Quinone formation from carcinogenic benzo[a]pyrene mediated by lipid peroxidation in phosphatidylcholine liposomes

The behavior of benzo[a]pyrene (B[a]P) during peroxidation of phosphatidylcholine (PC) liposomes initiated by an azo compound was investigated to examine the mechanism of quinone formation from carcinogenic B[a]P mediated by nonenzymatic lipid peroxidation occurring in vivo. B[a]P had a retarding effect on the peroxidation of polyunsaturated fatty acid moiety of PC. The major oxidation products which accumulated in the peroxidized liposomes were B[a]P 1,6-, 3,6-, and 6,12-quinone. Antioxidants acting as scavengers of chain-propagating lipid peroxy radicals effectively prevented not only lipid peroxidation but also B[a]P oxidation in the liposomal suspension. PC hydroperoxides, the primary products of PC oxidation, did not react with B[a]P in the absence of the azo compound, indicating that lipid peroxy radicals, not lipid hydroperoxides, are responsible for the formation of these quinones. The experiments using 18O2 gas and 18O-labeled methyl linoleate hydroperoxides demonstrated that B[a]P quinones are formed by incorporating molecular oxygen and their origin is partly due to the lipid peroxy radical. The mechanism proposed for the formation of B[a]P quinones mediated by peroxidation of membrane lipids involves a direct attack of the lipid peroxy radical on B[a]P and subsequent autocatalytic oxidation. Weak carcinogenic and noncarcinogenic pentacyclic aromatic hydrocarbons showed little reactivity to the lipid peroxy radical in the liposomes. Thus, the facility of the peroxidative attack on B[a]P may be related to the powerful carcinogenic activity of this substance.     

Peroxidatic oxidation of benzo[a]pyrene and prostaglandin biosynthesis.

The arachidonic acid dependent oxidation of benzo[a]pyrene to a mixture of 3,6-, 1,6-, and 6,12-quinones has been studied by using enzyme preparations from sheep seminal vesicles. Maximal oxidation is observed at 100 microM benzo[a]pyrene and 150 microM arachidonic acid. The arachidonic acid dependent oxidation is peroxidatic and utilizes prostaglandin G2 (PGG2), generated in situ from arachidonate, as the hydroperoxide substrate. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid is equivalent to PGG2 as a hydroperoxide substrate, but hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide are much poorer substrates. Arachidonic acid dependent benzo[a]pyrene oxidation by microsomal and solubilized enzyme preparations is markedly.     

Lipid peroxidation, antioxidant enzymes, and benzo[a]pyrene-quinones in the blood of rats treated with benzo[a]pyrene

The lipid peroxidation (as malondialdehyde, MDA), activities of superoxide dismutase (SOD) and catalase (CAT), and benzo[a]pyrene (BaP) metabolites were investigated in sera and erythrocytes of male Sprague-Dawley rats treated with BaP (20 mg per rat). MDA levels were significantly increased in sera (16.98+/-3.29 nmol/ml serum, P<0.05) 12 h after BaP treatment and persisted up to 96 h (13.80+/-1. 65 nmol/ml serum, P<0.05), but no significant change in NIDA levels was observed in erythrocytes. SOD and CAT activities were significantly increased in erythrocytes shortly after BaP exposure, and they were slightly decreased in sera, indicating an inverse correlation between lipid peroxidation and antioxidant enzyme activity. BaP and BaP-quinones (BaP-1,6-quinone and BaP-3,6-quinone) were measured in sera during the study period. A rapid increase of unmetabolized BaP was observed in sera (41.27+/-4.14 pmol/ml serum) 3 h after BaP treatment, reaching a peak at 6 h (48.56+/-4.62 pmol/ml serum) followed by a sharp decrease. Formation of the BaP-1, 6-quinone and BaP-3,6-quinone started in sera 3 h after BaP treatment, reached a peak at 24 h (7.23+/-1.02 pmol/ml serum) and 12 h (9.20+/-0.98 pmol/ml serum), respectively, and then decreased gradually. The time-dependent pattern of serum lipid peroxidation and the level of erythrocyte antioxidant enzymes were shown to be related to the concentrations of the BaP-quinone metabolites. These results suggest that BaP treatment, probably via the formation of BaP-quinones, oxidatively altered lipids and antioxidant enzymes in the blood, and might be associated with BaP-related vascular toxicity including carcinogenesis.     

Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Arsenic co-exposure potentiates benzo[a]pyrene genotoxicity

The reactive industrial chemicals acrylamide (AA) and N-methylolacrylamide (MAA) are neurotoxic and carcinogenic in animals, MAA showing a lower potency than AA. The causative agent in AA-induced carcinogenesis is assumed to be the epoxy metabolite, glycidamide (GA), which in contrast to AA gives rise to stable adducts to DNA. The causative agent in MAA induced carcinogenesis is so far not studied. The two AAs were studied in mice and rats using analysis of hemoglobin (Hb) adducts as a measure of in vivo doses and the in vivo micronucleus (MN) assay as an end-point for chromosome damage. Male CBA mice were treated by intraperitoneal (i.p.) injection of three different doses and male Sprague-Dawley rats with one dose of each AA. Identical adducts were monitored from the two AAs [N-(2-carbamoylethyl)valine] and the respective epoxide metabolites [N-(2-carbamoyl-2-hydroxyethyl)valine]. Per unit of administered amount, AA gives rise to higher (three to six times) Hb adduct levels than MAA in mice and rats. Mice exhibit, compared with rats, higher in vivo doses of the epoxy metabolites, indicating that AAs were more efficiently metabolized in the mice. In mouse the two AAs induced dose-dependent increases in both Hb adduct level and MN frequency in peripheral erythrocytes. Per unit of administered dose MAA showed only half the potency for inducing micronuclei compared with AA, although the MN frequency per unit of in vivo dose of measured epoxy metabolite was three times higher for MAA than for AA. No increase in MN frequency was observed in rat bone marrow erythrocytes, after treatment with either AA. This is compatible with a lower sensitivity of the rat than of the mouse to the carcinogenic action of these compounds.     

Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

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 some methylated benzo[a]pyrene derivatives

The mutagenicity of benzo[a]pyrene (BP) and a number of methylated derivatives towards Salmonella typhimurium has been tested. The most mutagenic derivative tested was 6-methylbenzo[a]pyrene which produced about twice the number of revertants as did BP, 11-Methylbenzo[a]pyrene was slightly more mutagenic than BP. All the other compounds tested (7-, 8-, 9- and 10-methylbenzo[a]pyrene and 7,8- and 7,10-dimethylbenzo[a]pyrene) were significantly less active than benzo[a]pyrene. With the exception of 6-methylbenzo[a]pyrene, these results closely parallel the known carcinogenicity of the methylated benzo[a]pyrenes, and support the view that metabolic activation of BP may involve the 7-10 positions which are blocked in the methylated compounds.     

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.     

Analysis of benzo[a]pyrene deactivation mechanisms in rats

The experimental data on the effects of a widespread carcinogen, benzo[a]pyrene (BP), on individual reactions of rats were treated using mathematical-statistical methods. The individual reactions were analyzed in dependence of doses and modes of administration (single or chronic). The analysis revealed a statistically significant correlation between life span and urinary content of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-BP) in rats treated with BP. The calculated regression equations revealed that the individual sensitivity to carcinogen in case of the BP single administration to rats is mainly determined by efficiency of excretion of the BP active forms out of the organism, whereas after chronic BP administration it is determined by mechanisms of enzymatic deactivation of BP.     

The formation of mutagenic derivatives of benzo[a]pyrene by peroxidising fatty acids

Benzo[a]pyrene (B[a]P) when incubated in the presence of peroxidising polyunsaturated fatty acids such as linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5) or docosahexaenoic acid (C22:6) was converted to oxidised products. Between 7% and 9% of the B[a]P was oxidised in one hour when incubated with arachidonic acid and docosahexaenoic acid. 1,6- 3,6- and 6,12-Quinone derivatives of B[a]P were identified by HPLC. The products of B[a]P oxidation were shown to be mutagenic when tested using Sister chromatid exchange (SCE) technique and the occurrence of SCEs in CHV79 cells was increased significantly. Lipid peroxides also induced SCEs in the absence of B[a]P and there was a positive correlation between the frequency of SCEs and the extent of lipid peroxidation. The results indicate that the oxidation of B[a]P mediated by the non-enzymic peroxidation of polyunsaturated fatty acids is likely to play a role in mutagenesis and, possibly, also in carcinogenesis.     

Free radicals derived from benzo[a]pyrene.

At least four different free radicals can be formed from benzo[a]pyrene under different reaction conditions, namely the 6-oxybenzo[a]pyrene radical, the benzo[a]pyrene anion and cation radicals and a radical from heated benzo[a]pyrene. The formation and esr spectra of these radicals have been studied with the aim of clarifying the nature of the radical species involved under different reaction conditions. Additionally the reactivity of the 6-oxybenzo[a]pyrene and the benzo[a]pyrene cation radicals towards several phenolic antioxidants have also been investigated.     

Binding of benzo[a]pyrene by purified cytochrome P-450c

Benzo[a]pyrene (BP) fluorescence-emission intensities in phospholipid micelles are quantitatively described over a broad range of lipid and BP concentrations by excitation that is linearly dependent upon BP concentration and an offsetting excimer quenching that is dependent upon the square of the BP concentration. The fluorescence of BP is quenched by the presence of cytochrome P-450c in proportion to the concentration of the cytochrome in the micelles and in accord with stoichiometric complex formation. Parallel optical titrations indicate a change in spin state of P-450c to a predominantly high-spin state that correlates directly with the percentage fluorescence quenching of complexed BP. Neither change occurs with five other purified forms of rat liver P-450 that have low activity in BP metabolism. N-Octylamine, a ligand that binds to the heme of P-450, competitively inhibits both the spin-state changes and the fluorescence quenching in equal proportion. The Kd for the interaction of BP with P-450c is exceptionally low (10 nM) relative to the Km for monooxygenation (ca. 1 microM). Decreasing the concentration of either dilauroylphosphatidylcholine or dioleoylphosphatidylcholine concomitantly increases the high-spin state (from 30% to 80%) of fully complexed P-450c and the fluorescence quenching (50-100%) of the complexed BP (half-maximal at 80 micrograms of lipid/mL). It is concluded that spin state and fluorescence quenching both reflect the same changes in the interaction of the BP with the P-450 heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of benzo[a]pyrene in human skin xenografts.

Although a human neonatal foreskin graft to a nude mouse has been shown to be morphologically intact for several months after establishment, the feasibility of using this system for carcinogenesis studies has not been widely investigated. In this study, we have investigated the metabolism of benzo[a]pyrene (BaP) in human skin xenografts after the topical application of different concentrations of [3H]BaP (0.5 microgram-10.0 micrograms and 20 muCi/graft) for 2 h and 1.0 micrograms [3H]BaP for various intervals of time up to 4 h. Significant amounts of different organic solvent soluble metabolites were observed in all the different samples. The increase in the amounts of the organic solvent soluble metabolites was linear over the 0.5 microgram to 5.0 micrograms/graft range. When 1.0 microgram [3H]BaP was applied to each graft, the maximum production of the organic solvent soluble metabolites was observed 10 minutes after treatment and it then decreased with time. Diols were the major metabolites detected in each of the experiments, followed by phenols, and then tetrols. The levels of water soluble glucuronide and sulfate conjugates were almost equivalent for each treatment over the same range of application of BaP per graft. The combined levels of these conjugates and the non-extractable organic soluble metabolites in the residue, generally ranged between 7-24% of the total metabolites in the various experiments.     

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.