Effect of a standardized complex mixture derived from coal tar on the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons in human cells in culture

A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, standard reference material (SRM) 1597, has been shown to initiate tumor formation in mouse initiation-promotion assays in our laboratory [(2001) Carcinogenesis 22 (7), 1077-1086]. To determine the effects of SRM 1597 on PAH activation in human cells, we investigated the PAH-DNA adduct formation in the human mammary carcinoma-derived cell line MCF-7. We examined the effects of SRM 1597 on the metabolic activation to DNA binding derivatives of two carcinogenic PAHs, the bay region containing benzo[a]pyrene (B[a]P) and the more carcinogenic fjord region containing dibenzo[a,l]pyrene (DB[a,l]P). PAH-DNA adduct analysis by 33P-postlabeling and reversed phase high-performance liquid chromatography revealed a significant decrease in the levels of both B[a]P and DB[a,l]P DNA adduct formation on cotreatment with SRM 1597 in comparison to cells exposed to B[a]P or DB[a,l]P alone. However, the inhibition of PAH-DNA adduct formation only occurred within the first 48 h of exposure in cells cotreated with SRM 1597 and B[a]P. In contrast, SRM 1597 significantly inhibited the level of DB[a,l]P DNA adducts throughout the 120 h of exposure. Induction of human cytochrome P450 (P450) enzymes 1A1 and P4501B1 on treatment with SRM 1597 was observed by immunoblots. These results suggest that the important factors in determining the carcinogenic activity of PAH within a complex mixture would depend on the ability of other components of the mixture to promote or inhibit the activation of carcinogenic PAH by the induction of P450 enzymes followed by the formation of DNA adducts.     

Urban dust particulate matter alters PAH-induced carcinogenesis by inhibition of CYP1A1 and CYP1B1.

The polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are well-studied environmental carcinogens, however, their potency within a complex mixture is uncertain. We investigated the influence of urban dust particulate matter (UDPM) on the bioactivation and tumor initiation of B[a]P and DB[a,l]P in an initiation-promotion tumorigenesis model. SENCAR mice were treated topically with UDPM or in combination with B[a]P or DB[a,l]P, followed by weekly application of the promoter 12-O-tetradecanoylphorbol-13 acetate. UDPM exhibited weak tumor-initiating activity but significantly delayed the onset of B[a]P-induced tumor initiation by two-fold. When cotreated with UDPM, DB[a,l]P-treated animals displayed no significant difference in tumor-initiating activity, compared with DB[a,l]P alone. Tumor initiation correlated with PAH-DNA adducts, as detected by (33)P-postlabeling and reversed-phase high-performance liquid chromatography. Induction of cytochrome P450 (CYP)1A1 and 1B1 proteins was also detected following UDPM treatment or cotreatment with B[a]P or DB[a,l]P, indicating PAH bioactivation. Further genotoxicity analyses by the comet assay revealed that cotreatment of UDPM plus B[a]P or DB[a,l]P resulted in increased DNA strand breaks, compared with PAH treatment alone. The metabolizing activities of CYP1A1 and CYP1B1, as measured by the 7-ethoxyresorufin O-deethylation (EROD) assay, revealed that UDPM noncompetitively inhibited CYP1A1 and CYP1B1 EROD activity in a dose-dependent manner. Overall, these data suggest that components within complex mixtures can alter PAH-induced carcinogenesis by inhibiting CYP bioactivation and influence other genotoxic effects, such as oxidative DNA damage. These data further suggest that in addition to the levels of potent PAH, the effects of other mixture components must be considered when predicting human cancer risk.     

DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by 32P-postlabeling

 Numerous wood preserving waste (WPW) sites in the United States pose genotoxic hazards. WPWs consist of complex mixtures containing toxic, including genotoxic, compounds which are derived from the preservatives coal tar creosote and pentachlorophenol (PCP) and other polychlorinated aromatics. The genotoxicity of WPW extracts, which has not been tested in mammals, cannot be evaluated on the basis of data for individual components because of possible compound interactions. Therefore, whole extracts need to be assayed. ³²P-postlabeling represents a powerful tool to determine DNA adduct formation by complex genotoxic mixtures, such as cigarette smoke, diesel exhaust, and coke oven and foundry emissions in experimental animals and humans. In the present study, a mouse bioassay was used in combination with ³²P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6×10⁶ DNA nucleotides in skin (both extracts) and one adduct in 3.2×10⁷ or 1.2×10⁷ DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the ³²P-labeled derivative of the reaction product of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) with N ² of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. The benzo[a]pyrene adduct levels in the five tissues correlated linearly with total adduct levels and thus represented a surrogate for the latter. Overall, the results suggest that DNA adducts in mouse tissues, as analyzed by ³²P-postlabeling, are suitable biomarkers and dosimeters of the genotoxicity of WPW extracts. Received: 26 September 1995/Accepted: 7 December 1995     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)-DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P-DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [³H] B[a]P, but increased the cellular discharge of [³H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20-500 μM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P-DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation.     

Influence of the metabolic properties of human cells on the kinetic of formation of the major benzo[a]pyrene DNA adducts

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. Some of them, including benzo[a]pyrene (B[a]P), are tumorigenic due to their ability to generate DNA adducts. In order to define potential biomarkers of B[a]P exposure, the aim of the study was to identify the major stable DNA adducts in B[a]P-treated human cells. The role played by cellular metabolism on the nature and frequency of the DNA lesions was investigated using keratinocytes (HaCat) and actively metabolizing hepatocytes (HepG2) cell lines. Quantification of DNA damage was carried out by HPLC coupled to tandem mass spectrometry, a sensitive method making possible the selective detection of the different potential stable DNA adducts of B[a]P. These include two adducts of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) pathway and three adducts of the radical cation pathway. The results indicate that incubation of cells with B[a]P induces almost exclusively the formation of BPDE DNA adducts on purine bases. The amount of DNA adducts generated in hepatocytes was found to be two orders of magnitude higher than that measured in keratinocytes. Interestingly, the level of the DNA adducts produced in the cells incubated with (+/-)-anti-BPDE was similar in the two cell lines, indicating that the difference observed upon incubation with B[a]P could be attributed to different kinetics of B[a]P metabolism. The repair rate of BPDE DNA adducts was identical in the two cell lines with a half-life estimated to be around 20 h. These data support the use of the stable BPDE DNA adducts, as relevant biomarkers of exposure to B[a]P.     

Role of retinoic acid in the modulation of benzo(a)pyrene-DNA adducts in human hepatoma cells: implications for cancer prevention

Carcinogen-DNA adducts could lead to mutations in critical genes, eventually resulting in cancer. Many studies have shown that retinoic acid (RA) plays an important role in inducing cell apoptosis. Here we have tested the hypothesis that levels of carcinogen-DNA adducts can be diminished by DNA repair and/or by eliminating damaged cells through apoptosis. Our results showed that the levels of total DNA adducts in HepG2 cells treated with benzo(a)pyrene (BP, 2 μM) + RA (1 μM) were significantly reduced compared to those treated with BP only (P = 0.038). In order to understand the mechanism of attenuation of DNA adducts, further experiments were performed. Cells were treated with BP (4 μM) for 24 h to initiate DNA adduct formation, following which the medium containing BP was removed, and fresh medium containing 1 μM RA was added. The cells were harvested 24 h after RA treatment. Interestingly, the levels of total DNA adducts were lower in the BP/RA group (390 ± 34) than those in the BP/DMSO group (544 ± 33), P = 0.032. Analysis of cell apoptosis showed an increase in BP + RA group, compared to BP or RA only groups. Our results also indicated that attenuation of BP-DNA adducts by RA was not primarily due to its effects on CYP1A1 expression. In conclusion, our results suggest a mechanistic link between cellular apoptosis and DNA adduct formation, phenomena that play important roles in BP-mediated carcinogenesis. Furthermore, these results help understand the mechanisms of carcinogenesis, especially in relation to the chemopreventive properties of nutritional apoptosis inducers.     

Effect of a dioxin-like PCB (CB 126) on the biotransformation and genotoxicity of benzo

Two experiments were performed to study the interaction between benzo[a]pyrene (BaP) and the planar, dioxin-like PCB congener 3,3′,4,4′,5-pentachlorobiphenyl (CB 126) in the flatfish dab (Limanda limanda). The first experiment involved four groups. Group I was treated with 10 μg/kg CB 126, group II was treated with 2 mg/kg BaP, group III was first treated with 10 μg/kg CB 126 and exposed to 2 mg/kg BaP 6 days later, and group IV was a control group. The second experiment was similar, except that the BaP dosage level was increased to 50 mg/kg. Pre-treatment with 10 μg/kg of CB 126 always caused the induction of hepatic cytochrome P450 1A (CYP1A), as measured by significant increases of the model reaction 7-ethoxyresorufin-O-deethylase (EROD) in microsomal preparations. Treatment of dab with BaP caused a significant EROD induction at the 50 mg/kg, but not at the 2 mg/kg level. Concurrent with EROD induction by either CB 126 or 50 mg/kg BaP, was a significant change in the biliary metabolite pattern in favour of 1-hydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene and towards a lower fraction of the procarcinogen BaP-7,8-dihydrodiol (7,8-DIOL). Pre-treatment with CB 126 did not cause an increase of hepatic BaP DNA adducts formed after treatment with either 2 or 50 mg/kg BaP. Glutathione S-transferase (GST) activities remained also unaffected by any of the treatments. The results of this study suggest that the pattern of BaP metabolites in bile depends on the level of CYP1A induction. Moreover, the concurrence of a potent CYP1A inducer and BaP does not necessarily lead to an increase in DNA adduct levels in liver tissue. The observation that the level of 7,8-DIOL is decreased despite a higher (CYP1A mediated) EROD activity explains, at least in part, the lack of induction of DNA adducts.     

Induction of CYP1A1 and CYP1B1 in liver and lung by benzo(a)pyrene and 7,12-d imethylbenz(a)anthracene do not affect distribution of polycyclic hydrocarbons to target tissue: role of AhR and CYP1B1 in bone marrow cytotoxicity

Polycyclic aromatic hydrocarbons (PAHs) (50 mg/kg, i.p.) selectively deplete mouse bone marrow (BM) hematopoietic cells through a process that is dependent on CYP1B1. 7,12-dimethylbenz(a)anthracene (DMBA), which forms greater amounts of dihydrodiol-epoxide-DNA adducts in BM, is much more effective in depleting BM cells than benzo(a)pyrene (BP). BM toxicity by BP is restored in congenic mice expressing a weakly responsive aryl hydrocarbon receptor (AhR(d) replaces AhR(b)). BP strongly induces CYP1A1 around the hepatic vein whereas DMBA produces a weaker diffuse response, paralleling differences in CYP1A1 protein. These responses are absent in AhR(d) mice. BP and DMBA broadly and equally induce CYP1A1 in the lung, while CYP1B1 is induced in bronchial blood vessels. In sternum, CYP1B1 is induced in BM and white fat, whereas CYP1A1 is induced only in brown fat. BP and DMBA levels were similar within blood, lung, and BM and did not rise in AhR(d) mice. In liver, selective decrease of BP was consistent with induced metabolism via CYP1A1, which nevertheless does not determine the blood levels and distribution to BM. Effective delivery of BP to BM is indicated by formation of BP-quinone DNA adducts and the effective induction of CYP1B1. The low formation of BP-dihydrodiol-epoxide-DNA adducts suggests effective AhR induction of BM detoxifying reactions that prevents their formation from dihydrodiols. These findings contrast with the substantial hepatic CYP1A1 contribution for PAHs previously seen for intragastric administration where first pass elimination limits the amount of PAHs reaching the BM.     

Mdm2 as a Sensitive and Mechanistically Informative Marker for Genotoxicity Induced by Benzo[a]pyrene and Dibenzo[a,l]pyrene

Mdm2 is an oncoprotein interacting with p53 and maintaininglow p53 levels in unstressed cells. Here we investigated theeffect of genotoxic compounds on Mdm2 phosphorylation levels.Employing the Mdm2 2A10 antibody and phosphatase treatment wefound that Mdm2 accumulated in HepG2 cells when exposed to lowconcentrations of genotoxic compounds such as mitomycin C, etoposide,5-fluorouracil, and benzo[a]pyrene (BP). The low-dose responseswere not accompanied by p53 accumulation and the effect of lowconcentrations of BP on Mdm2 was not affected by small interferingRNA for p53. In human lymphoblasts 10nM BP induced an Mdm2 response.Low concentrations of BP also induced binding of Mdm2 to chromatinin HepG2 cells, but no p53 binding or H2AX phosphorylation.The more mutagenic dibenzo[a,l]pyrene as well as higher BP concentrationsinstead induced H2AX and p53 Ser15 association with chromatin.Acrolein potentiated the effect of BP on p53 stabilization andchromatin binding. Taken together, these data suggest that (1)Mdm2 is a sensitive biomarker for certain types of genotoxicity,and (2) that polycyclic aromatic hydrocarbons-induced Mdm2 bindingto chromatin reflects repairable damage, whereas chromatin bindingof p53 Ser15 and H2AX indicates more persistent DNA damage.The analysis of Mdm2 and related endpoints might be useful forevaluating mutagenic potentials of DNA damages. It is suggestedthat patterns documented here can be used for separating BPdoses that induce readily repaired DNA adducts from doses thatoverwhelm this capacity.     

Cytochrome P450 (CYP1A) induction and DNA adducts in a rat hepatoma cell line (Fao), exposed to environmentally relevant concentrations of organic compounds, singly and in combinations

Cytochrome P4501A (CYP1A) induction and DNA adduct formation were evaluated in the rat hepatoma cell line Fao, as biomarkers of exposure to organic compounds. Cells were exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) or 3,3',4,4'-tetrachlorobiphenyl (TCB), and to combinations of B[a]P and TCB. Both B[a]P and TCB induced CYP1A proteins in a concentration-dependent relationship, up to concentrations of 10 and 1 μM, respectively, detected by Western blotting. DNA adducts, analyzed by (32)P-postlabeling, were found at the highest concentrations of B[a]P (1 and 10 μM). No adducts were found in cells exposed to 0.1 μM TCB alone. The cotreatment of TCB and B[a]P indicated an increase in DNA adduct formation, compared with B[a]P, but no further induction of CYP1A protein compared with TCB alone. This study suggests that Western blotting and (32)P-postlabeling might be suitable methods for detecting CYP1A protein induction and DNA adducts, respectively, after exposure to environmentally relevant concentrations of organic compounds.     

Epigallocatechin-3-gallate does not affect the activity of enzymes involved in metabolic activation and cellular excretion of benzo[a]pyrene in human colon carcinoma cells

Benzo[a]pyrene (B[a]P) and related procarcinogens found in cigarette smoke and roasted foodstuff require metabolic activation to build mutagenic DNA adducts that may cause tumor diseases like colorectal cancer. The major B[a]P-activating enzymes belong to the cytochrome-P450 (CYP)-1 family and are regulated by the aryl hydrocarbon receptor (AhR). Previous studies have indicated that an inhibition of AhR is accompanied with a reduced metabolic activation of B[a]P and therefore may act protective against carcinogenesis. We investigated if the green tea flavonoid (−)-epigallocatechin-3-gallate (EGCG), a known AhR inhibitor, is able to influence B[a]P-metabolizing and B[a]P-transporting enzymes in human Caco-2 colon carcinoma cells. Strikingly, treatment with EGCG did neither affect constitutive and B[a]P-inducible expression of CYP1A1 and UDP-glucuronosyltransferase (UGT)-1A1 nor overall CYP1 and UGT enzyme activities, indicating that EGCG does not antagonize the AhR in Caco-2 cells. Since flavonoids were also identified to enhance the activity of B[a]P-carrying transporter, we analyzed if EGCG exposure alters cellular excretion of B[a]P conjugates. In contrast to the positive control fisetin, EGCG did not affect cellular excretion of B[a]P metabolites. Our data provide evidence that EGCG does not alter the metabolism and transport of B[a]P in Caco-2 cells, and thus may not protect against procarcinogenic food contaminants.     

Sustained overexpression of CYP1A1 and 1B1 and steady accumulation of DNA adducts by low-dose, continuous exposure to benzo[a]pyrene by polymeric implants

Many carcinogenesis and tumorigenesis studies reported in the past several decades have relied upon bolus dose(s) of test compounds to determine their DNA damage and carcinogenic potential. The high doses are far from the human scenario where exposure is almost always to low doses and for long duration. In this study, we report a novel polymeric implant system that provides continuous ("24/7") exposure to low doses using benzo[a]pyrene (BP) as a model carcinogen. Cylindrical implants (1 cm length, 3.2 mm diameter; 10 mg BP/100 mg implant) prepared from polycaprolactone:F68 (9:1) showed controlled release in vitro for long duration. To determine the rate of release and biochemical effects in vivo, groups of female Sprague-Dawley rats received either no treatment or subcutaneous sham or BP implants (1 cm, 10% load) and were euthanized after 6, 15, 30, and 180 days; the average dose of BP by the implant route was 16.7 ± 3 μg/rat. For comparison, rats were also treated with a single bolus dose of BP intraperitoneally (10 mg/rat) and euthanized at 6, 15, and 30 days. DNA adducts analyzed by (32)P-postlabeling in the lung and liver increased steadily with time with levels reaching 31 ± 3 and 17 ± 6 adducts/10(9) nucleotides, respectively, after 25 weeks; the adduct burden in the mammary tissue initially increased but then declined with time presumably due to high cell turn over. In contrast, the bolus dose treatment showed the highest DNA adduct levels after 6 days, followed by a steady decline. The steady accumulation of tissue DNA adducts in the implant groups corroborates the sustained overexpression of CYP1A1 and 1B1, the cytochrome P450s involved in the conversion of BP to its electrophilic metabolites. In contrast, the overexpression of CYP1A1 and 1B1 resulting from the bolus dose of BP lasted only for a few days. This is the first demonstration revealing that low-dose, continuous exposure to environmental polycyclic aromatic hydrocarbons such as BP can render sustained expression of CYPs and steady accumulation of tissue DNA adducts. On the basis of our recent study in which we showed the presence of 17β-estradiol in the lung, the sustained overexpression of CYP1A1 and 1B1 due to continuous exposure to BP may increase the susceptibility to estrogen-mediated carcinogenicity.     

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.     

Inhibition of carcinogen-bioactivating cytochrome P450 1 isoforms by amiloride derivatives

We examined the effects of amiloride derivatives, especially 5-(N-ethyl-N-isopropyl)amiloride (EIPA), on the activity of cytochrome P450 (CYP) 1 isoforms, known to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BP), into mutagenic metabolites and whose cellular expression can be induced through interaction of PAHs with the arylhydrocarbon receptor. EIPA was found to cause a potent and dose-dependent inhibition of CYP1-related ethoxyresorufine O-deethylase (EROD) activity in both liver cells and microsomes. It also markedly reduced activity of human recombinant CYP1A1 enzyme through a competitive mechanism; activities of other human CYP1 isoforms, i.e. CYP1A2 and CYP1B1, were also decreased. However, EIPA did not affect BP-mediated induction of CYP1A1 mRNA and protein levels in rat liver cells, likely indicating that EIPA does not block activation of the arylhydrocarbon receptor by PAHs. Inhibition of CYP1 activity by EIPA was associated with a decreased metabolism of BP, a reduced formation of BP-derived DNA adducts and a diminished BP-induced apoptosis in liver cells. The present data suggest that amiloride derivatives, such as EIPA, may be useful for preventing toxicity of chemical carcinogens, such as PAHs, through inhibition of CYP1 enzyme activity.     

DNA damage caused by benzo(a)pyrene in MCF-7 cells is increased by verapamil, probenecid and PSC833

The aim of this study was to clarify whether pharmaceutical drugs capable of inhibiting ABC-transporters affect the toxicity of benzo(a)pyrene (BP). MCF-7 breast adenocarcinoma cells were cultured for 24 and 48 h with benzo(a)pyrene (1 microM) and the transporter inhibitors verapamil (0.125-100 microM), PSC833 (0.05-5 microM) or probenecid (0.05-2 mM). DNA binding of benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) was analyzed by synchronous fluorescence spectrophotometry and p53 protein by immunoblotting. BP metabolism was studied using thin layer chromatography (TLC). MTT assay and ATP quantitation were used for the analysis of cell viability. At 24 h there was no statistically significant increase in the DNA-adduct formation by any of the used inhibitors. However, at 48 h all of the inhibitors, in concentrations known to effectively block ABC transporters, increased the BPDE-DNA adduct formation 1.5 to 2-fold compared to adduct formation with BP only. PSC833 and verapamil also increased p53 protein expression at 48 h (p<0.05). Probenecid decreased glucuronidation of (3)H-BP metabolites. Other inhibitors did not decrease statistically significantly the overall formation of water-soluble metabolites. BP alone slightly decreased viability of cells at 48 h according to ATP quantitation as compared to vehicle treated controls (86.4+/-16.4%). Even though the used inhibitors showed some cytotoxicity, the combination of BP and inhibitors did not decrease cell viability in synergistic manner. According to these results certain pharmaceutical drugs may increase DNA damage caused by benzo(a)pyrene in MCF-7 cells at least partly through the inhibition of transporters. Taking into account the complex metabolism of BP and lack of specificity of the inhibitors used, it is likely that increased DNA damage seen in this study was the result of multiple interactions between the inhibitors, BP metabolism and the efflux of the compounds.     

Resveratrol,a natural aryl hydrocarbon receptor antagonist,protects lung from DNA damage and apoptosis caused by benzo[a]pyrene

Benzo[a]pyrene (BaP) is an agonistic ligand for the aryl hydrocarbon receptor (AhR) and a major environmental carcinogen implicated in the aetiology of lung cancer through the induction of benzo[a]pyrene diol epoxidation (BPDE) and BPDE-DNA adducts. Because BaP metabolization requires cytochrome P-450 1A1 (CYP1A1) induction through activation of the AhR, we hypothesized that resveratrol, a natural competitive inhibitor of AhR, could prevent these adverse effects of BaP on the lung. Balb-C mice were injected for 5 weeks with corn oil, BaP (5 mg kg(-1) week(-1)), resveratrol (50 mg kg(-1) week(-1)) or BaP + resveratrol. Immunohistochemistry was performed on lung sections for the determination of CYP1A1 protein, BPDE-DNA adducts and apoptosis. A semi-quantitative immunohistochemistry score (H score) was used for data analysis. Mice exposed to BaP had a significant induction of lung BPDE-DNA adducts when compared with controls (H scores: control, 26, interquartile range 18-33; BaP, 276, interquartile range 269-288; P < 0.01). The BPDE-DNA adduct induction by BaP was abrogated significantly by resveratrol (H score: BaP + resveratrol, 103, interquartile range 96-113). A similar pattern was found by immunohistochemistry for apoptosis (H scores: control, 121, interquartile range 102-137; BaP, 288, interquartile range 282-292, P < 0.05; BaP + resveratrol, 132, interquartile range 121-141, P = NS) and CYP1A1 (H scores: control, 170.3, interquartile range 164-175; BaP, 302.3, interquartile range 291-315, P < 0.05; BaP + resveratrol, 200.7, interquartile range 174-215, P = NS). Western blotting confirmed that resveratrol prevented BaP-induced CYP1A1 expression. This increase in CYP1A1 expression in response to BaP administration most likely causes BaP metabolism, BPDE-DNA adduct formation and subsequent apoptosis. All BaP-induced effects could be prevented by resveratrol, suggesting a possible chemopreventive role for this natural phytoalexin against the development of lung cancer.     

On the bioactivation and genotoxic action of fluoranthene

Fluoranthene (FA) was studied with respect to possible mechanisms of its high mutagenicity but low carcinogenicity, in comparison with the corresponding properties of benzo[a]pyrene (BaP), and with regard to the synergism of these two compounds shown by van Duuren and Goldschmidt (J Natl Cancer Inst 56, 1976, 1237). FA and BaP activated by S9 from Aroclor 1254 (PCB)-treated rats induce HPRT mutations in CHO cells with about equal effectiveness at the same exposure doses, which also lead to the same frequencies of repairable DNA adducts, enzyme-induced strand breaks being used as an indirect measure of adducts to DNA. FA was also shown to be an efficient inducer of SCE in human peripheral lymphocytes cocultivated with PCB-treated HepG2 cells or with liver cells from PCB-pretreated rats. For the induction of SCE, FA and BaP were shown to act additively. From metabolic studies with liver microsomes from C57B1/6 mice it is concluded that, whereas BaP induces the metabolism of BaP to the mutagenic epoxide, neither BaP nor FA is able to induce the metabolism of FA. In mutation experiments with V79 cells (XEM2) constitutive for P450 IA1 activity, BaP 7,8-diol but not FA 2,3-diol provokes a high frequency of HPRT mutations. In cells constitutive for P450 IA2 enzymatic activity FA and BaP are but weakly mutagenic and practically nonmutagenic, respectively. Due to the additivity of the genotoxic effects of FA and BaP, induction of an error-prone condition by the latter compound seems to be excluded. It therefore appears that for an explanation of the difference in carcinogenic potency between FA and BaP, indications that BaP but not FA acts as a promoter should be studied further.