Metabolism of benzo(a)pyrene by isolated hepatocytes and factors affecting covalent binding of benzo(a)pyrene metabolites to DNA in hepatocyte and microsomal systems

Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrene-treated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxides) and of epoxide derivatives of BP-9-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:1 to 3:1 between 30 and 100 μm BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated.     

Metabolism of benzo(a)pyrene by duck liver microsomes

The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.     

The microsomal metabolism of benzo(a) pyrene phenols.

Analysis of repetitive scan difference spectra of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxybenzo(a)pyrene, oxygen, and NADPH shows the formation of products with absorbance in the 400–450 nm region. Based on the chromatographic retention time, absorbance, and fluorescence spectra, the two major products of 9-hydroxybenzo(a)pyrene metabolism may be diphenols. The existence of spectral intermediates which resemble phenols rather than quinones during the steady-state metabolism of 3-hydroxybenzo(a)pyrene strongly indicates that either the major product is a diphenol which slowly oxidizes to yield 3,6-quinone and/or that an active quinone reductase exists in liver microsomes.     

Metabolic activation of benzo(a)pyrene by human amniotic fluid

The in vitro activation of benzo(a)pyrene was studied in amniotic fluid from ten 4-month pregnant women. Benzo(a)pyrene monooxygenase and epoxide hydrolase activities were in the same range in amniotic fluid as in human liver. Glutathione epoxide transferase activity was markedly lower than in hepatocytes. Human amniotic fluid also catalyzed the formation of hydrocarbon metabolites mutagenic to Salmonella typhimurium TA98 (Ames system). Profiles of amniotic fluid aromatic hydrocarbons from non smokers exhibited low benzo(a)pyrene concentration (less than 0.1 ng/ml).     

The metabolism of benzo[a]pyrene phenols by rat liver microsomal fractions

The oxidative metabolism of benzo[a]pryrene (B[a]P) phenols catalyzed by liver microsomes in vitro leads to multiple products. High-pressure liquid chromatography analysis of the organic-soluble products formed indicates that regardless of the animal pretreatment regime, 3-hydroxy-B[a]P is metabolized to the 3,6-quinone and to a hydroxylated derivative tentatively identified as 3,9-dihyroxy-B[a]P. However, the distribution of products obtained with 9-hydroxy-B[a]P varied with animal pretreatment. A maximum of three distinct metabolites was obtained when the 9-phenol was metabolized in vitro with microsomes from phenobarbital-pretreated rats and the tentative 3,9-dihydroxy derivative was a common metabolite for all pretreatment regimes. Physical characterization, including mass spectrometry, indicates that all three products have an extra oxygen atom incorporated into their molecular structure from molecular oxygen. Studies utilizing specific inhibitors of the cytochrome P-450-dependent monooxygenase clearly suggest that the formation of dihydroxy or phenol-oxide derivatives is catalyzed by the hemoprotein, cytochrome P-450. These metabolites of the benzo[a]pyrene phenols are most likely related to the putative phenol-oxides of benzo[a]pyrene which have been demonstrated to alkylate DNA and protein. Repetitive scan difference spectrophotometric analysis of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxy-B[a]P, NADPH, and oxygen shows the conversion of the phenols into products which absorb in the region from 400 to 500 nm. During and after the steady state of the reaction, it can be seen that certain of the hydroxy compounds produced are in equilibrium with their respective quinone form and may be involved in an oxygen-coupled redox cycle.     

Metabolism and covalent binding of benzo[alpha]pyrene in human peripheral lung

Short-term organ cultures of peripheral lung from lung cancer patients metabolise benzo[alpha]pyrene to ethylacetate-soluble metabolites, which covalently bind to tissue macromolecules. The nature and quantities of metabolites formed and the extent of covalent binding are dependent upon the time of incubation, the substrate concentration and interindividual variability in the metabolic activity of the lung. Individuals whose lungs rapidly metabolise the carcinogen exhibit more extensive further metabolism of primary metabolites and higher levels of covalent binding. Certain striking differences in the relative retention in the tissue or release into the extra-cellular medium of different metabolites have been found as illustrated by the observation that the ratio of 7,8-dihydro-7,8-dihydroxybenzo[alpha]-pyrene to 9,10-dihydro-9,10-dihydroxybenzo[alpha]pyrene was always significantly higher in the tissue than in the extracellular medium.     

Metabolism of benzo(a)pyrene by microsomes from tissues of pregnant and fetal hamsters

Pretreatment of hamsters with benzo (a) pyrene (BaP) greatly increased the $\text{in vitro}$ metabolism of BaP by lung microsomes from pregnant hamsters, and had less effect on the metabolism of BaP by liver microsomes. The production of various metabolites of BaP by lung microsomes was increased to different extents: 3-hydroxy-BaP (3-OH-BaP) was one of the major metabolites; the metabolic yields of 9, 10-dihydrodihydroxy-BaP (9, 10-diol) and 7,8-diol were increased more than that of the 4,5-diol. In the case of liver microsomes, only the yields of 9,10-diol and 7,8-diol were increased over the control levels. The presence of cyclohexene oxide in the incubation mixtures decreased the production of the diols. Basal-level enzyme activities in placental, fetal liver, and fetal skin microsomes in metabolizing BaP were very low. Pretreatment of pregnant hamsters with BaP induced BaP-metabolizing enzymes in fetal tissue 2–3 fold.     

Binding of benzo(a)pyrene to hepatic cytosolic protein enhances its microsomal oxidation

Sephadex G-100 gel permeation chromatography of rat liver cytosol saturated with ¹⁴C-benzo(a)pyrene (BP) resulted in two peaks of protein bound radioactivity. Glutathione-S-transferase (GST) activity (towards 1-chloro 2,4-dinitrobenzene as substrate) was eluted as a single major peak which coincided with one peak of protein bound BP. Oxidation of protein bound BP (GST rich fractions) by microsomes from control or 3-methylcholanthrene treated rats was significantly enhanced as compared to ethanol suspended BP. The formation of oxidized products from the protein-bound BP was dependent on incubation time and microsomal protein concentration, required NADPH and was inhibited by monooxygenase inhibitors α-napthoflavone, 1-benzylimidazole, metyrapone and SKF 525A. Coemergence of BP binding-protein with GST suggests that the soluble protein could be one of the glutathione-S-transferases.     

Liver microsomal DT diaphorase: noninvolvement in hydroxylation of benzo(a)pyrene.

A highly purified reconstituted system isolated from the microsomes of 3-methylcholanthrene-treated rats consisting of cytochrome P-448, NADPH-cytochrome $\text{c}$ reductase and synthetic dilauroyl phosphatidylcholine had no DT diaphorase activity, but hydroxylated benzo[a]pyrene at a faster rate than microsomes from 3-methylcholanthrene-treated rats. DT diaphorase purified from liver microsomes of 3-methylcholanthrene-treated rats when added to this reconstituted system did not stimulate or inhibit benzo[a]pyrene hydroxylation, nor could it replace or NADPH-cytochrome $\text{c}$ reductase in supporting the reaction. We therefore conclude that microsomal DT diaphorase is not involved in microsomal hydroxylation of benzo[a]pyrene to its phenolic products despite the observation that both DT diaphorase activity and the hydroxylation of benzo[a]pyrene are induced by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin     

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.     

Metabolism and mutagenic activation of benzo(a)pyrene by subcellular fractions from mussel (Mytilus galloprovincialis) digestive gland and sea bass (Discenthrarcus labrax) liver

1. The formation of B(a)P diols, phenols and bacterial mutagens by mussel subcellular fractions is dependent on NADPH whereas B(a)P quinones, the major metabolites, appear to be produced by radical reactions and chiefly in the absence of NADPH.2. B(a)P metabolism in sea bass liver fractions is totally dependent on NADPH and insensitive to radical scavengers excepted tocopherol inhibition of B(a)P mutagenesis.3. The 9–10 and 7–8 epoxides formed by sea bass microsomes have a high affinity for EH which readily metabolized all those epoxides to diols.4. Alpha-naphtoflavone inhibits sea bass B(a)P metabolism at high concentration (100 μM) whereas it increases it at low concentration (20 μM).     

Induction,modification and inhibition of rat liver microsomal benzo(a)pyrene hydroxylase; Correlation with the S-9-mediated mutagenicity of benzo(a)pyrene

The effects of various pretreatments $\text{in vivo}$ (3MC, PB, 2 and 4FAA) and of various inhibitors $\text{in vitro}$ (7,8 BF, SKF525A and MN ^R) on the activity of rat liver microsomal BP hydroxylase were analyzed and correlated with the S-9 mediated mutagenicity of BP. 3MC is the only treatment which both induces and modifies the hydroxylase activity; it also specifically increases the enzyme mediated mutagenicity. Miconazole ^R which inhibits all the tested microsomal preparations, also reduces the mutagenicity mediated by all the S-9 preparations whereas the inhibitory effects of 7,8 BF and SKF525A are limited respectively to enzyme preparations from 3MC induced and control or PB treated rats.     

Metabolism of benzo(a)pyrene in rat pleural mesothelial cells in culture

Cultures of Rat pleural mesothelial cells obtained after isolation of parietal cells metabolized benzo(a)pyrene to organosoluble and hydrosoluble derivatives. This property is well maintained since it was observed with cells at 6th passage as well as after 39 passages.     

乳杆菌吸附苯并芘的特性

[目的]探讨植物乳杆菌(Lactobacillus plantarum)121和戊糖乳杆菌(Lactobacillus pentosus)ML32的苯并芘吸附作用与机制.[方法]采用高效液相色谱检测菌体对苯并芘的吸附率.[结果]菌株121和ML32对苯并芘的吸附率分别为65.9%和64.9%,这种吸附特性与菌体活力无关,随培养时间延长、温度提高以及细胞浓度的上升而增加.菌株121和ML32的吸附率在pH 4和5时达到最大,分别为87.6%和89.0%.当培养液中Ca2+或Mg2+浓度大于0.05mol/L时,菌体吸附率与盐离子浓度呈正相关.苯洗脱会导致乳杆菌所吸附的苯并芘减少90%.经碱性蛋白酶、中性蛋白酶、溶菌酶及TCA和SDS等方法处理后,菌体吸附率上升,且不易被苯去除.在胆盐及胃酸环境下,两株菌的吸附率均提高至70%以上,而胰蛋白酶的存在仅对菌株121的吸附率有较大影响.[结论]两株乳杆菌可以通过吸附作用从环境中清除苯并芘,其吸附效果与细菌细胞壁的结构和组成有关.     

Involvement of cytochrome b5 in the hepatic microsomal metabolism of benzo(a)pyrene

Ethanol consumption decreased the specific content of microsomal cytochrome b5 in both chow-and liquid diet-fed hamsters while cytochrome P450 levels were unchanged in chow-fed animals and increased in liquid diet-fed animals. Microsomes from animals receiving ethanol in their drinking water exhibited decreased rates of microsomal aryl hydrocarbon hydroxylase activity and postmitochondrial supernatant mediated mutagenicity of benzo(a)pyrene. In contrast, microsomes from hamsters receiving ethanol in liquid diets showed no changes in either of these two activities. When the observed rates of 7,8 and 9,10 diol formation per nmole P450 for chow-fed animals are plotted vs. the b5/P450 ratio a positive correlation was observed suggesting that cytochrome b5 participates directly in the microsomal metabolism of benzo(a)pyrene.     

Differential activities of rat and human lung glutathione S-transferase isoenzymes towards benzo(a)pyrene epoxides

The isoenzymes of human and rat lung glutathione S-transferase (GST) differ among themselves in their activities towards the epoxides of benzo(a)pyrene (BP). The Ya' and Yc-type subunits of rat lung GST exhibit maximum activities towards BP-4,5-oxide and BP-7,8-oxide suggesting that these two subunits are preferentially involved in the detoxification of highly reactive epoxides and diol-epoxides of polycyclic aromatic hydrocarbons (PAH). The studies with human lung GST isoenzymes indicate that BP-4,5-oxide, and BP-7,8-oxide are preferred substrates for the cationic (pI 8.3) form of the enzyme. Identification of compounds which can selectively induce these isoenzymes of GST could prove useful as inhibitors of PAH induced neoplasia.