar in both cell lines andwere dependent on CdSO₄ concentration and induction time. MaximalCYP1A1 activities were obtained in 4–6 h with 5–7µM CdSO₄. BPD-induced cytotoxicity and hypoxanthine phosphoribosyltransferase mutagenicity were both quantitatively correlatedwith the level of CYP1A1 activity and were greater in DNA repair-deficientcells than in DNA repair-proficient cells. The results suggestthat modestly induced CYP1A1 activity is a risk factor in polycyclicaromatic hydrocarbon-induced carcinogenesis.     

Effects of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on human small airway epithelial cells and the protective effects of myo-inositol

Benzo[a]pyrene (B[a]P), a tobacco-derived carcinogen, induceslung tumors in rodents through its carcinogenic metabolite,anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(B[a]PDE). Tumorigenesis is inhibited by dietary myo-inositolin the post-initiation phase. However, little is known abouthow B[a]PDE and myo-inositol affect normal human lung cells.We addressed this question using untransformed human small airwayepithelial (SAE) cells. SAE cell viability decreased <50%in parallel to an increase of apoptotic cells (>20%) 2 daysafter the cells were treated for 1 h with B[a]PDE (>100 nM).In contrast, the cell number and viability were not alteredin A549 human lung cancer cells by B[a]PDE treatment up to 10µM with <5% apoptotic cells and <10 U/l LDH in themedium. SAE cells retain the features of basal cells in serum-free,low Ca²⁺ (4 nM) medium up to 4–5 passages, but in serum-supplementedor serum-free, high Ca²⁺(1 mM) cultures, they differentiateinto non-ciliated epithelial cells expressing Clara cell secretoryprotein (CCSP). A non-toxic, physiologically relevant dose ofB[a]PDE (1 nM) partially inhibited serum and Ca²⁺-induced SAEcell differentiation. This effect was abolished by wortmannin,a phosphatidylinositol-3 kinase (PI-3K) inhibitor, and PD98059,a mitogen activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor,but not by SB202190, a p38 MAPK inhibitor, or melittin, a proteinkinase C inhibitor. Myo-inositol (10–100 µM) didnot alter growth or differentiation of untreated SAE or A549cells, but reversed the inhibitory effect of B[a]PDE on serumand Ca²⁺-induced SAE cell differentiation when supplementedto the culture after B[a]PDE treatment. This myo-inositol actionwas not altered by PD98059, wortmannin or melittin, but waspartially suppressed by SB202190. Collectively, these resultsindicate that B[a]PDE inhibits serum-induced SAE cell differentiation,possibly involving activating signals through a PI-3K/MEK1 mediatedMAPK pathway, whereas myo-inositol protects SAE cells againstthis inhibitory effect of B[a]PDE perhaps through both PI-3K/MEK1and p38 MAPK pathways.     

In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene

Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclicaromatic hydrocarbon with potent mutagenic and carcinogenicactivity. In order to acquire an understanding of the mutagenicpathways of CPP, we studied mutations induced by this chemicalin human cells. Four independent cultures of a human cell lineexpressing cytochrome P450 CYP1A1 (cell line MCL-5) were treatedwith CPP, and mutants at the hypoxanthine phosphoribosyltransferase(HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance.The kinds and positions of the mutations were analyzed usingthe combination of high-fidelity polymerase chain reaction (hifi-PCR)and denaturing gradient gel electrophoresis (DGGE). The thirdexon of the HPRT gene was amplified from the 6TG-resistant cellsusing the hifi-PCR and the amplified fragment was subsequentlyanalyzed by DGGE to separate mutant sequences from the wild-typesequence. Mutant bands were excised from the gel, amplifiedusing PCR and sequenced. Sixteen different mutations were identifiedand consisted mostly of the G to T and A to T transversions.Other mutations identified included G to A and A to G transitions,a G to C transversion, and a single G deletion. Of these mutations,six occurred within a run of six guanines. The predominanceof transversions involving a guanine or an adenine observedwith CPP is similar to the data previously reported for theracemic mixture of benzo[a]pyrene (B[a]P), suggesting that themechanisms of mutation induced by CPP may be similar to thoseinduced by B[a]P.     

Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants

Cytochrome P450 1A1 (CYP1A1) plays a key role in the metabolism of carcinogens, such as benzo[a]pyrene (B[a]P) and metabolites to ultimate carcinogens. Three human allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were coexpressed by coinfection of baculovirus-infected insect cells with human NADPH-P450 reductase. These recombinant enzymes (in microsomal membranes) were used to analyze whether CYP1A1 polymorphisms affect catalytic activities towards B[a]P and B[a]P-7,8-dihydrodiol. The complete spectrum of phase I metabolites, including the tetrahydrotetrols resulting from hydrolysis of the ultimate carcinogen, B[a]P-7,8-dihydrodiol-9,10-epoxide, was examined by HPLC. Wild-type enzyme showed the highest total metabolism of B[a]P, CYP1A1.2 was approximately 50%, and CYP1A1.4 approximately 70%. Km values for all metabolites with CYP1A1.2 were generally significantly lower than with wild-type enzyme (e.g. B[a]P-7,8-diol formation: 13.8 microM for wild-type, 3.5 microM for CYP1A1.2 and 7.7 microM for CYP1A1.4). Addition of epoxide hydrolase markedly increases the relative diol-to-phenol activities by all three variants. However, CYP1A1.4 exhibits the greatest efficiency to produce diol species. Each variant produced the diol epoxides from B[a]P-7,8-dihydrodiol. CYP1A1.1 exhibited with 10.4 pmol/min/pmol CYP1A1 the greatest total rate for 7,8-diol metabolites followed by CYP1A1.2 (7.2 pmol/min/pmol CYP1A1) and CYP1A1.4 (5.5 pmol/min/pmol CYP1A1). All enzyme variants produced about three times more diol epoxide 2-derived metabolites than diol epoxide 1-derived ones, whereby both rare allelic variants exhibited statistically significantly increased formation of diol epoxide 2. This study showed that the three CYP1A1 variants had different enzyme kinetics properties to produce both the diol metabolites from B[a]P and the ultimate mutagenic species diol epoxide 2 from B[a]P-7,8-dihydrodiol, which must be considered in the evaluation of individual susceptibility to cancer.     

Identification of benzo[a]pyrene-7,8-dione as an authentic metabolite of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in isolated rat hepatocytes

Dihydrodiol dehydrogenase (DD) has been shown to catalyze theoxidation of (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-diol) to yield benzo[a]pyrene-7,8-dione (BPQ) in uninducedfortified rat liver S100 fractions but the formation of BPQhas not been observed in whole cells. In these studies [³H]BP-diolwas incubated with isolated hepatocytes from uninduced ratsfor 0–20 min at 37°C. Organic-extractable radioactivityin the cell media accounted for 20% of the total [³H]BP-dioladded. Reverse phase (RP)-HPLC analysis of this fraction revealedthe formation of an unknown metabolite that co-chromatographedwith an authentic synthetic standard of BPQ. The identity ofthe unknown metabolite was further established by: (i) co-chromatographywith synthetic BPQ under both RP- and normal phase-HPLC conditionsusing diode array detection, which indicated that the metaboliteshared UV/ vis spectral identity with standard BPQ; and by (ii)electron impact mass spectrometry of the unknown metabolitewhich gave the same parent and fragment ions as the syntheticstandard. The formation of BPQ by isolated hepatocytes was foundto be 0.50 nmol/3x10⁶ cells/10 min, and represented 7% of thetotal organic-soluble metabolites in the extracellular media.Its formation was abolished by the addition of indomethacin,a competitive inhibitor of DD, indicating that this enzyme wasresponsible for BPQ formation. Other organic-soluble metabolitesformed corresponded to BP-tetraols (hydrolysis products of theanti-and syn-diol epoxides). Examination of the aqueous phaseof the extracellular media indicated that a large portion ofBP-diol was converted to glucuronide and sulfate conjugates.Under the conditions employed BP-tetraols and BPQ were formedto an equal extent implying that in hepatocytes isolated fromuninduced rats, DD and CYP1A1 contributed equally to the metabolismof BP-diol.     

Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of benzo[a]pyrene

3-Hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol)wag isolated from arylsulfatase/ß-glucuronidase-treatedbile of rats to which 3-hydroxybenzo[a]pyrene (3-OH-BP) hasbeen administered. This triol was investigated for mutagenicityin Salmonella typhimurium (reversion to histidine prototrophyof strains TA 97, TA 98, TA 100 and TA 1537) and in V79 Chinesehamster cells (acquisition of resistance to 6-thioguanine).When no exogenous metabolizing system was added the triol wasinactive, while 3-OH-BP showed weak mutagenic effects with allfour bacterial strains. In the presence of NADPH-fortified postmitochondrialsupernatant fraction (S9 mix) of liver homogenate from Aroclor1254-treated rats, the mutagenicity of 3-OH-BP was potentiated,and the triol was activated to a mutagen(s). In the presenceof S9 mix, the triol was 5—18 times more mutagenic than3-OH-BP in strains TA 97, TA 100 and TA 1537, but both compoundsshowed similar mutagenic potencies with strain TA 98. Thesestrain differences strongly suggest that the mutagenicity of3-OH-BP in the S9 mix-mediated test was not exclusively dueto metabolites of 3-OH-BP-7, 8-diol. Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol), like the triol, showed mutagenic effects onlyin the presence of S9 mix. Strain TA 1537 was reverted by thetriol but not by the diol. In the other bacterial strains thediol was more mutagenic than the triol, the difference in potencybeing largest in strain TA 100 (2.5-to 10-fold, depending onthe experimental conditions). In V79 cells, the diol was a potentmutagen, while the triol showed only very weak mutagenic effects.However the triol was more cytotoxic than the diol. High cytotoxicityof the triol was observed even in the absence of S9 mix. Theresults of the present study demonstrate that metabolites of3-OH-BP-7, 8-diol) are biologically-active derivatives of benzo[a]pyrene.Comparison of the mutagenic effectiveness in different bacterialstrains also reveals that metabolites of 3-OH-BP-7, 8-diol andof BP-7, 8-diol substantially differ in the kind of geneticalterations they evoke.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.      

Tumor-initiating activity and carcinogenicity of dibenzo[a,l]pyrene versus 7, 12-dimethylbenz[a]anthracene and benzo[a]pyrene at low doses in mouse skin

Dibenzo[a,l]pyrene (DB[a,l]P) is an extremely potent carcinogenthat may be present in environmental samples. Dose-responsestudies were conducted at low doses in mouse skin by initiation-promotionand repeated application to compare its activity to that of7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P),DB[a,l]P-8,9-dihydrodiol and DB[a,l]P-11,12-dihydrodiol. FemaleSENCAR mice were initiated with 1 or 0.25 nmol of DB[a, l]P,DMBA, B[a]P or DB[a,l]P-11,12-dihydrodiol and promoted withphorbol ester acetate. At 1 nmol, DB[a, l]P induced 2.6 tumors/mouse,whereas DB[a,l]P-11,12-dihydrodiol and DMBA induced 0.17 and0.29 tumors/mouse respectively. At the low dose, DB[a,l]P induced0.79 tumors/mouse, but the other two compounds were virtuallyinactive. B[a]P, tested only at 1 nmol, was inactive. Thesethree compounds, as well as DB[a,l]P-8,9-dihydrodiol, were testedby repeated application twice weekly for 40 weeks at 1 and 4nmol per dose. In addition, DB[a,l]P, DMBA and B[a]P were alsotested at 8 nmol. At 8 and 4 nmol, DB[a,l]P induced malignanttumors in 91 and 70% of mice respectively. At 4 nmol DB[a, l]P-11,12-dihydrodiolelicited only benign tumors in 36% of mice. At 4 nmol DMBA inducedtwo carcinomas in one mouse and at 8 nmol it induced one papillomaand one sebaceous gland adenoma. B[a]P and DB[a,l]P-8,9-dihydrodiolwere inactive at all doses tested. These results demonstratethat DB[a, l]P is a much more potent carcinogen than DMBA, thearomatic hydrocarbon previously considered to be the most potent.Combination of these results with previous comparisons of DB[a,l]P,DB[a,l]P-11,12-dihydrodiol, DMBA and B[a]P at higher doses (E.L.Cavalieri et al. (1991) Carcinogenesis, 12, 1939–1944)shows clearly the interference of toxicity with the tumorigenicityof DB[a,l]P and its 11,12-dihydrodiol.     

Rosemary components inhibit benzo[a]pyrene-induced genotoxicity in human bronchial cells

The commonly used spice and flavouring agent, rosemary, derivedfrom the leaves of the plant Rosmarinus officinalis L., displaysantioxidant properties in foods and in biological systems. Moreover,in animal models rosemary components were found to inhibit theinitiation and tumour promotion phases of carcinogenesis. Inthis work, we studied the mechanisms by which rosemary componentsblock initiation of carcinogenesis by the procarcinogen benzo[a]pyrene(B[a]P) in human bronchial epithelial cells (BEAS-2B). Wholerosemary extract (6 µg/ml) or an equivalent concentrationof its most potent antioxidant constituents, carnosol or carnosicacid, inhibited DNA adduct formation by 80% after 6 h co-incubationwith 1.5 µM B[a]P. Under similar conditions, cytochromeP450 (CYP) 1A1 mRNA expression was 50% lower in the presenceof rosemary components, and CYP1A1 activity was inhibited 70–90%.The observed reduction of DNA adduct formation by rosemary componentsmay mostly result from the inhibition of the activation of benzo[a]pyreneto its ultimate metabolites. Carnosol also affected expressionof the phase II enzyme glutathione-S-transferase which is knownto detoxify the proximate carcinogenic metabolite of B[a]P.Treatment of BEAS-2B cells with carnosol (1 µg/ml) for24 h resulted in a 3- to 4-fold induction of GST     

CYP1A1 genotype-selective inhibition of benzo[a]pyrene activation by quercetin

Epidemiological studies suggest that food rich in quercetin and naringin may protect against certain types of lung cancer, and that genotype dependent inhibition of cytochrome P450 1A1 (CYP1A1)-mediated bioactivation of procarcinogens could be the underlying mechanism. We studied the inhibitory effects of quercetin and naringin on the terminal bioactivation step of benzo[a]pyrene (B[a]P), a member of the major class of lung carcinogens. This reaction (epoxidation of (+/-)-trans-7,8-dihydro-7,8-dihydroxy-B[a]P to the ultimate carcinogenic product, (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2)) was examined using three of the most common allelic variants of human CYP1A1, namely wild-type CYP1A1.1, CYP1A1.2, and CYP1A1.4. Quercetin potently inhibited diolepoxide 2 formation by all CYP1A1 types with IC(50) values between 1.6 and 7.0 microM. The differences between the wild-type enzyme and the variants were statistically highly significant (P < 0.01). Enzyme kinetics revealed quercetin as a mixed-type inhibitor of CYP1A1.1, CYP1A1.2, and CYP1A1.4 with K(i) values of 2.0, 6.4, and 9.3 microM, respectively. Naringin inhibited diolepoxide 2 formation only slightly. Our data support the hypothesis that quercetin may have a stronger chemopreventive effect in individuals carrying wild-type compared with variant CYP1A1 genes. Future studies should consider the influence of P450 polymorphisms on both procarcinogen activation and its inhibition to facilitate the development of genotype-specific chemoprevention regimes.     

Transformation of rat tracheal epithelial cells by benzo[a]pyrene and its metabolites

Benzo[a]pyrene (B[a]P) and its 7,8-dihydrodiol form were metabolically activated to malignantly transform a non-tumorigenic rat tracheal epithelial cell line in culture. The proximal carcinogenic metabolite of B[a]P, diolepoxide I, was more efficient in transforming the epithelial cell line than the intermediate or parent compound, even at a 50- or 100-fold lower concentration, respectively. Inoculations of the transformed cell lines into immunosuppressed isogenic recipients produced differentiated carcinomas similar to those which occur in humans.     

Secondary metabolites of benzo[a]pyrene: 3-hydroxy-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, a biliary metabolite of 3-hydroxybenzo[a]pyrene in the rat

Rats administered 3-hydroxybenzo[a]pyrene (50 mg/kg, i.p.),excrete via the bile metabolites which, after treatment withß-glucuronidase and aryl sulphatase, yield, in additionto 3-hydroxybenzo[a]pyrene, 3-hydroxy-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene(3-OH-BP-7, 8-diol) and a minor, highly labile, metabolite tentativelyidentified as 3,5-dihydroxybenzo[a]pyrene. These novel metabolitesare readily isolated in a pure state via preparative layer chromatography.The structure of the 3-OH-BP-7, 8-diol was revealed by its u.v.,proton magnetic resonance and mass spectral properties. Itshydroxyl functions are in a predominantly quasi-diequatorialconformation.     

DNA adduct formation and repair in hamster and rat tracheas exposed to benzo[a]pyrene in organ culture

Syrian golden hamsters are much more susceptible than Wistarrats to the induction of tracheal tumors by benzo[a]pyrene (B[a]P).To investigate whether this difference is reflected in the patternof DNA adduct induction and removal, tracheas from either specieswere isolated and exposed to B [a]P (5 µg/ml) in organculture. At various time- points B[a]P-DNA adducts were quantifiedby ³²P-postlabeling; unscheduled DNA synthesis (UDS) and cellproliferation were determined by [³H]thymidine incorporationduring the 18 h before sampling. In an induction-repair experimenttracheas were exposed to B[a]P for 2 days, and cultured foranother 4 days without B[a]P After 2 days of exposure totalB[a]P-DNA adduct levels were 10 times higher in hamster comparedto rat tracheas. In hamster tracheas one major adduct was formed(95%), namely the adduct between (+)-anti-benzo[a]pyrene diolepoxideand deoxy guanosine (BPDE-N²dG). In rat tracheas BPDE-N²dG comprised60% of the total B[a]P-DNA adduct level. The other major adductfound in rat tracheas is probably derived from interaction ofsyn-BPDE and deoxyadenosine. During exposure to B[a]P in hamstersthe adduct level increased to 36 ± 19 adducts/10⁶ nucleotides(add/10⁶n) on day 2. Two days after removal of B[a]P-the B[a]P-DNAadduct level had decreased to 60% of that on day 2; there wasno further decrease in the B[a]P-DNA adduct level, despite considerablecell proliferation at the end of the 6 day culture period. UDSincreased during exposure to B[a]P and decreased after removalof B[a]P. In rats removal of B[a]P did not lead to a decreasein the B[a]P adduct level, which agreed with the observed absenceof UDS. In a second experiment tracheas were exposed to B[a]Pcontinuously for 15 days. In hamster tracheas the total B[a]P-DNAadduct level increased from 11 ± 0.7 add/10⁶n after 1day of exposure to 105 ± 2 add/10 after 15 days; alsoUDS increased with increasing exposure until day 11. Cell proliferationwas low at the end of the culture period. In rat tracheas noprogressive increase in the B[a]P-DNA adduct level was seen,UDS was not increased and cell proliferation had increased significantlyat the end of the exposure period. The extent of adduct inductionin the trachea of the two species corresponded with the differentsusceptibilities to B[a]P-induced tumor formation.     

Comparative tumorigenicity of benzo[a]pyrene, 1-nitropyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine administered by gavage to female CD rats

Agents that are ubiquitous in the environment and are knowninducers of mammary cancer in rodents can be regarded as potentialcauses of human cancer and need to be evaluated more completely.Therefore, the purpose of this study was to determine underidentical conditions the relative carcinogenic potency in themammary glands of rats of benzo[a]pyrene (B[a]P), 1-nitropyrene(1-NP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyidne (PhIP).Thirty-day-old female CD rats were gavaged once weekly for 8weeks with B[a]P, 1-NP or PhIP. Each compound was given at 50µmol/rat/week in 0.5 ml trioctanoin for a total dose of400 µmoul/rat. Forty-one weeks after the last carcinogenadministration, rats were killed. In the 1-NP-treated rats,treatment elicited primarily benign tumors. In contrast, theB[a]P- and PhIP-treated rats developed both malignant and benigntumors. The incidence of adenocarcinomas in rats treated withB[a]P or PhIP was comparable and significantly higher than thatin animals receiving trioctanoin only. The incidence of benigntumors (fibroadenomas, desmoplastic adenomas and adenomas) observedin animals treated with B[a]P or 1-NP was comparable and significantlyhigher than that in animals given PhIP or trioctanoin. Thisis the first report describing the carcinogenic activity ofPhIP, given by gavage, in the mammary gland of CD rats and rankingthe carcinogenic potency observed under identical conditions,of three agents (B[a]P     

Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts and increased p53 protein in mouse skin

p53 protein expression has been shown to increase in responseto DNA damage in cell culture systems. We have Studied p53 expressionand benzo[a]pyrene (B[a]P) -induced DNA-damage in the form ofbenzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts as measuredby synchronous fluorescence spectrophotometry (SFS) in B[a]P-treatedC57BL/6 mouse skin. Polyclonal murine antibody CM5, which iscomparable to human CM1, detecting both wild-type and mutatedprotein, was used. BPDE-DNA adducts reached their maximum at24 h after all dosage regimens, but were very well detectablealso at 12 and 48 h after the treatment, while no adducts weremeasurable at 1 week and thereafter. P53 expression was seenin 9/17(53%) skin samples from mouse treated with 500 µgof B[a]P 12–48 h after the treatment, while all 25 (100%)cases of similarly treated mouse skins were negative after 30weeks of the treatment. Only one positive sample of total 11was found among mice treated with repeated 62.5 µg dosesand this was 24 h after the last treatment. After one 62.5 µgdose all mice were negative. This is the first report of anassociation of p53 protein with DNA damage in vivo and givessupport for the putative function of p53 in cellular defensemachinery towards chemical damage.     

Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1

Transgenic cell lines were constructed to study dynamic competition between activation versus detoxification of benzo[a]pyrene (B[a]P) and its metabolites. Transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone, or expressing hCYP1A1 in combination with human glutathione S-transferase P1 (hGSTP1), were used to determine how effectively GST protects against macromolecular damage or mutagenicity of B[a]P or its enantiomeric dihydrodiol metabolites (+)-benzo[a]pyrene-7,8-dihydrodiol [(+)B[a]P-7,8-diol] and (-)-benzo[a]pyrene-7,8-dihydrodiol [(-)-B[a]P-7,8-diol]. Mutagenicity of B[a]P at the hprt locus was dose- and time-dependent in cells that expressed hCYP1A1. Mutagenicity was reduced in cells further modified to co-express hGSTP1. Dose-response and time-course studies indicated that mutagenicity was reduced up to 3-fold by hGSTP1 expression, compared with cells expressing hCYP1A1 alone. Mutagenicity induced by the B[a]P 7,8-dihydrodiols was also dose-dependent, and was reduced 2- to 5-fold by hGSTP1. Expression of hGSTP1 reduced B[a]P adducts in total cellular macromolecules by 3.8-fold, which correlated with the reduction in B[a]P mutagenicity and with reduction in the formation of the proximate metabolite B[a]P 7,8-dihydrodiols from B[a]P. However, measurement of total B[a]P metabolites bound to DNA isolated from cells incubated with [3H]-B[a]P revealed only 12, 33 and 24% reduction at 12, 24 and 48 h, respectively, by GSTP1 expression. Nevertheless, (32)P-post-labeling analysis demonstrated nearly total prevention of the known B[a]P-DNA adduct, N2-guanine-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), in cells co-expressing hGSTP1. This adduct, thought to be the most mutagenic of the stable B[a]P adducts, accounts for 15% or less of the total DNA adducts observed. These results indicate that the reduction in hCYP1A1-mediated B[a]P mutagenesis by hGSTP1 is probably largely due to prevention of the N2-guanine-BPDE adduct. However, the significant fraction (30-40%) of this mutagenesis and the majority of the total DNA binding that are not prevented together suggest formation by hCYP1A1 of a subset of mutagenic metabolites of B[a]P that are not effectively detoxified by hGSTP1.     

Comparison of DNA adduct formation in mice fed coal tar or benzo[a]pyrene

Coal tar is a complex mixture containing hundreds of compounds,including the carcinogenic poly cyclic aromatic hydrocarbon,benzo[a]pyrene. In order to compare the metabolic activationof a single carcinogen versus a complex mixture containing thecarcinogen, we determined the DNA adduct profiles in B6C3F₁mice fed doses of coal tar or benzo[a]pyrene at concentrationscorresponding to the amount of benzo[a]pyrene found in the respectivecoal tar treatments. DNA adduct formation was quantified inliver, lungs and forestomach by ³²P-postlabeling and was foundto increase as a function of dose in each tissue with both coaltar and benzo[a]pyrene. In mice fed benzo[a]pyrene, a majoradduct was detected with the same elution characteristics byTLC and HPLC as the major adduct, 10ß-(deoxyguanosin-N²-yl)-7ß,8     

Persistence of benzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene in Fischer 344 rats: time distribution of total metabolites in blood, urine and feces

A comparison of the rates of elimination of [³H]benzo[a] pyrene(BaP) and 7,8-dihydro-7,8-diol-[³H]benzo[a]pyrene (BPD), aftersubcutaneous injection into Fischer 344 rats, shows they areboth eliminated at about the same rates and with the same patternover at least 7 days post-exposure. The end-rate of combinedurinary and fecal excretion was {small tilde}40 nmol/day. About20% of the injected BaP and {small tilde}3% of the injectedBPD remained at the site of injection for at least 9 days. Theremainder was distributed throughout the animal. If the rateof excretion continued at the observed steady-state rates, theBaP and BPD could persist for up to 40 days for each milligramof injected substance. The concentration of excretion productswere highest during day 1 and day 2 following exposure, decreasedexponentially to a concentration of {small tilde}0.5 µM(mixed metabolites) by day 5 following exposure, and then continuedto be excreted at that rate. Feces contained the highest totalamounts of radioactivity, which were {small tilde}2- to 4-foldhigher than the amounts in urine and {small tilde}15- to 50-foldhigher than in total blood. The conversion of organic ³H to³H₂O during the experimental period indicates.that whole-bodyphenol(quinone) formation was significant for BaP metabolism,but was much less for BPD metabolism. When BaP was injected,both blood and urine contained water-soluble, volatile tritiumcounts (³H₂O). Injection of BPD resulted in volatile ³H₂O inurine but not in blood. The persistence of BaP and BPD metabolitesin skin, blood, urine and feces compartments indicates thereis a substantial reservoir of the chemical(s) that could beused to replenish repaired or discarded DNA adducts.