In vitro influence of molybdenum on benzo[a]pyrene metabolism in hepatic and pulmonary rat microsomes

Our study presents the in vitro molybdenum influence on benzo[a]pyrene (BaP) microsomal metabolism. Addition of various concentrations of different molybdenum salts [MoS2, MoCl5, (NH4)6Mo7O24 . 4H2O] to liver and lung microsomal fractions of rats previously treated with 3-methylcholanthrene produces a decrease in the different BaP metabolites assessed by high-performance liquid chromatography (HPLC) analysis. This inhibition varies, depending on the considered metabolite and in relation to both the molybdenum level and the origin of the microsomal suspension. The minimum effective concentration is 0.26 and 0.52 mM Mo from liver and lung, respectively. The inhibitory potencies of the +5 (chloride) and +6 (ammonium heptamolybdate) molybdenum compounds are comparable; that of the sulfide is lower.     

Studies on the ability of rat lung and liver microsomes to facilitate transfer and metabolism of benzo[a]pyrene from diesel particles

Little is known about the bioavailability of inhaled organic compounds that are associated with particles. It is known that certain particle-associated organic compounds, such as polycyclic aromatic hydrocarbons (PAH) adsorbed on diesel soot particles, are retained in the lung longer than PAH inhaled in pure form. If such particle-associated compounds are available for tissue interaction, their prolonged retention may result in an increased potential for a toxic effect. To determine the factors affecting the bioavailability of particle-associated PAH, we have studied the ability of microsomes to facilitate transfer of benzo[a]pyrene (B[a]P) adsorbed on the surface of diesel exhaust soot particles to the microsomes and the ability of the microsomes to metabolize the transferred B[a]P. Our results indicate that rat lung and liver microsomes were able to facilitate the transfer of small amounts of B[a]P from diesel particles (less than 3%), but only a fraction of the amount transferred (1-2%) was metabolized. Under the same incubation conditions without soot, free B[a]P was extensively metabolized by microsomes, principally to B[a]P-9,10-diol. Lung microsomes were about twice as effective as liver microsomes for the transfer of the B[a]P. The ability to transfer B[a]P to the microsomes was independent of metabolism or the presence of protein, but was related to the lipid content of the microsomal fraction. There was no metabolism of the B[a]P coated on diesel particles as analyzed by high-performance liquid chromatography. These findings suggest that microsomes are able to enhance the slow transfer of only a small amount of B[a]P from diesel particles in a form that can be metabolized. However, over a long period of time, this slow release might be significant.     

Hydrogen peroxide-supported oxidation of benzo [a]pyrene by rat liver microsomal fractions

In the presence of liver microsomes from phenobarbital-pretreated rats, hydrogen peroxide oxidized benzo [a]pyrene to a number of biologically significant products at a rate that was approximately 20 per cent as fast as that seen by us and others with NADPH and oxygen. As with NADPH-dependent reactions [J. Capdevila, R. W. Estabrook, and R. A. Prough, Archs. Biochem. Biophys.200, 186 (1980)], the hydrogen peroxide-dependent reactions resulted in the production of relatively large quantities of dihydrodiols as metabolites. This was in marked contrast to the product distribution observed when cumene hydroperoxide was utilized as a cosubstrate (foregoing reference). The formation of the various organic-soluble metabolites was dependent on the presence of functional liver microsomal cytochrome P-450 in the reaction mixture. Approximately 48 per cent of the benzo[a]pyrene metabolites, however, was observed to be bound to microsomal protein, and inhibition of cytochrome P-450 function, by metyrapone or N-octylamine did not affect the extent of covalent binding of the hydrocarbon to the microsomal protein. The differences noted during benzo[a]pyrene metabolism using hydrogen peroxide strongly suggest that at least two distinct mechanisms exist to account for the oxidation of the hydrocarbon, i.e. epoxidation and one-electron oxidation reactions.     

Influence of chlordecone and mirex exposure on benzo[a]pyrene metabolism of rat-liver microsomes

The metabolism of benzo[a]pyrene (BP) in hepatic microsomes isolated from rats exposed to chlordecone or mirex was compared to that of untreated rats and rats treated with 3-methylcholanthrene (3-MC) or phenobarbital (PB). Treatment with chlordecone resulted in a two- to three-fold increase in cytochrome P-450 content but the BP-hydroxylase activity per mg microsomal protein was unaffected. Addition of alpha-naphthoflavone (alpha-NF) or chlordecone caused changes in BP-hydroxylase activity indicating that chlordecone-induced cytochromes P-450 were similar to control. H.p.l.c. analyses of BP metabolites confirmed this similarity. Treatment with mirex caused a two-fold induction of cytochrome P-450, and BP-hydroxylase activity expressed per mg microsomal protein was increased 1.3-fold. Addition of chlordecone or alpha-NF caused changes in BP-hydroxylase activity, indicating differences between control and mirex-induced cytochromes P-450. H.p.l.c. analyses of BP metabolites confirmed this difference. Treatment with chlordecone or mirex increased microsomal epoxide hydrolase activity three-fold. Chlordecone accumulated in hepatic nuclei.     

Lithium-induced increase in the metabolism of benzo[a]pyrene and drugs in rat liver.

Administration of lithium chloride (2.5 mEq/kg/day) to rats for 4 or 12 days increased the rates of hepatic hydroxylation of benzo[a] pyrene, antipyrine and zoxazolamine by 100, 114 and 56 per cent, respectively, and the rate of hepatic conjugation of 4-methylumbelliferone with glucuronic acid by 19 per cent. Liver lithium levels were highly correlated with plasma lithium levels but not with hepatic benzo[a] pyrene hydroxylase activities. In vitro addition of lithium to liver 10,000 g supernatants did not affect the hydroxylation of benzolalpyrene, suggesting that the lithium-induced increase in hepatic drug hydroxylation is not due to enzyme activation and is probably a result of lithium-mediated enzyme induction.     

Effects of organic solvent vehicles on benzo[a]pyrene metabolism in rabbit lung microsomes

In order to study the metabolism of benzo[a]pyrene (BP), it must be dissolved in an organic solvent vehicle for delivery to the tissue. We studied the effects of five organic solvent vehicles, i.e. dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, and ethyl acetate, on benzo[a]pyrene hydroxylase activity and the BP metabolite profile in rabbit lung microsomes. Fluorescence detection of 3- and 9-OH-BP was used to evaluate benzo[a]pyrene hydroxylase activity, and the BP metabolite profile was obtained by HPLC analysis. All solvent vehicles inhibited benzo[a]pyrene hydroxylase in a dose-dependent manner. When the smallest volume of each solvent (10 microliter/ml reaction mixture) was employed, the resulting enzyme activities as related to solvent type, from highest to lowest, were DMSO greater than or equal to methanol greater than ethanol greater than or equal to acetone greater than ethyl acetate. HPLC analysis of BP metabolites formed in the presence of the five solvent vehicles showed that production of all metabolites was greatest when DMSO was used and that linearity of product formation was retained longer with DMSO. The metabolites produced when DMSO was used as the solvent were BP-9,10-diol, BP-4,5-diol, BP-7,8-diol, BP-1,6-quinone, BP-3,6-quinone and 3-OH-BP. A similar metabolite profile was obtained when reactions were carried out with methanol as the solvent vehicle, although the magnitude of production was less than with DMSO. When acetone was used, there were greater amounts of BP-4,5-diol and BP quinone formation and lesser amounts of 3-OH-BP formed than with DMSO or methanol. When ethanol or ethyl acetate was used as a solvent, BP-9,10-diol and 3-OH-BP were the only metabolites produced. These results indicate that all solvent vehicles studied inhibit benzo[a]pyrene hydroxylase from rabbit lung microsomes in a dose-dependent manner and that the magnitudes and types of metabolites formed are highly dependent upon the specific solvent used as the vehicle. The study also indicates that DMSO is probably the solvent vehicle of choice for study of BP metabolism in rabbit lung microsomes.     

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.     

Biotransformation of benzo[a]pyrene and 7-ethoxyresorufin and heme-staining proteins in microsomes from human fetal liver and placenta

Microsomal proteins from human fetal livers and mid-gestational and term placentas were stained for heme in an attempt to detect multiple forms of cytochrome P-450. In fetal liver microsomes five protein bands staining for heme in the mol. wt region 46,000-60,000 were found. In the placentas two bands were seen in the region 46,000-52,000. Fetal liver and placental microsomes were assayed for metabolism of benzo[a]pyrene (B[a]P and 7-ethoxyresorufin (7-EOR). The B[a]P metabolites were separated using high performance liquid chromatography. Following incubations with fetal liver microsomes, in general only phenols were detectable, while after incubations with mid-gestational as well as term placentas from smoking women, the 9,10-, 4,5- and 7,8-dihydrodiols were also formed. No quinones were detected. Placental microsomes from non-smoking women did not catalyse the formation of B[a]P metabolites. The 7-EOR O-de-ethylase activity was in the same range (2-5 pmol/min x mg microsomal protein) in the fetal livers as in the mid-gestational placentas. The activities were somewhat higher in the placentas originating from smokers. No correlation between enzymatic activities in vitro and intensity of any specific protein band was observed for the fetal livers of placentas studied.     

Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line.

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 microM. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca2+ in relation to the time-course for induction of microsomal ethoxyresorufin-0-deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 microM BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 microM BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 microM BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 microM BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca2+ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.     

Menadione and cumene hydroperoxide induced cytotoxicity in biliary epithelial cells isolated from rat liver

Biliary epithelial cells (BEC) and parenchymal cells isolated from normal rat liver were exposed in vitro to a number of toxic compounds. BEC were found to be highly sensitive to concentrations of menadione (100 microM) and cumene hydroperoxide (10 microM) that are usually not effective as toxic agents in hepatocytes under normoxic conditions. On the other hand, BEC were not affected by concentrations of carbon tetrachloride or 7-ethoxycoumarin that are known to exert toxic effects on hepatocytes. The development of both menadione- and cumene hydroperoxide-induced toxic injury in BEC followed a common and time-correlated pattern, and included a strong depletion of GSH, depletion of protein thiols and an increase in the extent of cell death. The damage induced by cumene hydroperoxide was found to be independent of lipid peroxidative processes and was prevented by a pre-incubation with desferrioxamine. The cytotoxicity of menadione was further exacerbated by dicoumarol but was not prevented by desferrioxamine or promethazine. The mechanisms underlying BEC injury and death induced by the quinone and by the organic hydroperoxide are discussed in relation to the known biochemical characteristics of BEC.     

A comparative study of benzo[a]pyrene hydroxylase and epoxide hydrolase derived from isolated rat hepatic nuclei and microsomes

Several properties of benzo[a]pyrene hydroxylase (AHH) and epoxide hydrolase (EH) in isolated rat hepatic nuclei were investigated. Comparisons were made with microsomal AHH and EH in order to determine whether the nuclear enzymes were distinct from those of endoplasmic reticulum origin. The ratio of EH to AHH in the nuclei of adult male rats was 4.5 compared to 1.6 in the microsomes. Phenobarbital increased AHH in nuclei from immature males and females. However, the increase were markedly lower than those observed in the microsomes. In contrast, 3-methylcholanthrene increased nuclear AHH to a greater extent than the microsomal enzyme. The age and sex dependence, the inhibition by SKF 525-A, the stimulatory and inhibitory effects of 7,8-benzoflavone and the decrease produced by trans-stilbene oxide administration were the same for both nuclear and microsomal AHH. Phenobarbital, l-alpha-acetyulmethadol, and trans-stilbene oxide increased nuclear EH. The increases were approximately 3-fold lower than observed in the microsomes. EH activity in both nuclei and microsomes was inhibited by 1,2-epoxy-3,3,3-trichloropropane. The data indicate that both the constitutive and induced form(s) of AHH in isolated nuclei and endoplasmic reticulum have a number of common properties. The constitutive form(s) of microsomal and nuclear EH also appear to be similar. However, AHH and EH in isolated nuclei differ from their microsomal counterparts in quantitative inducibility. These differences in inducibility support the suggestion that AHH and EH are located in isolated nuclei rather than resulting from contamination by endoplasmic reticulum.     

Effect of low-level short-term o-xylene inhalation of benzo[a]pyrene (BaP) metabolism and BaP-DNA adduct formation in rat liver and lung microsomes

o-Xylene is a commonly used solvent that alters mixed-function oxidase (MFO) activity in an organ- and isozyme-specific pattern following intraperitoneal (ip) administration. Similar MFO alterations have been observed after ip or inhalation exposure to other methyl benzenes. These MFO alterations shifted the metabolism of the carcinogen benzo[a]pyrene (BaP) toward formation of toxication metabolites in lung. The purpose of this study was to determine whether o-xylene inhalation caused similar MFO changes and whether these alterations were reflected in altered BaP metabolism and BaP-DNA adduct formation. o-Xylene (300 ppm, 6 h) decreased the activity of arylhydrocarbon hydroxylase (AHH) in lung. CYP2B1 activity (benzyloxyresorufin O-dealkylase; BROD), which is responsible for metabolism of BaP to relatively nontoxic metabolites, was decreased in lung, as was, to a lesser extent, CYP1A1 (ethoxyresorufin O-dealkylase; EROD), which is responsible for metabolism of BaP to reactive/toxic metabolites. The BROD/EROD ratio, an indirect indicator of the pattern of BaP toxication/detoxication, was decreased in lung, suggesting that BaP metabolism is shifted toward toxication. No MFO alterations were observed in liver. In lung microsomes, o-xylene increased formation of 7,8-BaP-diol, while 9,10-BaP-diol, 3-OH BaP, and 9-OH BaP were decreased. In liver, o-xylene increased 9-OH BaP formation, while 4,5- and 9,10-diols as well as total diols were decreased. The toxication/detoxication ratios for BaP individual and total metabolite groups were increased in lung microsomes and unaltered in liver. The major BaP-DNA adduct, BaP diol epoxide-N2-deoxyguanosine, was increased in lung but decreased in liver microsomes from o-xylene-exposed rats. Four minor BaP-DNA adducts were formed in lung and three in liver, only one of which (liver adduct 3) was decreased. The o-xylene-induced increase in BaP adduct formation in lung and decrease in liver indicate that coexposure to organic solvents such as the methyl benzenes may alter the carcinogenesis of BaP, or other PAHs, in an organ-specific fashion.     

Chemopreventive effects of aloe against genotoxicity induced by benzo[a]pyrene

Chemopreventive effects of aloe against benzo[a]pyrene (BaP) mutagenicity were investigated in the Salmonella typhimurium bacterial mutation assay, the chromosome aberration assay using Chinese hamster ovary (CHO) cells, and the mouse micronuclei test using bone-marrow cells. In the bacterial assay, aloe produced a concentration-dependent decrease in the number of mutant colonies induced by BaP. The chromosome-damaging responses of BaP in CHO cells were abolished by treatment with aloe, approximately to the level seen in control. In the in vivo mouse bone-marrow micronuclei test, pretreatment of aloe 24 h prior to BaP treatment reduced the frequency of micronucleated polychromatic erythrocytes. In the cells of CHO and bone marrow treated with aloe, glutathione (GSH) levels were shown to be higher and extracellular discharge rate increased as incubation time with aloe rose. MDR1 and MRP2 gene were more expressed in Hepa c cells than in NTCC cells, but there was no change in BCRP gene expression. The antimutagenic effects of aloe were statistically significant and concentration dependent. These results demonstrated that aloe might exert chemopreventive effects against BaP-induced mutagenicity.     

Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-cyclodextrin reduces bioavailability of orally administered [3H]benzo[a]pyrene in the rat

The excretion and plasma kinetics of total radioactivity were studied following single oral administration of [(3)H]benzo[a]pyrene after multiple oral administration of beta-cyclodextrin at 0, 5, 50, or 500 mg/kg/day. The AUC and C(max) values in male and female rats following administration of [(3)H]benzo[a]pyrene in combination with 5 to 500 mg/kg beta-cyclodextrin were considerably lower than that in rats administered [(3)H]benzo[a]pyrene alone. At all dose levels of beta-cyclodextrin, the excretion of total radioactivity was almost entirely via feces, with <2% recovered in urine, demonstrating either that absorption of the orally administered dose was low or that, for any absorbed material, biliary excretion was the main route of excretion. However, following administration of vehicle, up to 5% of the administered radioactivity was recovered in the urine, suggesting that absorption may have been reduced by the presence of beta-cyclodextrin in the intestine. At all dose levels of beta-cyclodextrin, there was minimal retention of radioactivity in the carcase at the end of the collection period. Beta-cyclodextrin did not affect the apparent terminal half-life of radioactivity. Therefore, the reduced systemic exposure of rats to radioactivity in the presence of beta-cyclodextrin is likely related to a reduced oral bioavailability.     

Metabolism of benzo[a]pyrene in the combined rat liver--lung perfusion system

The influence of the insertion of a liver into the perfusion circuit of a lung on the availability of benzo[a]pyrene and benzo[a]pyrene metabolites to the lung was examined. Perfused lungs from 5,6-benzoflavone pretreated rats release high quantities of free benzo[a]pyrene metabolites and conjugates into the perfusion medium. The insertion of a liver taken from an untreated rat reduces the concentration of unmetabolized substrate and of free diol, quinone and phenol metabolites to less than 20% of the concentrations found in the absence of the liver. When the liver of a 5,6-benzoflavone-pretreated rat is used, substrate depletion is not much greater than in the experiments with control livers; however, the concentration of free metabolites is further reduced to one third. In lung tissue, only very low levels of benzo[a]pyrene and greatly reduced levels of free and conjugated metabolites are found when a 5,6-benzoflavone-induced liver had been present during perfusion. These findings can explain the protective effect of the liver on covalent binding of benzo[a]pyrene metabolites to pulmonary macro-molecules observed in previous experiments with the combined liver-lung perfusion model [Klaus et al., Biochem. Biophys. Res. Commun., 105 (1982) 596].     

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.