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.     

3-Hydroxybenzo(a)pyrene as a biomarker of dermal exposure to benzo(a)pyrene

The aim of this study was to determine the percutaneous absorption flux of BaP (20 μg/cm² in ethanol) and the usefulness of urinary 3-OHBaP as a bio-indicator of dermal exposure to BaP. The percutaneous absorbed dose and absorption flux were estimated by comparison with intravenous administration of BaP (0.01 and 0.05 mg/kg in Cremophor^®) as reference way. A percutaneous absorption flux of 0.37 μg/cm²/h was determined by killing groups of rats, following exposure time of 4.5 and 24 h. [¹⁴C] skin content was 3.1 μg/cm², after 24 h exposure to BaP. Total urinary 3-OHBaP accounted for 0.4% of the real absorbed dose, which was fourfold higher than the percentage of an intravenous dose excreted as 3-OHBaP. This finding reveals that percutaneous absorption of BaP, based on the ratio of urinary excretion of 3-OHBaP following percutaneous exposure compared to percutaneous absorption following intravenous administration of BaP, is overestimated in the rat. In vitro, BaP was intensively metabolised by rat skin. Unchanged BaP and 3-OHBaP in receptor fluid accounted for 50 and 30% of the total radioactivity. This percutaneous first past effect of BaP in rats could, in part, explain the higher urinary excretion ratio of 3-OHBaP compared to the value based on intravenous administration of BaP. Conversely, BaP was largely lower metabolised as 3-OHBaP during percutaneous absorption by humans, so BaP absorption flux should be overestimated to a lesser extent in humans than in rats.     

Combined effects of curcumin and piperine in ameliorating benzo(a)pyrene induced DNA damage

The present study was planned to investigate the antigenotoxic effects of curcumin and piperine separately and in combination against benzo(a)pyrene (BaP) induced DNA damage in lungs and livers of mice. Male Swiss albino mice received curcumin (100 mg kg⁻¹ body weight) and piperine (20 mg kg⁻¹ body weight) separately as well as in combination orally in corn oil for 7 days as pretreatments and subsequently, 2 h after, BaP was administered orally in corn oil (125 mg kg⁻¹ body weight). A single dose of BaP to normal mice increased the level of 8-oxo-2′-deoxyguanosine (8-oxo-dG) content and % DNA in the comet tail in the lungs and liver. Pretreatments of curcumin and curcumin plus piperine before administration of single dose of BaP significantly decreased the levels of 8-oxo-dG content and % DNA in the comet tail in both the tissues. Moreover, the genoprotective potential of curcumin plus piperine was significantly higher as compared to curcumin alone against BaP induced DNA damage.     

Synergistic effects of piperine and curcumin in modulating benzo(a)pyrene induced redox imbalance in mice lungs

The objective of the present study was to evaluate the effects of curcumin alone and with adjuvant piperine against benzo(a)pyrene (BaP) induced oxidative stress in lungs of male Swiss albino mice. Mice were pretreated either with curcumin (100?mg/kg body weight), or piperine (20?mg/kg body weight), and in combination of both for one week, followed by single dose of benzo(a)pyrene (125?mg/kg body weight) treatment. Treatment with benzo(a)pyrene resulted in increased levels of lipid peroxides (LPO), protein carbonyl content (PCC) and with consequent decrease in the levels of tissue antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and reduced glutathione (GSH), which however, were increased significantly following curcumin treatment, but the increase was more pronounced when piperine was used as an adjuvant. BaP treatment alone did not alter significantly the GST activity. Pretreatment with curcumin increased the GST activity in BaP treated group, which was enhanced further upon synergistic treatment with piperine and curcumin. Therefore, combined administration of curcumin and piperine shall prove to be more effective in attenuating BaP induced toxicity.     

Synergistic effects of piperine and curcumin in modulating benzo(a)pyrene induced redox imbalance in mice lungs

The objective of the present study was to evaluate the effects of curcumin alone and with adjuvant piperine against benzo(a)pyrene (BaP) induced oxidative stress in lungs of male Swiss albino mice. Mice were pretreated either with curcumin (100 mg/kg body weight), or piperine (20 mg/kg body weight), and in combination of both for one week, followed by single dose of benzo(a)pyrene (125 mg/kg body weight) treatment. Treatment with benzo(a)pyrene resulted in increased levels of lipid peroxides (LPO), protein carbonyl content (PCC) and with consequent decrease in the levels of tissue antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and reduced glutathione (GSH), which however, were increased significantly following curcumin treatment, but the increase was more pronounced when piperine was used as an adjuvant. BaP treatment alone did not alter significantly the GST activity. Pretreatment with curcumin increased the GST activity in BaP treated group, which was enhanced further upon synergistic treatment with piperine and curcumin. Therefore, combined administration of curcumin and piperine shall prove to be more effective in attenuating BaP induced toxicity.     

Chemopreventive efficacy of mangiferin against benzo(a)pyrene induced lung carcinogenesis in experimental animals

Chemoprevention has emerged as a very effective preventive measure against carcinogenesis. Several bioactive compounds present in fruits and vegetables have revealed their cancer curative potential on carcinogenesis. Tumor markers correlate strongly with prognosis on tumor burden. Glycoprotein and membrane ATPases play an important role in carcinogenesis. Hence this study was launched to evaluate the effect of mangiferin on the changes in glycoprotein components, ATPases and membrane lipid peroxidation in control and lung carcinoma bearing mice. A significant increase in the levels of glycoproteins, membrane ATPases and membrane lipid peroxidation were observed in animals with lung carcinoma. On administration of mangiferin, these changes were reverted back to near normal levels. The increased levels of glycoprotein components found in lung carcinoma were also significantly decreased in mangiferin treated. Overall, the above data shows that the anticancer effect of mangiferin is more pronounced when used as an chemopreventive agent rather than as a chemotherapeutic agent against B(a)P induced lung carcinogenesis.     

Antioxidant and anticancer efficacy of hesperidin in benzo(a)pyrene induced lung carcinogenesis in mice

Summary  Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of cancer. Several bioactive compounds present in fruits and vegetables have revealed their cancer curative potential on lung cancer. Hesperidin is one such naturally occurring flavonoid widely found in citrus fruits. The aim of the present study is to divulge the chemopreventive nature of hesperidin during benzo(a)pyrene (B(a)P) induced lung cancer in Swiss albino mice. Administration of B(a)P (50 mg/kg body weight) to mice resulted in increased lipid peroxides (LPO), lung specific tumor marker carcinoembryonic antigen (CEA) and serum marker enzymes aryl hydrocarbon hydroxylase (AHH), gamma glutamyl transpeptidase (GGT), 5′nucleotidase (5′ND) and lactate dehydrogenase (LDH) with concomitant decrease in the levels of tissue antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), vitamin E and vitamin C. Hesperidin supplementation (25 mg/kg body weight) significantly attenuated these alterations thereby showing potent anticancer effect in lung cancer. Further the antiproliferative effect of hesperidin was confirmed by histopathological analysis and proliferating cell nuclear antigen (PCNA) immunostaining. Overall, these findings substantiate the chemopreventive potential of hesperidin against chemically induced lung cancer in mice.     

Cardiac arrhythmogenic action of benzo(a)pyrene in the rabbit

Rabbits treated with benzo(a)pyrene developed cardiac arrhythmias when exposed by inhalation to 8100 ppm trichloroethylene or 15000 ppm halothane to a greater extent and at lower doses of epinephrine challenge than did controls. Benzo(a)pyrene and 3-methylcholanthrene both increased the metabolism of trichloroethylene, but 3-methylcholanthrene did not increase its cardiotoxic effect. The basis of the arrythmogenic action of benzo(a)pyrene appears to be unrelated to its ability to induce xenobiotic metabolism.     

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     

Epidermis: the major site of cutaneous benzo(a)pyrene and benzo(a)pyrene 7,8-diol metabolism in neonatal BALB/c mice

The metabolism of benzo(a)pyrene (BP) and benzo(a)pyrene-7,8-diol (BP-7,8-diol) by microsomes prepared from whole skin, dermis, and epidermis of neonatal BALB/c mice pretreated with topically applied 3-methylcholanthrene (MCA) was compared. In control animals, microsomes prepared from epidermis showed higher rates of metabolism of BP and BP-7,8-diol (1.4-2.6-fold) than did microsomes prepared from whole skin or dermis. A single topical application of MCA increased the rate of metabolism of BP and BP-7,8-diol in microsomes prepared from whole skin, dermis, and epidermis. The greatest increase occurred in the epidermis. The in vivo covalent binding of [3H]BP, [3H]BP-7,8-diol, and 7,12-[3H]dimethylbenz(a)anthracene ([3H]DMBA) to DNA was found to be greater in epidermis (8.7-15.4-fold) than in whole skin or in dermis. A single topical application of MCA to BALB/c mice enhanced the in vivo binding of [3H]BP, [3H]BP-7,8-diol and [3H]DMBA to DNA of whole skin, dermis, and epidermis more than 2-fold. Exposure of Salmonella tester strains TA98 and TA100 to 2-aminoanthracene, a skin carcinogen, in the presence of an epidermal metabolic activation mixture resulted in a greater mutagenic response when compared to activation mixtures derived from whole skin or dermis. These results indicate that epidermis is the major site of polycyclic aromatic hydrocarbon metabolism and of enzyme-mediated covalent binding of polycyclic aromatic hydrocarbon carcinogens to DNA in skin of BALB/c mice and that topically applied MCA has maximum enzyme induction effects in this skin compartment.     

Effects of benzo(e)pyrene and benzo(a)pyrene on P-glycoprotein-mediated transport in Caco-2 cell monolayer: a comparative approach.

The previous studies from our laboratory reported that benzo(a)pyrene (Bap) influenced efflux transport of rhodamine 123 (Rho-123) by induction of P-glycoprotein (P-gp) in Caco-2 cells. The present study investigated whether induction of P-gp and the enhanced efflux transport of Rho-123 were caused by benzo(e)pyrene (Bep), which has a structure similar to Bap, but is not a carcinogenic compound. In Caco-2 monolayer exposed to 50 microM Bep for 72 h, the ratio of the apparent permeability coefficient (P(app)) of Rho-123 efflux increased significantly compared to that of the control monolayer. Similarly, a significant increase in expression of MDR1 mRNA and of P-gp at the protein level were detected by RT-PCR and by Western blot analysis, respectively, in Caco-2 cells exposed to Bep, compared to that of the control. Caco-2 cells exposed to Bep showed oxidative stress that was detected by fluorescence microscopy using aminophenyl fluorescein. However, the oxidative stress was weaker compared with that of Bap. The cellular GSH content was decreased to 80% or 59% of control cells, respectively, in Caco-2 cells exposed to either Bep or Bap. Our results further show that Bep or Bap-induced P-gp in Caco-2 cells might have been the result of oxidative stress rather than DNA damage.     

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     

苯并(a)芘致癌的表观遗传学作用机制研究进展

近年研究发现,环境致癌物苯并(a)芘的致癌机制除遗传毒性外,可引起全基因组甲基化减低、抑癌基因甲基化升高及原癌基因甲基化降低,亦可引起微RNA表达升高或降低、长链非编码RNA表达升高、组蛋白磷酸化水平升高、组蛋白甲基化和乙酰化失衡等表观遗传学变化。这些改变既可引起基因表达异常、染色体结构异常和不稳定性增加直接致癌,又可以引起相应的遗传毒性改变,如基因突变、基因损伤修复异常、细胞凋亡和细胞周期阻滞等协同致癌,被认为是苯并(a)芘致癌可能的表观遗传学机制。上述研究为进一步揭示苯并(a)芘引起的环境相关性疾病和职业病的发生机制及防治策略提供了科学依据。     

Excretion of 3-hydroxy-benzo(a)pyrene and mutagenicity in rat urine after exposure to benzo(a)pyrene

3-hydroxy-benzo(a)pyrene (3-OH-B(a)P) and mutagenic activity in rat urine were determined after the oral administration of benzo(a)pyrene given in three repeated doses of 10, 20 and 50 mumol kg-1. The procedure for the determination of 3-OH-B(a)P consisted of enzymic hydrolysis, separation and HPLC-analysis. The mutagenic activity of concentrated urine samples was assayed with the Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. The urinary excretion of 3-OH-B(a)P and mutagens showed a correlation and both increased dose-dependently during the sampling period of 6 days. Data indicated that 3-OH-B(a)P can be regarded as a reliable representative of all urinary (pre)-mutagens derived from benzo(a)pyrene and exposure of rats to benzo(a)pyrene could be detected with greater sensitivity by the HPLC assay of 3-OH-B(a)P than with the non-specific mutagenicity assay.     

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.     

The effect of paraquat on the mutagenicity of benzo(a)pyrene

Elevated activities of superoxide dismutase (SOD) were detected in histidine-requiring strains of Salmonella typhimurium after the bacteria were preincubated for 1 h at 37 degrees C with S-9 mix and paraquat (methylviologen, PQ2+) at 10(-4) M. A fivefold increase in SOD level was found for strains TA 98 and TA 100. These elevated levels of SOD activity were correlated with a significant reduction of the mutagenicity of metabolically activated benzo(a)pyrene (B(a)P) in these tester bacteria when evaluated in a preincubation assay system. A 69.0-92.5% and 23.5-66.9% reduction was noticed when 0.5-4.0 micrograms per plate of B(a)P was used in TA 98 and TA 100, respectively. However, exogenous superoxide dismutase at 10-100 micrograms ml-1 added to top agar had no significant effect on the number of revertants produced by activated B(a)P. These data indicate a major role of intracellular superoxide anion in promoting mutagenicity of B(a)P.     

Capsaicin as an in vitro inhibitor of benzo(a)pyrene metabolism and its DNA binding in human and murine keratinocytes

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the active principle of capsicum fruits, such as hot peppers, is a known inhibitor of substance P. This substance was also found to be a potent in vitro inhibitor of human and murine epidermal metabolism of benzo(a)pyrene (BP) and the enzyme-mediated binding of BP metabolites to DNA. In both untreated and 3-methylcholanthrene-treated neonatal rat epidermal microsomes, capsaicin resulted in a dose-dependent inhibition of aryl hydrocarbon hydroxylase activity with an I50 value of 3.0 X 10(-4)-3.6 X 10(-4) M. A Lineweaver-Burk plot of the inhibition of aryl hydrocarbon hydroxylase activity suggested that the inhibition is of the noncompetitive type with Ki value of 50 microM. Capsaicin also inhibited BP metabolism and the binding of 3H-BP to DNA in BALB/c mouse and human keratinocytes. The formation of BP-7,8-diol was also substantially diminished in both systems in the presence of capsaicin (180-300 microM). Our results indicate that the substance P inhibitor, capsaicin, is also an inhibitor of epidermal BP metabolism and DNA binding of its metabolites. Therefore, in addition to its neurological effects, capsaicin may represent a new category of compound with anti-carcinogenic effects.