Glucuronidation of benzo[a]pyrene in hamster embryo cells

The formation of water-soluble metabolites of tritium-labeled benzo[a]pyrene (BP) by cultured hamster embryo cells was studied. The ratio of the radioactivity in the aqueous phase to that in the organic phase increased with the incubation period. After incubation for 48 h with 3.75 nmol/ml of [3H] BP in the medium more than 90% of the 3H-radioactivity was found in the aqueous phase, whereas with 10-fold more BP about half the radioactivity remained in the organic phase. The main metabolites extracted from the medium at 37.5 nmol/ml BP with ethyl acetate by high pressure liquid chromatography (HPLC) were 9,10-diol and 7,8-diol; but after treatment of the medium with beta-glucuronidase the main oxygenated metabolites were phenols, the amount of 9-OH BP being more than that of 3-OH BP. beta-Glucuronidase also released 9,10-diol and 7,8-diol, but most of these diols were in the free form in the medium. The medium from cells treated with 3.75 nmol/ml BP has a quantitatively different profile, and most of the radioactivity obtained by extraction with organic solvent and digestion with beta-glucuronidase was eluted in the regions of phenols. These results show that in hamster embryo cells BP is mainly metabolised to conjugates of phenols with glucuronic acid.     

Inhibition of the mutagenicity of benzo[a]pyrene in the V79/HGPRT system by bioantioxidants

In the presence of metabolic activation (S9 microsomal fraction of mouse-liver homogenate) the mutagenicity of benzo[a]pyrene (BP) in Chinese hamster V79 cells was inhibited by the phenolic bioantioxidants (BA) Dibunol (2,6-di-tert-butyl-4-methylphenol-D) and 5-methylresorcine(5-MR). The mixture BP + D and BP + 5-MR at molar ratios of 1:1 and 1:85 respectively showed no mutagenic activity compared to the control. One can assume that D and 5-MR inhibited BP-induced mutagenesis by binding the free radicals of BP metabolites with the formation of less active phenolic derivatives and also by linkage with cytochrome P-450, which prevents further metabolic activation of BP.     

Metabolism of benzo[A]pyrene in hamster embryo cells. Effect of the concentration of benzo[A]pyrene on its metabolism.

The metabolism of benzo[a]pyrene (BP) by hamster embryo cells was studied. The production of water-soluble metabolites, shown to be conjugates with glucuronic acid, depended on BP concentration. With increased BP concentration the amount of glucuronic acid conjugates increased, but the proportion of conjugates in BP or its metabolites present in the medium decreased. The metabolites extracted with ethylacetate were trans-7,8-dihydrodiol-BP (7,8-dihydrodiol) and trans-9,10-dihydrodiol-BP (9,10-dihydrodiol), but large peaks of phenolic metabolites were found by high pressure liquid chromatography (HPLC) after digesting the medium with beta-glucuronidase. Therefore, BP is metabolized to oxygenated forms, and of these, most of the phenolic metabolites and parts of the dihydrodiols are conjugated with glucuronic acid. The proportions of dihydrodiols to phenols, estimated by HPLC after beta-glucuronidase digestion, decreased when the BP concentration was decreased. The results suggest that dihydrodiols are less readily glucuronidated than phenols and so may be metabolized further to metabolites other than glucuronic acid conjugates.     

Inhibition of benzo[a]pyrene-induced mutagenesis in Chinese hamster V79 cells by hemin and related compounds

The inhibitory effects of hemin and related compounds on the mutagenicity of benzo[a]pyrene (BP) were investigated in Chinese hamster V79 cells co-cultivated with X-irradiated hamster embryo cells. Mutant V79 cells were selected by their resistance to ouabain. The mutation frequency induced by BP was substantially inhibited dose dependently by hemin. The mutagenicity of BP (1 microgram/ml) on V79 cells was reduced to 6.5% by hemin, 52% by biliverdin, 73% by protoporphyrin and 85% by chlorophyllin at the highest concentration of the compounds tested (15 microM).     

Transient DNA lesions induced by benzo[a]pyrene in Chinese hamster ovary cells

Alkaline sucrose gradient sedimentation analysis was used to detect DNA lesions induced by benzo[a]pyrene B(a)P in Chinese hamster ovary cells. The number of lesions detected immediately following treatment with 10(-4) M B(a)P was related directly to the duration of treatment. When treated cells were incubated in a B(a)P-free medium, the majority of lesions disappeared rapidly and could no longer be detected 15 min following treatment. These data indicate that a population of B(a)P-induced DNA lesions may be removed by a rapid DNA-repair process. The transient nature of such lesions should be considered when assays for DNA damage or repair are designed and interpreted.     

Mutagenicity and metabolism of dimethylnitrosamine and benzo[alpha]pyrene in tissue homogenates from inbred Syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[alpha]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SSLak, LSH/SlLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagans. Dimethylnitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[alpha]pyrene as substrate. MC does not induced AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC. Activity of DMND and conversion of DMN to mutagen are correlated (r = 0.59) in hamster liver. Microsomes of hamster liver are more effective than those from mouse in converting DMN to mutagen, despite similar DMND activities in livers from the two species.     

Metabolism of benzo[A]pyrene and the related enzyme activities in hamster embryo cells.

The rate of metabolism of benzo[a]pyrene (BP) and changes in related enzyme activities in cultured hamster embryo cells during successive subculture were studied. The activity of aryl hydrocarbon hydroxylase (AHH) was the highest when embryo cells were first dispersed in tissue culture flasks and decreased during subsequent passages. On the other hand, UDP-glucuronyl transferase activity increased gradually during successive subculture. Treatment of the cells with 13 nmol/ml of benz[a]anthracene (BA) for 24 h increased the activity of AHH but not that of UDP-glucuronyl transferase. The metabolism of BP was measured in cells of the passages 1, 3 and 7; metabolism of BP was most efficient in cells in passage 3 and their formation of glucuronic acid conjugates of BP, one of the major metabolites found in the medium, was 3- and 10-fold more than those of cells in passages 1 and 7, respectively. Analysis of BP-metabolites extracted from the medium with ethylacetate showed that the main metabolites were 9,10-diol and 7,8-diol. Phenols and quinones were released by treatment of the medium with beta- glucuronidase and their amounts were larger than those of diols at all passages. These results show that in hamster embryo cells in early passage, BP is metabolized to conjugates of phenols with glucuronic acid.     

The irreversible binding of benzo[a]pyrene to rat liver macromolecules in vivo and in vitro: effects of agents that influence benzo[a]pyrene metabolism

The present study was carried out to determine the effects of agents that influence benzo[a]pyrene (BP) metabolism in vitro on the irreversible binding of BP to rat hepatic macromolecules in vivo. The irreversible binding of [3H]BP was found to be both dose and time dependent after its intraperitoneal administration to male Wistar rats. The SKF 525-A, at doses of 50 and 75 mg/kg, ip 3 h before BP, decreased the level of binding from control by 31 and 34%, respectively. At 35 mg/kg, SKF-525-A had no effect. Diethyl maleate (0.6 mL/kg, ip) and cysteine (150 mg/kg, ip), 30 and 5 min before BP, respectively, did not alter the binding of BP from control. Oral methadone treatment, previously shown to increase selectively epoxide hydrase activity in male Wistar rats, also failed to alter the amount of BP bound to hepatic macromolecules. 3-Methylcholanthrene (20 mg/kg per day, ip, for 2 days) administered 24 h before BP, decreased the level of binding from control by 30%. Parallel in vitro studies were carried out with the various agents used in vivo.     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Mutagenicity of vinyl chloride, vinylidene chloride and chloroprene in V79 Chinese hamster cells.

The mutagenicity of vinyl chloride, vinylidene chloride (1,1-dichloroethylene) and chloroprene (2-chloro-1,3-butadiene) was tested in V79 Chinese hamster cells in the presence of a 15 000 x g liver supernatant from phenobarbitone-pre-treated rats and mice. Mutations in terms of 8-azaguanine and ouabain resistance were induced in a dose-related fasion by exposure to vapour of vinyl chloride in the presence of liver supernatant from phenobarbitone-pretreated rats. Vapours of vinylidene chloride and chloroprene induced a dose-related toxicity in the presence of liver supernatant from phenobarbitone-retreated rats, but these two compounds were not mutagenic in V79 Chinese hamster cells under the present assay conditions. The results are discussed with regard to the metabolic activation of the compounds and to the correlation with their carcinogenicity in man and experimental animals.     

Covalent binding of benzo[a]pyrene to dna in fish liver

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ($\text{Parophrys vetulus}$) force fed tritiated benzo[a]pyrene. No statistically signïficant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 10⁶ nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.     

Liver microsomal electron transport systems. Properties of a reconstituted, NADH-mediated benzo[a]pyrene hydroxylation system.

An enzyme system from rat liver microsomes which catalyzes the NADH-mediated hydroxylation of benzo[a]pyrene has been reconstituted. The essential microsomal components of this NADH-dependent pathway were NADH-cytochrome b₅ reductase, cytochrome b₅, cytochrome P-448 and, phosphatidyl choline. Highly purified NADPH-cytochrome c reductase containing small amounts of deoxycholate stimulated this NADH-mediated pathway supported by 0.2 mm NADH whereas boiled reductase had little effect. Part of this stimulation could be attributed to hydroxylation of benzo[a]pyrene via a second pathway; i.e., NADPH-cytochrome c reductase in combination with cytochrome P-448 and phosphatidylcholine also supported a low rate of NADH-dependent hydroxylation. The mechanism of the remaining stimulation is not known. However, the effect of NADPH-cytochrome c reductase on the reconstituted cytochrome b₅-dependent pathway was not unique; high concentrations of deoxycholate also stimulated this pathway, perhaps by facilitating the transfer of electrons from NADH-cytochrome b₅ reductase to cytochrome b₅. The addition of NADPH-cytochrome c reductase to the cytochrome b₅-dependent reconstituted system also affected the apparent K_m of NADH for benzo[a]pyrene hydroxylation. In the absence of NADPH-cytochrome c reductase, the apparent K_m of NADH was 1.3 μm while in its presence a low (1.3 μm) and a high (1700 μm) K_m were observed, consistent with the affinities of the two flavoproteins for NADH. Our results also indicate that the relative contribution of the pathway due to NADPH-cytochrome c reductase in combination with phosphatidyl choline and cytochrome P-448 to the overall rate of NADH-supported benzo[a]pyrene hydroxylation in microsomes would be greatly dependent on the concentration of NADH chosen. The rate of benzo[a]pyrene hydroxylation by these reconstituted components was almost 10-fold greater with 10 mm NADH than with 0.2 mm NADH, a result consistent with the reduction of NADPH-cytochrome c reductase by high concentrations of NADH.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells treated in vitro with non-k-region dihydrodiols of 7-methylbenz[a]anthracene and benzo[a]pyrene

Studies were carried out on the incidence of sister-chromatid exchanges induced in Chinese hamster ovary cells by in vitro treatment with the polycyclic aromatic hydrocarbons 7-methylbenz[a]anthracene and benzo[a]pyrene and with related K-region and non-K-region dihydrodiols. Appreciable increases in the incidence of sister-chromatid exchanges were apparent in cells treated with non-K-region dihydrodiols: the most active compounds were 3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and the effects were dose-dependent. The parent hydrocarbons and the related K-region dihydrodiols induced some sister-chromatid exchanges but they were considerably less active than these two non-K-region diols. The results suggest that this system may usefully be applied to studies aimed at determining which dihydrodiols are important in the metabolic activation of the carcinogenic polycyclic hydrocarbons. These and other results also infer that Chinese hamster ovary cells possess some intrinsic ability to metabolize such compounds in the absence of exogenous activation systems.     

Mutational specificity of benzo[a]pyrene diolepoxide in monkey cells

Benzo[a]pyrene diolepoxide (BPDE) is thought to be the major mutagenic and carcinogenic intermediate in benzo[a]pyrene metabolism in mammalian cells. In order to test the mutagenic specificity of this compound in mammalian cells, we have used the pZ189 shuttle vector system to identify and analyze point mutations induced when DNA treated in vitro with BPDE is replicated in monkey cells. We find that point mutations occur almost exclusively at G.C base pairs; G.C----T.A and G.C----C.G transversions and single base pair deletions occur most frequently. This pattern is consistent with the known preferential covalent binding of BPDE to G residues.     

Effects of pretreatment with benzo[a]pyrene on the stereochemical selectivity of metabolic activation of benzo[a]pyrene to DNA-binding metabolites in hamster embryo cell cultures

The proportions of individual benzo[a]pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600). To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing [3H]BaP. After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group. Cells pretreated for 24 h with BaP and then exposed to [3H]BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to [3H]BaP for 5 h. In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to [3H]BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct. Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced [3H]uridine incorporation into RNA by more than 65% at all times. These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells. The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE.     

Mutagenicity, cytotoxicity and DNA crosslinking in V79 Chinese hamster cells treated with cis- and trans-Pt(II) diamminedichloride.

The mutagenicity and cytotoxicity of cis- and trans-Pt(II) diamminedichloride (PDD) were examined in V79 Chinese hamster lung cells and compared with effects on DNA measured by alkaline elution. DNA--protein crosslinks and DNA interstrand crosslinks were detected following doses of cis-PDD which reduced cell survival 80--90% and which produced a mutant frequency of 3 X 10(-4) at the HGPRT locus. Equitoxic doses of trans-PDD were much less mutagenic than cis-PDD. At equitoxic doses, trans-PDD produced more DNA-protein crosslinking than did cis-PDD, but interstrand crosslinking for the two isomers was comparable. Hence, the interstrand crosslink could be the cytotoxic lesion produced by these Pt compounds whereas neither of these DNA lesions are necessarily mutagenic. The mutagenesis produced by cis-PDD could be due to crosslinks of a different type than those produced by trans-PDD or it may be due to monofunctional damage.     

Comparison of the in vitro mutagenicity and metabolism of dimethylnitrosamine and benzo[a]pyrene in tissues from inbred mice treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

Homogenates of liver, lung, kidney, stomach, small intestine and colon from 8 strains of mice were compared for their ability to metabolize benzo[a]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains CF1, AKR/J, AU/SsJ, DBA/2J, SWR/J, A/J, C3H/HeJ, and C57BL/6J were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. The effects of these drugs on organ weight and on the amounts of DNA, S-10 protein, and microsomal protein per unit weight of tissue are reported. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. For each organ there was an optimal balance between amount of tissue homogenate and concentration of test compound for maximal yield of revertants. A sensitive radiometric assay of DMN demethylase (DMND) is described which permits measurement of the enzyme in liver, lung and kidney. DMN at 1 mM is used as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured in all tissue using BP as substrate. AR and MC are very good inducers of AHH activity in livers of mice classified as aromatic hydrocarbon responsive, but not in those classified as hydrocarbon nonresponsive. Responsiveness is strain-specific and genetically regulated. Metabolism of BP to mutagens by liver homogenates was correlated with extent of AHH induction. This dimorphism of response of AHH to inducers was present, but less pronounced, in non-hepatic tissues. Basal activities of AHH and DMND were correlated in livers and lungs from untreated mice. DMND activities were increased less than 2-fold by PB, MC or AR treatments. Metabolism of DMN to mutagens was not closely correlated with DMND activities. Strain of mouse, type of tissue and test substance are important variables in assessing the potential effect of microsomal enzyme-inducing agents on the metabolism of mutagenic substances.