Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Hematite (Fe(2)O(3)) enhances benzo[a]pyrene genotoxicity in endotracheally treated rat,as determined by Comet Assay

Since epidemiological studies have firmly implied the co-exposition between iron oxides and polycyclic aromatic hydrocarbons (PAH) as potential etiological factor involved in the excess of mortality by lung cancer in miners, experimental studies have been performed to investigate the role of iron particles on benzo[a]pyrene (B[a]P)-induced lung pathogenesis. In the present study, the alkaline single-cell gel electrophoresis (SCGE; Comet Assay) was used to measure DNA single-strand breaks in four cell types (alveolar macrophages, lung cells, peripheral lymphocytes and hepatocytes) of OFA Sprague-Dawley rats 24h after endotracheal administration of a single dose of an iron oxide (hematite; Fe(2)O(3)) (0.75mg) or B[a]P (0.75mg) or B[a]P (0.75mg) coated onto hematite particles (0.75mg). No damage was observed in cell from the four investigated organs in rats treated with iron oxide alone, while a statistically significant increase in DNA damage was observed compared with control animals in all tested cell types of rats treated with B[a]P alone or in association with hematite. The highest levels of damage were observed in lung cells and peripheral lymphocytes; the levels of damage in alveolar macrophages and hepatocytes were increased, but to a lesser extent compared with the first two cell types.The main finding was to notice a statistically significant increase of the damage in all organs of rats treated with B[a]P coated onto hematite (approximately two-fold increases; P<0.001), versus B[a]P alone. The current study shows that iron particles increase the genotoxic properties of B[a]P in the respiratory tract of endotracheally treated OFA Sprague-Dawley rats. Hence, our data may contribute to explain the excess mortality by lung cancer in epidemiological studies and overall why exposures to B[a]P coated onto Fe(2)O(3) particles resulted in higher toxicity in rodents compared with exposure to B[a]P alone.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites

The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have studied the integrated metabolism of BP in vivo in germ-free rats given (14)C-labeled BP. Urinary metabolites were separated into groups according to acidity using lipophilic ion exchangers. The groups were analyzed by mass spectrometry and were further fractionated by high-performance liquid chromatography. The fraction of urinary metabolites previously shown to contain N-acetylcysteine and glucuronic acid conjugates was found to contain derivatives of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. These compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surprisingly, ring opening is a major pathway for metabolism of BP in the germ-free rat. The dicarboxylic acid may be excreted in urine as an ester glucuronide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3, 4-dicarboxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.     

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.     

Mechanism of antimutagenicity of aquatic plant extracts against benzo[a]pyrene in the Salmonella assay

The mechanism of antimutagenicity of water extracts of grass-wrack pondweed (Potamogeton oxyphylus Miquel), curled pondweed (Potamogeton crispus L.) and smartweed (Polygonum hydropiper L.) towards benzo[a]pyrene mutagenicity in Salmonella typhimurium was investigated. The antimutagenic components in the aquatic plants were water-soluble, heat-resistant and had a high molecular weight; chlorophyll did not play an important role.     

A fluorometric-HPLC assay for quantitating the binding of benzo[a]pyrene metabolites to DNA

A nonradiometric method is presented for quantitating low levels of benzo[a]pyrene (BP) derivatives that are covalently bound to the DNA of BP-treated mice. This method consists of hydrolyzing the DNA with acid to liberate the BP-adducts in the form of the isomeric tetrols of BP. These tetrols have fluorescence quantum yields of ～0.7 in deoxygenated solution at 298 K. Hence they are easily quantitated, following HPLC separation, by means of fluorescence detection. The sensitivity of the method is such that one bound BP residue per 10⁷ bases can be detected in 100 μg of DNA.     

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.     

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.     

A benzo[a]pyrene -7,8-dihydrodiol-9,10-epoxide is the major metabolite involved in the binding of benzo[a]pyrene to DNA in isolated viable rat hepatocytes

Benzo[a]pyrene is metabolised by isolated viable hepatocytes from both untreated and 3-methylcholanthrene pretreated rats to reactive metabolites which covalently bind to DNA. The DNA from the hepatocytes was isolated, purified and enzymically hydrolysed to deoxyribonucleosides. The hydrocarbon-deoxyribonucleoside products after initial separation, on small columns of Sephadex LH-20, from unhydrolysed DNA, oligonucleotides and free bases, were resolved by high pressure liquid chromatography (HPLC). The qualitative nature of the adducts found in both control and pretreated cells was virtually identical; however pretreatment with 3-methylcholanthrene resulted in a quantitatively higher level of binding. The major hydrocarbon-deoxyribonucleoside adduct, found in hepatocytes co-chromatographed with that obtained following reaction of the diol-epoxide, (±)7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with DNA. Small amounts of other adducts were also present including a more polar product which co-chromatographed with the major hydrocarbon-deoxyribonucleoside adduct formed following microsomal activation of 9-hydroxybenzo[a]pyrene and subsequent binding to DNA. In contrast to the results with hepatocytes, when microsomes were used to metabolically activate benzo[a]pyrene, the major DNA bound-product co-chromatographed with the more polar adduct formed upon further metabolism of 9-hydroxybenzo[a]pyrene. These results illustrate that great caution must be exercised in the extrapolation of results obtained from short-term mutagenesis test systems, utilising microsomes, to in vivo carcinogenicity studies.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

The effect of norharman on the metabolism of benzo[a]pyrene by rat-liver microsomes in vitro in relation to its enhancement of the mutagenicity of benzo[a]pyrene

The effect of norharman on the metabolism of benzo[a]pyrene by rat-liver microsomes was studied. Separation of the metabolites into hydrophilic and hydrophobic fractions showed that norharman inhibited the conversion of hydrophobic metabolites to hydrophilic ones.Analysis of the hydrophobic metabolites by high-pressure liquid chromatography showed that norharman also inhibited the disappearance of benzo[a]pyrene itself. However, large amounts of hydrophobic metabolites, such as phenol, quinones and diols, were formed in the presence of norharman, and formation of the strong mutagen 7,8-dihydroxybenzo[a]pyrene was increased 10-fold by norharman. The increase in formation of this compound may be one of the chief reasons why norharman enhances the mutagenicity of benzo[a]pyrene on Salmonella typhimurium.     

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.     

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     

Modulation of the cytotoxicity and mutagenicity of benzo[a]pyrene and benzo[a]pyrene 7,8-diol by glutathione and glutathione S-transferases in mammalian cells (CHO/HGPRT assay)

Biologically reactive metabolites of benzo[a]pyrene (BP) and benzo[a]-pyrene 7,8-diol (BP-diol), formed by the mixed-function oxidase (MFO) system, are substrates for conjugation and detoxication by glutathione (GSH) when catalyzed by glutathione S-transferases (GSHT). We have investigated the detoxication of BP- and BP-diol-induced cytotoxicity and mutagenicity with GSH by supplementing the S9 mix used in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) assay with GSH (6.5 mM) or GSH plus GSHT. The addition of GSH to the S9 mix resulted in a reduction of BP- and BP-diol induced cytotoxicity. GSH plus GSHT eliminated BP-induced cytotoxicity and reduced the mutagenicity of BP. GSH inhibited the mutagenicity at low (essentially non-lethal) concentrations of BP-diol, but did not do so at toxic concentrations. GSH plus GSHT inhibited the cytotoxicity and mutagenicity of BP-diol at concentrations not affected by GSH alone. These studies indicate that biochemical mechanisms of detoxication can affect the biological activity of a carcinogen, such as BP or BP-diol as profoundly as bioactivation by the MFO system.     

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.