Separation of water-soluble metabolites of benzo[a]pyrene formed by cultured human colon.

A method has been developed to separate conjugated metabolites of benzo[a]pyrene into three major fractions: sulfate esters, glucuronides and glutathione conjugates. In cultured human colon, formation of sulfate esters and glutathione conjugates is the major conjugation pathway, while formation of glucuronides accounts for only 6 per cent of the water-soluble metabolites. Hydrolysis of the sulfate esters with arylsulfatase and the glucuronides with β-glucuronidase released metabolites of benzo[a]pyrene that were extractable with organic solvent. Separation of these metabolites by high-pressure liquid chromatography indicated that trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene,7,8,9, 10-tetrahydro-7,8,9, 10-tetrahydroxybenzo[a]pyrene and trans-9, 10-dihydro-9, 10-dihydroxybenzo[a]pyrene were the major substrates for UDP-glucuronic acid transferase, while trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene were the major substrates for sulfotransferase in cultured human colon.     

Studies on the metabolism and excretion of Benzo(a)pyrene in isolated adult rat hepatocytes

[³H]Benzo(a)pyrene is metabolised by isolated rat hepatocytes to both ethyl acetate-soluble metabolites, which co-chromatograph with 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene and its sulphate ester, benzo(a)pyren-3-yl-hydrogen sulphate, and to water-soluble metabolites. Hydrolysis of the water-soluble metabolites with β-glucuronidase release ethyl acetate-soluble metabolites which co-chromatograph with 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 4,5-dihydro-4,5-dihydrobenzo(a)pyrene, 9,10-dihydroxybenzo(a)pyrene (9,10-catechol) and 3-hydroxybenzo(a)pyrene. During the incubation significant differences in the distribution of metabolites between the cells and the extracellular medium are observed. Initially the cells produce predominantly ethyl acetate-soluble metabolites, which are only partly released into the extracellular medium, but at later times in the incubation a greater percentage of the metabolites are further metabolised to water-soluble conjugates which are very readily released from the cells. Individual ethyl acetate-soluble metabolites show significant distributional differences. Monohydroxybenzo(a)pyrenes accumulates intracellularly and only low amounts are released into the medium. Sulphate esters of monohydroxybenzo(a)pyrenes such as benzo(a)pyren-3-yl-hydrogen sulphate also accumulate intracellularly, although to a lesser extent than the monohydroxybenzo(a)pyrenes. 4,5-Dihydro-4,5-dihydroxybenzo(a)pyrene and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene are distributed more evenly between cells and medium whereas 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene is found mainly in the medium. Significant amounts of radioactivity are bound irreversibly to cellular macromolecules.     

Linoleate-dependent co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by rat cytosolic lipoxygenase

1. Co-oxygenation of ¹⁴C-labelled benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol was studied in rat lung cytosol, using linoleic acid as a co-substrate. Covalently bound and soluble metabolites were quantified by radiometry and?h.p.l.c., respectively.

2. The co-oxygenation resulted in the production of reactive metabolites capable of protein binding as well as a series of soluble derivatives.

3. Co-oxygenation of benzo(a)pyrene yielded primarily a significant amount of benzo(a)pyrene-6,12-dione while benzo(a)pyrene-7,8-dihydrodiol led to a significant amount of benzo(a)pyrene-trans-anti-tetrol.

4. Their production was abolished by addition of 25 μM of the lipoxygenase inhibitor and antioxidant NDGA.

5. It is postulated that the lineoleic acid peroxyl radicals, formed by rat lung lipoxygenase, initiate the one-electron oxidation of benzo(a)pyrene to its quinones, and epoxidation of benzo(a)pyrene-7,8-diol to the ultimate carcinogenic benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide.     

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [¹⁴C]benzo[a]pyrene (3 μmol/kg) to rabbits, 30% of the ¹⁴C dose appeared in bile and 12% in urine, within six hours.

2. Biliary and urinary metabolites were mainly conjugated; <12% of the ¹⁴C was extractable with ethyl acetate, but after treatment with β-glucuronidase or aryl sulphatase 30–40% became extractable.

3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of ¹⁴C in bile and 24% of ¹⁴C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total <10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total <4%).

4. The proximate carcinogen, the 7,8-diol, was not detected in any extract.

5. After intraduodenal administration of biliary metabolites of [¹⁴C]benzo[a]pyrene (approx. 0·3 μmol), ¹⁴C was excreted in the bile (21% dose) and urine (14%) within 23?h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.     

Metabolism of benzo[a]pyrene in isolated human scalp hair follicles.

Human scalp hair follicles contain an enzyme system that metabolizes the carcinogen benzo[a]pyrene. The major ethyl acetate soluble metabolites are 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene,9,10-dihydro-9,10-dihydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene. Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO), an inhibitor of epoxide hydratase, prevents the formation of the dihydrodiols. The overall metabolism can be inhibited by the addition of alpha-naphthoflavone. The metabolism of benzo[a]pyrene in a cell culture of human scalp hair follicles has also been investigated. The results show that the activity of arylhydrocarbon hydroxylase (AHH) and epoxide hydratase (EH) is maintained in culture.     

Hydroxylation of benzo[a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450: Effect of 7,8-benzoflavone,butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (?)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene[(?)trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM₂ and a concomitant 3-fold enhancement in microsomal benzo[a]pyrene hydroxylase activity, whereas the conversion of (?)trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM₂ exhibited the highest activity and specificity toward benzo[a]pyrene and the lowest activity toward (?)trans-7,8-diol. Conversely, P-450LM₄ was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (?)trans-7, 8-diol but not of benzo[a]pyrene. Homogeneous P-450LM₄ preferentially Catalyzed the oxygénation of (?)trans-7,8-diol, but was largely ineffective with benzo[a]pyrene. Partially purified P-450LM₇ lacked substrate specificity, for it metabolized both benzo[a]pyrene and (?)trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[a]pyrene hydroxylation by P-450LM₄ and phenobarbital-induced microsomes, as well as (?)trans-7,8-diol metabolism by P-450LM₄ and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM₄ and LM₂ with benzo[a]pyrene and (?)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM₂ and LM₄, respectively.     

Benzo(a)pyren-3-yl hydrogen sulphate,a major ethyl acetate-extractable metabolite of benzo(a)pyrene in human,hamster and rat lung cultures

Benzo(a)pyrene is metabolised by human bronchial epithelium to ethyl acetate-extractable metabolites which co-chromatograph with 9,10-dihydro-9,10-dihydroxybenzo(a) pyrene and 7,8-dihydro-7,8-dihydroxybenzo(a) pyrene, whereas little 4,5-dihydroxybenzo(a)pyrene and 3-dydroxybenzo(a)pyrene are formed. Similar results are obtained with human lung except that a major ethyl acetate-soluble metabolite (X) is observed. X has been identified as benzo(a)pyren-3-yl hydrogen sulphate on the basis of enyymic and acid hydrolysis experiments, incorporation of [³⁵S]sulphate and its u.v. and fluorescence spectra which were similar to those of the synthetic metabolite. The u.v. absorption spectrum of benzo(a)pyrene-3-yl hydrogen sulphate is comparable with the X₁ metabolite of benzo(a)pyrene, one of the principal metabolites of unestablished identity found in earlier in vivo studies. The biological activity of the sulphate ester of 3-hydroxybenzo(a)pyrene is of interest as this metabolite could be extremely persistent in man because of its physico-chemical properties, which may prevent its excretion in the urine and bile.     

The mechanistic plurality of cytochrome P-450 and its biological ramifications

1. The mechanistic plurality of the microsomal cytochrome P-450 enzyme system is illustrated by studies of the oxidative metabolism of benzo[a]pyrene, 3-hydroxybenzo[a]pyrene and arachidonic acid.

2. Rat liver microsomal metabolism of benzo[a]pyrene or 3-hydroxy-benzo[a]pyrene, supported by cumene hydroperoxide, generates benzo[a]pyrene quinones via molecular oxygen-dependent and -independent pathways.

3. Arachidonic acid is metabolized by rat liver microsomal fractions to a variety of oxygenated products, including cis-trans diene conjugated monohydroxy-acids, epoxy-acids as well as ω- and ω — 1 -oxidation products. The chemistry of the different reaction products is discussed in terms of the possible mechanisms responsible for their formation and the role of the haemoprotein during catalysis.

4. An integrated view for the reaction cycle of cytochrome P-450 is presented.     

Metabolism and cytotoxicity of benzo(a)pyrene in the human lung tumour cell line NCI-H322

1. The human lung tumour cells NCI-H322 metabolized benzo(a)pyrene (BP) at a rate of 160 pmol/10⁶ cells/h for at least 8 h. About 30% of the total metabolites were water-soluble, 30% of which were conjugates with glutathione. The water-soluble fraction also contained BP sulphates but no BP glucuronides.

2. The cytotoxic potency of BP and its metabolites, 3-hydroxybenzo(a)pyrene (3-OH-BP) and (±) anti-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (7,8-diol-BP), differed by an order of magnitude with the ranking 7,8-diol-BP > BP > 3-OH-BP. The cytotoxicity of BP was not detected at the end of a 24 h treatment period but became increasingly apparent at later times. In contrast, the cytotoxicity of 3-OH-BP was observed immediately after the treatment period and did not increase greatly thereafter. 7,8-Diol-BP caused both ‘immediate’ and ‘late’ effects.

3. The time course and concentration dependence suggested that the cytotoxicity of BP in NCI-H322 cells was not attributable to the formation of 3-OH-BP, but more likely resulted from the formation of other products such as 7,8-diol-BP.     

Metabolism of (+)-trans-benzo[a]mpyrene-7,8-dihydrodiol by 3-methylcholanthrene-induced rat liver homogenates

Using a new sensitive reverse-phase HPLC assay with on-line radioactivity detector, metabolism of (+)-trans-benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol) to the ultimate carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) was studied using 3-methylcholanthrene-induced rat liver homogenates. The results demonstrate that the stereoselectivity of B[a]PDE formation is a function of the concentration of the cellular constituents in the incubation media. At more dilute concentrations of the homogenate, the ratio of anti- to syn-B[a]PDE was the highest and decreased as the homogenate protein was increased in the incubation medium. However, there was a marked and parallel decrease of free B[a]PDE and DNA-bound radioactivity with increasing concentrations of cellular constituents in the incubation medium. The decreased DNA-bound radioactivity appears to be due to the preferential binding of B[a]PDE to glutathione and to proteins as the homogenate concentration was increased in the incubation media. These results indicate that liver homogenates, while apparently preserving the function of microsomes, present additional opportunities to study the interrelationship among cytochrome P450 monooxygenase activity, water-soluble conjugates, and binding of B[a]P diol metabolites to macromolecules in the study of benzo[a]pyrene-induced carcinogenesis.     

Exposure of two species of deposit-feeding amphipods to sediment-associated [3H]benzo[a]pyrene: Uptake,metabolism and covalent binding to tissue macromolecules

Two species of deposit-feeding marine gammaridian amphipods, Rhepoxynius abronius and Eohaustorius washingtonianus, were exposed to sediment-associated [³H]benzo[a]pyrene (BaP) at 12±1°C. Concentrations of BaP-derived radioactivity increased with time in both E. washingtonianus and R. abronius, and the levels of radioactivity were similar in both species after 7 days of exposure. A significantly (P<0.05) lower proportion (51%) of unconverted BaP was present in R. abronius than in E. washingtonianus (73%) at 1 day. The proportion of unconverted BaP decreased with time in R. abronius (30% at 7 days), but did not vary significantly with time in E. washingtonianus. Reverse-phase high-pressure liquid chromatography (HPLC) of organic solvent-soluble metabolites released from β-glucuronidase and arylsulfatase treated tissue extracts of amphipods showed the presence of metabolites such as 7,8-dihydroxy-7,8-dihydroBaP (BaP-7,8-diol), 9,10-dihydro-9,10-dihydroxyBaP (BaP-9,10-diol), 3-hydroxyBaP and 9-hydroxyBaP. The ratio of BaP-7,8-diol to BaP-9,10-diol obtained from normal-phase HPLC was 1.2 for R. abronius and 0.7 for E. washingtonianus. Moreover, the level of covalent binding of BaP intermediates to macromolecules was significantly (P≤0.01) higher at 7 days in R. abronius (7.00±0.98 pmol BaP equivalents/g-protein) than in E. washingtonianus (4.08±0.51 pmol/g-protein). Thus, using BaP as a representative of sediment-associated polynuclear aromatic hydrocarbons, it was shown that although both amphipod species accumulated similar concentrations of BaP-derived radioactivity, R. abronius converted a higher proportion of BaP into potentially toxic intermediates.     

Disposition and metabolic fate of benzo[a]pyrene in the common carp

The biological fate of intraperitoneally administered benzo[a]pyrene (BP) was investigated in the wild strain of common carp (Cyprinus carpio) maintained in flowing water. The tissue distribution of BP-derived radioactivity was determined at 24 and 72 h after treatment. Bile contained high concentrations of BP-derived radioactivity (7.9 μg BP equivalents/g bile at 72 h), which amounted to 25% of the radioactivity found in the fish at this time point. The major components of bile collected at 72 h were glucuronides (54%), sulfates (12%) and unmetabolized BP (14%). The potentially genotoxic metabolite BP-7,8-dihydrodiol and its glucuronide represented 0.7 and 20%, respectively, of the radioactivity of the bile at 72 h. High concentrations of BP-derived radioactivity were also present in liver and gonads (1,060 and 843 ng equivalents of BP/g tissue at 24 h, respectively). Muscle contained 10% of the BP-derived radioactivity found in the fish (46 ng eq/g at 24 h). No significant change was found between 24 and 72 h in the concentrations or proportional amounts of BP-derived radioactivity except in liver, in which the proportional amount decreased from 19 to 10% of the total found in the fish. In muscle, unmetabolized BP represented 95% of the radioactivity that could be extracted with ethyl acetate and characterized by HPLC. At 24 h BP-7,8-diol represented 0.6% of this extractable radioactivity in muscle and 1.0% of the extractable (0.4% of the total) radioactivity in liver. These data indicate that carp tissues produce significant amounts of BP-7,8-diol and its glucuronide, potentially capable of being converted (after hydrolysis of the glucuronide) to active metabolites capable of alkylating DNA and other macromolecules. However, only relatively small amounts of covalently bound radioactivity were found (amounting to 4% and 7% of the total radioactivity present in muscle and liver, respectively).     

Comparison of benzo(a)pyrene metabolism by testicular homogenate and the isolated perfused testis of rat following 2,3,7,8-tetrachlorodibenzo-P-dioxin treatment

Benzo(a)pyrene (BP) metabolism was studied in the cell free testicular homogenate and in the isolated perfused rat testis 72 h following tetrachlorodibenzo-P-dioxin (TCDD). The BP concentration for both metabolic systems was 2 × 10^–7 M. BP metabolites were extracted from testicular homogenate, perfusate and testicular tissue and subjected to high-pressure liquid Chromatographic analysis. The ratio of various BP metabolites in the cell free homogenates ranged from 3.5 to 164 times those of the isolated perfused testis, and the total BP metabolites in the cell free system of either control or TCDD-induced testis were 16 times that of the intact isolated perfused testis. The major BP metabolites in the organic extractable phase from the isolated perfused testis and the testicular homogenate were BP dihydrodiols and BP phenols, respectively.The ratio of water soluble metabolites to organic soluble metabolites in the homogenate and the isolated perfused testis is 1.1 and 3.0 respectively. Therefore, in the intact isolated testis, water soluble BP metabolites are formed three times greater than those of the organic soluble BP metabolites, and thus suggests that specific conjugating enzyme activities in the intact testis are greater than those of the homogenates. The magnitude of various BP metabolites in either the homogenates or the isolated perfused testis of TCDD treated rats ranged from 1.4 to 2.2 times their respective controls, except 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene in the isolated perfused testis was not altered by TCDD treatment. In conclusion, the isolated perfused rat testis is metabolically active and capable of biotransforming PAH. This system better reflects metabolic capability of the intact organ in the rats than do cell free homogenate system. The isolated perfused testis system retains the integrity of the complex biological organization of tissues, cell types, and enzymes in testicular metabolism and may provide data that may aid in the prediction of germ cell mutation as well as toxicity.The Abbreviations Used are PAH polycyclic aromatic hydrocarbons - BP benzo(a)pyrene - 9,10-diol 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene - 7,8-diol 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene - 4,5-diol 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene - 3-OH 3-hydroxybenzo(a)pyrene - 9-OH 9-hy-droxybenzo(a)pyrene - 7-OH 7-hydroxybenzo(a)pyrene - 12-OH 12-hydroxybenzo(a)pyrene - 1,6-quinone benzo(a)pyrene-1,6-dione - 3,6-quinone benzo(a)pyrene 3,6-dione - 6,12-quinone benzo(a)pyrene 6,12-dione - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 7,8-diol 9,10-epoxide 5,7-t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene - HPLC high pressure liquid chromatography - TCDD 2,3,7,8-tetrachlorodibenzo-P-dioxin - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - NADP⁺ nicotinamide adenine dinucleotide - AHH aryl hydrocarbon hydroxylase - EH epoxide hydrolase - GSH-T glutathione transferase - HPRT hypoxanthine phosphoribosyltransferase     

The metabolism of benzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene by short-term organ cultures of hamster lung

The metabolism of benzo(a)pyrene and two of its metabolites 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (7,8-dihydrodiol) and 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene (9,10-dihydrodiol) to both ethyl acetate-soluble and water-soluble metabolites has been studied using short-term organ cultures of hamster lung. Benzo(a)pyrene is metabolised to ethyl acetate-soluble metabolites which co-chromatograph with 9,10-dihydrodiol, 7,8-dihydrodiol and benzo(a)pyren-3-yl hydrogen sulphate but little or no 3-hydroxybenzo(a)pyrene and 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene (4,5-dihydrodiol) are detected. After culture with benzo(a)pyrene, the amount of 9,10-dihydrodiol in the medium is 9-fold greater than the amount of 7,8-dihydrodiol. Benzo(a)pyrene is also metabolised by short-term organ cultures of hamster lung to water-soluble metabolites, which on hydrolysis with β-glucuronidase yield metabolites co-chromatographing with 3-hydroxybenzo(a)pyrene, quinones, 4,5-dihydrodiol and 7,8-dihydrodiol. However little or no 9,10-dihydrodiol is detected. Both 7,8- and 9,10-dihydrodiols are metabolised by cultures of hamster lung to an ethyl acetate-soluble metabolite which co-chromatographs and has similar fluorescence excitation and emission spectra to 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxybenzo(a)pyrene (7,8,9,10-tetrahydrotetrol). More 7,8,9,10-tetrahydrotetrol is formed from 7,8-than 9,10-dihydrodiol. A major route for metabolism of 7,8-dihydrodiol is conversion into water-soluble metabolites, which on hydrolysis with β-glucuronidase yield an ethyl acetate-soluble metabolite co-chromatographing with 7,8-dihydrodiol. However only small amounts of water-soluble metabolites are observed after short-term organ culture with 9,10-dihydrodiol. The amount of covalent binding after short-term organ culture with 7,8-dihydrodiol was greater than that with 9,10-dihydrodiol and benzo(a)pyrene. This was in agreement with the many observations showing the high biological activity of the further metabolite of 7,8-dihydrodiol, i.e. 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide. These results however, also raise the possibility of a role for 9,10-dihydrodiol as a precursor of active metabolites.     

Comparison of benzo(a)pyrene metabolism in isolated perfused rat lung and liver

The metabolism of 1 mM benzo(a)pyrene was studied in isolated perfused lung and liver of 5,6-benzoflavone-pretreated rats. Benzo(a)pyrene metabolism by the liver was more rapid than by the lung, but total metabolite formation in the lung at the end of a 120-min perfusion period was comparable to that in the liver. Lung perfusate was characterized by high concentrations of free metabolites, with diols outweighing phenols; in liver perfusate free metabolite concentrations were low, and large quantities of metabolites were found as conjugates in the bile at the end of perfusion. The tissue concentrations of free diols and phenols including the precursors of the main DNA-binding secondary metabolites were higher in the lung than in the liver. These findings explain the similar level of covalent binding in perfused lung and liver previously described (Klaus et al. 1982).Abbreviations Used BP benzo(a)pyrene - 9,10-diol 9,10-dihydro9,10-dihydroxy-benzo(a)pyrene - 4,5-diol 4,5-dihydro-4,5-dihydroxy-benzo(a)pyrene - 7,8-diol 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene - 9-OH 9-hydroxy-benzo(a)pyrene - 3-OH 3-hydroxybenzo(a)pyrene - tetrols 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy-benzo(a)pyrenes - BF 5,6-benzoflavone - TLC thin-layer chromatography - HPLC high-pressure liquid chromatography     

Vitamin K epoxide reductase activity in the metabolism of epoxides

The importance of vitamine K epoxide reductase for the metabolism of a range of structurally diverse epoxides has been investigated. Vitamin K₁ epoxide is reduced by rat liver microsomes at a rate of 0.47 nmoles/g liver/min. The rate of menadione oxide reduction is not significantly higher than the non-enzymatic reduction rate. No measurable reduction of benzo[a]pyrene 4,5-oxide, benzo[a]pyrene 7,8-oxide, phenanthrene 9,10-oxide, styrene 7,8-oxide, and dieldrin has been detected, nor could trichothecene T-2 toxin inhibit reduction of vitamin K₁ epoxide. Thus, vitamin K epoxide reductase is very specific for vitamin K₁ epoxide. Taking into account the range of structurally diverse epoxides investigated and the high specific activities of microsomal epoxide hydrolase and cytosolic glutathione transferase for these epoxides it may be concluded that vitamin K epoxide reductase, in all likelihood, generally does not significantly contribute to the control of epoxides metabolically formed from xenobiotics.     

The intestinal metabolism and DNA binding of benzo[a]pyrene in guinea-pigs fed normal,high-fat and high-cholesterol diets

1. Strains of intestinal bacteria were capable of deconjugating benzo[a]pyrene metabolites in vitro. The hydrolysis products, and other primary oxidative metabolites of benzo[a]pyrene, were stable to further degradation by the strains tested.

2. Cytochromes P-450 and b5 were detectable in the mucosa of the guinea-pig small intestine, but not in the mucosae of the colon or rectum. The concentrations were unaltered by administration of benzo[a]pyrene and/or the feeding of high-fat or high-cholesterol diets.

3. Benzo[a]pyrene hydroxylase was measurable in the mucosa of the upper intestine, but was present in the lower gut only at very low levels in some animals. The activity was inducible, by oral administration of benzo[a]pyrene, in the small intestinal mucosa of guinea-pigs fed normal diet but not in those fed high-fat and high-cholesterol diets.

4. Low levels of covalent binding of ³H to DNA of liver and gut mucosa were obtained in guinea-pigs dosed orally with ³H-benzo[a]pyrene. Comparison with data for animals given ³H₂O suggested that approx. one quarter of the binding was probably due to ³H exchange during metabolism. The feeding of high-fat and high-cholesterol diets did not increase this binding. Guinea-pigs fed high-fat and high-cholesterol diets excreted a greater proportion of an oral dose of ³H-benzo[a]pyrene in urine, and less in faeces than animals fed a normal diet.

5. Due to the low, and apparently non-inducible, levels of benzo[a]pyrene hydroxylase activity and of covalent binding in the colonic mucosa, the administration of benzo[a]pyrene to guinea-pigs fed high-fat or high-cholesterol diets appears unlikely to provide a novel animal model for studies on mechanisms of colon carcinogenesis.     

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.     

Mutagenicity and in vivo disposition of biliary metabolites of benzo(a)pyrene

Rabbits and rats administered [3H]benzo(a)pyrene (BP; 40 mumol/kg, i.v.) excreted, via the bile, metabolites which increased reverse gene mutation frequency in Salmonella typhimurium TA98 when incubated with beta-glucuronidase. Glucuronic acid conjugates of BP 4,5-diol, BP 1,6-, 3,6- and 6,12-quinones were detected in rat bile with low levels of 3- and 9-OH BP and BP 7,8- and 9,10-diols. In rabbits BP 9,10-diol was the major aglycone along with smaller amounts of BP 1,6- and 3,6-quinones, BP 4,5- and 7,8-diols and 3- and 9-OH BP. Qualitatively similar metabolic profiles were found when animals were given 3 mumol/kg [3H]BP. When 3H-labelled biliary metabolites, which contained the mutagenic component, were administered intraduodenally to rats, radioactivity reached the systemic circulation but DNA adducts were not detectable (less than 0.03 pmol/mg DNA) in tissues (intestinal wall, liver and lung) exposed to the reabsorbed metabolites.     

The uptake and distribution of [3H]benzo[a]pyrene in the Northern pike (Esox lucius). Examination by whole-body autoradiography and scintillation counting

The uptake and distribution of the polyaromatic hydrocarbon benzo[a]pyrene in Northern pike (Esox lucius) were investigated by whole body autoradiography and scintillation counting. [³H]Benzo[a]pyrene was administered either in the diet or in the water. The levels of this xenobiotic employed corresponded to levels found in moderately polluted water. The uptake and distribution of this compound and its metabolites were followed from 10 hr to 21 days after the initial exposure. The autoradiography patterns observed here with both routes of administration suggest, as expected, that benzo[a]pyrene is taken up through the gastrointestinal system and the gills, metabolized in the liver, and excreted in the urine and bile. Other findings indicate that the gills may not be a major route of excretion for benzo[a]pyrene and its metabolites in the Northern pike; that benzo[a]pyrene may be taken up from the water directly into the skin of this fish; that benzo[a]pyrene and its metabolites are heterogeneously distributed in the kidney of the Northern pike; and that very little radioactivity accumulates in the adipose tissue. With scintillation counting, uptake of radioactivity from the water was found to occur rapidly in all organs, reaching a plateau in most cases after about 0.8 days. The concentrations of radioactivity in different organs ranged between 50 (many organs) and 80,000 (gallbladder + bile) times that found in the surrounding water. Since most of the radioactivity recovered in different organs of the pike after 8.5 days of exposure was in the form of metabolites, we feel that metabolism may play an important role in the bioconcentration of xenobiotics in fish.