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.     

Variations among untreated rabbits in benzo(a)pyrene metabolism and its modulation by 7,8-benzoflavone

The metabolism of benzo(a)pyrene by rabbit liver microsomes can be stimulated or inhibited by 7,8-benzo(a)flavone (ANF) depending on the distribution of specific P-450 enzymes present within the microsomes. Treatment of rabbits with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or rifampicin leads to an increase of hepatic microsomal metabolism of benzo(a)pyrene. ANF stimulates the rate of benzo(a)pyrene metabolism catalyzed by microsomes isolated from rabbits treated with rifampicin by 3-fold. In contrast, ANF moderately inhibits the activity of microsomes from TCDD-treated rabbits. Variations in the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits apparently reflect differences in the expression of P-450 1, a constitutive form of P-450. Thus, the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits, which varies from 0.40 to 1.5 nmol/min/mg of protein, is directly correlated with the microsomal concentration of P-450 1. The metabolism of benzo(a)pyrene by microsomes containing high concentrations of P-450 1 is inhibited by a monoclonal antibody specific for this cytochrome to approximately the rate exhibited by microsomes with a low concentration of P-450 1. The benzo(a)pyrene activity stimulated by ANF in microsomes with a low concentration of P-450 1 is not inhibited by the monoclonal antibody. The activity of P-450 1 is inhibited by ANF at concentrations that stimulate other constitutive forms of P-450. Thus, ANF produces offsetting effects on benzo(a)pyrene metabolism in microsomes from untreated animals by stimulating the activity of at least one cytochrome and inhibiting P-450 1-mediated activity.     

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.     

Distribution and macromolecular binding of benzo[a]pyrene in SENCAR and BALB/c mice following topical and oral administration

When benzo[a]pyrene (BaP) is used as the initiator in initiation-promotion assays, the topical route of administration has been shown to produce a greater epidermal tumor incidence than do other routes of administration, particularly the oral route. In addition, different strains of mice exhibit varying degrees of susceptibility to two-stage epidermal tumorigenesis using BaP. The SENCAR strain is known to be far more sensitive to epidermal tumor formation following BaP initiation than are other strains, such as the BALB/c strain. To investigate the possible contribution of distribution and binding to DNA in such route and strain differences, the distribution and macromolecular binding of [3H]BaP was examined in the skin, liver, lung, and stomach of SENCAR and BALB/c mice following topical or oral administration of BaP at time periods ranging from 0.5 to 48 h. Levels of labeled material in skin were higher, and the binding of BaP to epidermal DNA was greater following topical administration than following oral administration for mice of both strains. The much greater binding of BaP to epidermal DNA following topical administration may account for the much greater epidermal tumor incidence in mice following topical administration of BaP. Following topical administration, BALB/c mice had generally higher levels of labeled material in whole skin than did SENCAR mice, and the binding of BaP to epidermal DNA at 48 h was greater in BALB/c mice than in SENCAR mice following either route of administration. Thus, the known differences between these two strains in susceptibility to epidermal tumor formation when BaP is used as an initiator cannot be explained on the basis of differences in tissue distribution or the amount of binding to epidermal DNA at the time periods examined.     

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 microM of the lipoxygenase inhibitor and antioxidant NDGA. 5. It is postulated that the linoleic 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.     

Prenatal induction of benzo(a)pyrene hydroxylases in mice

1. Benzo(a)pyrene hydroxylase (BPH) activity was measured in homogenates of fetal liver (day 18) or of whole-embryos of mice on day 9, 10 or 12 of gestation after maternal pretreatment with B(a)P on 3 consecutive days. A³H-liberation assay with³H-B(a)P labelled either generally or at the 6-position was used. The values obtained with the embryonic/fetal tissues were compared with those found in maternal liver. 2. Three oral doses of 17.5 mg B(a)P/kg body wt were found to just significantly induce BPH in maternal liver. An induction was observed after pretreatment with 24 mg B(a)P/kg body wt in 9-, 10-or 12-day-old whole-embryos, but the V_max reached was only 10–20% (1% on day 9) of that of adult non-induced liver. The K_m (6-hydroxylation) for all tissues tested were in the same range (600–900 nM). The induction was demonstrable in embryos at tissue levels about one order of magnitude lower than those required for induction in maternal liver. 3. Treatment with 25 mg B(a)P/kg body wt on 3 consecutive days was required to induce BPH in fetal liver on day 18 of gestation. The required B(a)P tissue concentrations were about one half of those necessary for induction in maternal liver. 4. Among a variety of other polycyclic hydrocarbons only chrysene showed an inducing potency similar to that of B(a)P in adult and fetal liver. For all compounds tested there was no correlation found in the inducing potency between adult and fetal liver (e.g. coronene). 5. The doses required to induce BPH in the maternal or fetal liver or in whole embryos of rodents are significantly higher (mg range) than those of usual average human exposure or those taken up by smokers (ng range).Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo(a)pyrene - BPH benzo(a)pyrene hydroxylases - PAH polycyclic aromatic hydrocarbons     

Ovarian toxicity of benzo(a)pyrene and metabolites in mice

The effect of intraovarian injection of benzo(a)pyrene (BP) or one of three metabolites: +7,8-oxide (7,8-O), (-)-dihydrodiol (DHD), and (+)-diol-epoxide-2 (DE2) on ovarian volume, weight, and follicle number was investigated in DBA/2N (D2), C57BL/6N (B6), and (DBA/2N x C57BL/6N)F1 (F1) mice. Female mice, 6 to 8 weeks old, were treated by injection into the right ovary with the indicated compound (10 micrograms in 1 microL DMSO). The left ovary was untreated. Two weeks following treatment both ovaries were removed, fixed in Bouin's medium, serially sectioned, and stained with hematoxylin and eosin. Right ovarian weight was decreased in D2 mice treated with BP (P less than 0.01 and DHD (P less than 0.01). Left ovarian weight was increased in D2 mice treated with DE2 (P less than 0.05). BP decreased right ovarian volume in D2 (P less than 0.01) and F1 (P less than 0.01) mice. 7,8-O decreased right ovarian volume in D2 mice (P less than 0.05). DHD decreased right ovarian volume in D2 (P less than 0.01) and F1 (P less than 0.05) mice. DE2 decreased right ovarian volume in D2 (P less than 0.01) and F1 (P less than 0.01) mice. Left ovarian volume was increased in B6 (P less than 0.01) and D2 (P less than 0.05) mice treated with DE2. The number of small follicles was decreased in D2, B6, and F1 mice treated with DE2 (P less than 0.01). BP and DHD also decreased small follicle number in D2 and F1 mice (P less than 0.01). The number of growing follicles was decreased in B6, D2, and F1 mice treated with DE2 (P less than 0.01). Treatment with DHD decreased the number of growing follicles in D2 mice (P less than 0.05). The number of antral follicles was reduced in F1 mice treated with BP (P less than 0.05), DHD (P less than 0.01), and DE2 (P less than 0.01). The number of antral follicles was also reduced in B6 mice treated with DE2 (P less than 0.01) and in D2 mice treated with DHD (P less than 0.05) and D2 mice treated with DE2 (P less than 0.01). These experiments suggest that toxic effects to one ovary may result in compensatory hypertrophy of the contralateral ovary. Morphometric analysis of the ovary, including ovarian volume, represents a useful objective measure of ovarian toxicity.     

小鼠苯并(a)芘的急性免疫毒性

<正> 苯并(a)芘(B(a)P),是煤焦油、煤烟及其它燃料不完全燃烧的产物,不仅污染了人类的生产、生活环境,给人类健康带来严重危害,而且具有致癌作用。关于B(a)P ip的免疫毒性,国内外未见报道。本文观察了B(a)P对小鼠体液免疫、细胞免疫及巨噬细胞功能的影响。 材料与方法 LACA佰♀健康小鼠,8～10周龄,体重22～25g,由北京医科大学实验动物部提供。实验分200,100,50 mg/kg三组,同时设溶剂对照组,染毒途径为一次ip。B(a)P,Sigma和Fluka公司生产。溶于玉米油或橄榄油,在磁力搅拌器上避光搅拌4～6h,使     

Anticlastogenic activity of thymoquinone against benzo(a)pyrene in mice.

Thymoquione (TQ), the main constituent of the volatile oil of Nigella sativa seeds, has been shown to protect mice against benzo(a)pyrene [B(a)P]-induced forestomach carcinogenesis. The present investigation was undertaken to study the possible chemopreventive activity of TQ, supplemented in the drinking water, against B(a)P-induced chromosomal aberrations (CAs) in mouse bone marrow cells. Male Swiss albino mice received TQ (0.01% in drinking water) daily for 28 days. The daily dose of TQ was estimated to be 10mg/kg based on the calculated average daily water consumption by mice. From day 9, the carcinogen, B(a)P, was given by gastric intubation at dose level of 50mg/kg on alternative days for a total of 8 doses. On day 29, all mice were transferred to a normal drinking tap water. Control groups received corn oil vehicle, TQ alone or B(a)P alone. All mice were sacrificed at 12 weeks after the end of the treatment. Chromosome preparations were made of bone marrow. Cytogenetic end points screened were the frequencies of CAs and damaged cells induced. Daily intake of TQ after and before or during exposure to B(a)P significantly reduced the frequencies of CAs and damaged cells compared to the highly clastogenic activity of B(a)P alone.     

Benzo[a]pyrene metabolism in mouse liver. Association of both 7,8-epoxidation and covalent binding of a metabolite of the 7,8-diol with the Ah locus

The 7,8-epoxidation of benzo[a]pyrene, and the 9,10-epoxidation of benzo[a]-pyrene trans-7,8-dihydrodiol coupled with covalent binding of the highly reactive diol-epoxide, are two key P-450-mediated reactions believed to be important in cancer initiation, mutagenesis and teratogenesis. New assays for these two reactions were developed with mouse liver microsomes. These two activities have apparent Km values (approximately 6 microM) similar to that of aryl hydrocarbon hydroxylase activity. Twenty-six individual 3-methylcholanthrene-treated Ahb/Ahd and Ahd/Ahd progeny of the (C57BL/6N)(DBA/2N) F1 X DBA/2N backcross were studied. Both of the newly described activities appear to represent P-450 protein(s) that are responsible for aryl hydrocarbon hydroxylase activity and that are coordinately controlled by the Ahb allele.     

Epidermal benzo[a]pyrene metabolism and DNA-binding in Balb/C mice: Inhibition by ellagic acid

1. Topical application of ellagic acid, a common plant phenol, to control or to 3-methylcholanthrene (3-MC) pretreated Balb/C mice, resulted in significant inhibition of hepatic and epidermal microsomal aryl hydrogen hydroxylase activity, and of benzo[a]pyrene (BP) binding to epidermal and hepatic DNA in vivo.

2. In vitro addition of ellagic acid (0·25 mM) to epidermal microsomal incubation systems from either control or 3-MC-treated animals resulted in 62–75% inhibition of BP binding to calf thymus DNA.

3. These studies suggest that ellagic acid could prove useful in understanding and/or modulating polyaromatic hydrocarbon carcinogenesis.     

Epidermal benzo[a]pyrene metabolism and DNA-binding in Balb/C mice: inhibition by ellagic acid

Topical application of ellagic acid, a common plant phenol, to control or to 3-methylcholanthrene (3-MC) pretreated Balb/C mice, resulted in significant inhibition of hepatic and epidermal microsomal aryl hydrogen hydroxylase activity, and of benzo[a]pyrene (BP) binding to epidermal and hepatic DNA in vivo. In vitro addition of ellagic acid (0.25 mM) to epidermal microsomal incubation systems from either control or 3-MC-treated animals resulted in 62-75% inhibition of BP binding to calf thymus DNA. These studies suggest that ellagic acid could prove useful in understanding and/or modulating polyaromatic hydrocarbon carcinogenesis.     

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     

Trichloroethylene induced cutaneous irritation in BALB/c hairless mice: histopathological changes and oxidative damage

Trichloroethylene (TCE), a colorless and volatile organic solvent, has long been a major chemical hazard during occupational exposure because of its extensive use in industry. Exposure to TCE can cause significant skin lesions, but the effect of TCE on skin irritation has received little attention. We therefore investigated the effect of TCE on skin irritation and oxidative stress using hairless mice. BALB/c hairless mice were subjected to acute and cumulative topical TCE treatment. Skin reactions were evaluated by visual inspection, histopathology examined by microscopy and oxidative stress assessed by measurement of malondialdehyde (MDA) levels, superoxide dismutase (SOD) activities and nitric oxide (NO) production. Under acute and cumulative TCE irritation, the skin developed erythema and edema, and the predominant histopathological features were hyperkeratosis, spongiosis and inflammatory cell infiltrates. In parallel to these morphological changes, acute TCE irritation also concentration-dependently increased MDA levels and inhibited SOD activities of the skin. However, in cumulative irritation, the MDA levels and SOD activities were initially elevated with increased TCE concentrations, but thereafter reduced with further concentration increments; the linear dose-response relationship was not preserved. TCE also concentration-dependently increased NO production both in acute and cumulative irritation. These results suggest that TCE is capable of producing skin irritation effect in vivo, with histopathological changes characterized by hyperkeratosis, spongiosis and inflammatory cell infiltrates. Moreover, oxidative stress may be associated with the clinical manifestations and histopathological abnormalities in TCE-induced skin irritation.     

Soy isoflavones inhibits the genotoxicity of benzo(a)pyrene in Swiss albino mice

Dietary factors are considered important environmental risk determinants for various diseases. Isoflavones are one of the biologically active polyphenolic plant constituents that possess potential chemopreventive properties against a wide variety of chronic diseases. In the present study we have evaluated the antimutagenic potential of soy isoflavones against benzo(a)pyrene (B[a]P) (125 mg/ kg) induced genotoxicity in Swiss albino mice. The effect of soy isoflavones was studied by in vivo bone marrow chromosomal aberration and micronuclei induction test. Using an alkaline unwinding assay we monitored the DNA strand breaks. Two doses of soy isoflavones (20 and 40 mg/kg b.wt) were given orally for seven days prior to the administration of B[a]P. Soy isoflavone inhibited the genotoxicity of B[a]P in terms of chromosomal aberration and micronucleus formation. DNA strand break levels in only B[a]P treated group remained significantly high from the control values (P < 0.001). The pretreatment of soy isoflavone showed gradual reduction in strand breaks significantly (P < 0.001) and dose dependently. Soy isoflavone pretreatment also decreased cytochrome P450 (CYP) content. The activity of CYP was also decreased dose dependently by pretreatment with soy isoflavone. The chemopreventive effect of soy isoflavone on the inhibition of CYP activity and DNA integrity mediate the possible mechanism of inhibition of genotoxicity.     

Comparative metabolism of benzo(a)pyrene by ovarian microsomes of various species

Knowledge of the ability of the female reproductive system to metabolize polycyclic aromatic hydrocarbons (PAHs) is critical to the diagnosis and management of female infertility and for risk assessment purposes. The PAHs are a family of widespread pollutants that are released into the environment from automobile exhausts, cigarette smoke, burning of refuse, industrial emissions, and hazardous waste sites. In exposed animals, PAHs become activated to reactive metabolites that interfere with target organ function and as a consequence cause toxicity. The extent of susceptibility to PAH exposure may depend on the ability of animals to metabolize these chemicals. The present study has been undertaken to assess whether any differences exist among mammals in the metabolism of benzo(a)pyrene (BaP), a prototypical PAH compound. Microsomes isolated from the liver and ovaries of rats, mice, goats, sheep, pigs, and cows were incubated with 5 μM BaP. Postincubation, samples were extracted with ethyl acetate and analyzed for BaP/metabolites by reverse‐phase HPLC with fluorescence detection. The rate of metabolism (pmol of metabolite/min/mg protein) was found to be more in liver than in ovary in all the species studied (P < 0.05). The differences in metabolite concentrations were statistically significant (P < 0.0001) among the various species in both organs studied. Multiple species comparison also revealed that the differences were statistically significant (P < 0.001) between rodents (rat and mouse) and higher mammals (ewe, sow, and cow). Even among the higher mammals, in a majority of the cases, the differences in metabolite concentrations were significantly different (P < 0.001) both in ovary and liver. The BaP metabolites identified were 4,5‐diol; 7,8‐diol; 9,10‐diol; 3‐hydroxy BaP; and 9‐hydroxy BaP. The rodent microsomes produced considerably higher proportion of BaP 4,5‐diol and 9,10‐diol than did cow, sow, goat, and sheep. However, microsomes from higher mammals converted a greater proportion of BaP to 3‐hydroxy and 9‐hydroxy BaP, the detoxification products of BaP. Overall, our results revealed a great variation among species to metabolize BaP. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.