AHR/ROS-mediated mitochondria apoptosis contributes to benzo[a]pyrene-induced heart defects and the protective effects of resveratrol.

Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon, is widely present in the environment. BaP-induced heart defects have been frequently reported, but the underlying molecular mechanisms remain elusive. Here, we found that BaP increased heart malformations in zebrafish embryos in a concentration-dependent manner, which were attenuated by supplementation with either CH223191 (CH), an aryl hydrocarbon receptor (AHR) inhibitor, or N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger. While CH and NAC both inhibited BaP-induced ROS generation, NAC had no effect on BaP-induced AHR activation. We further demonstrated that BaP increased mitochondrial ROS, decreased mitochondrial membrane potential, and caused endogenous apoptosis, with all these effects being counteracted by supplementation with either CH or NAC. Resveratrol (RSV), a natural AHR antagonist and ROS scavenger, also counteracted the heart malformations caused by BaP. Further experiments showed that RSV attenuated BaP-induced oxidative stress, mitochondrial damage and apoptosis, but had no significant effect on AHR activation. In conclusion, our findings show that BaP induces oxidative stress via AHR activation, which causes mitochondria-mediated intrinsic apoptosis, resulting in heart malformations in zebrafish embryos, and that RSV had a protective effect against BaP-induced heart defects mainly by inhibiting oxidative stress rather than through antagonism of AHR activity.

Resveratrol ameliorates benzo(a)pyrene-induced testicular dysfunction and apoptosis: involvement of p38 MAPK/ATF2/iNOS signaling.

Benzo(a)pyrene [B(a)P] is an environmental toxicant that alters the steroidogenic profile of testis and induces testicular dysfunction. In the present study, we have investigated the molecular signaling of B(a)P and the ameliorative potential of the natural aryl hydrocarbon receptor (AhR) antagonist and antioxidant, resveratrol, on B(a)P-induced male reproductive toxicity. Studies showed that B(a)P treatment resulted in p38 MAPK activation and increased inducible nitric oxide synthase (iNOS) production along with testicular apoptosis and steroidogenic dysfunction. Resveratrol cotreatment maintained testicular redox potential, increased serum testosterone level and enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3ßHSD, 17ßHSD) and prevented subsequent onset of apoptosis. Resveratrol cotreatment resulted inhibition of testicular cytochrome P4501A1 (CYP1A1) expression, which is the major B(a)P metabolizing agent for BPDE-DNA adduct formation. Resveratrol also significantly decreased the B(a)P-induced AhR protein level, its nuclear translocation and subsequent promoter activation, thereby decreased the expression of CYP1A1. Resveratrol also down-regulated B(a)P-induced testicular iNOS production through suppressing the activation of p38 MAPK and ATF2, thus improved the oxidative status of the testis and prevented apoptosis. Our findings cumulatively suggest that resveratrol inhibits conversion of B(a)P into BPDE by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Chemoprotective effect of all-trans retinoic acid (ATRA) on oxidative stress and lung metastasis induced by benzo(a)pyrene.

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of cancer. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A under the family retinoids, derived by irreversible oxidation of retinol (vitamin A), the parent compound for all natural retinoids. The aim of the present study is to divulge the chemopreventive and chemoprotective nature of ATRA during benzo(a)pyrene (B(a)P) induced lung cancer development in BALB/c mice. Administration of B(a)P (50 mg/kg body weight) to mice resulted in increased lipid peroxides (LPO), lipid hydroperoxides (LOOH) and nitric oxide (NO) with concomitant decrease in the levels of tissue anti-oxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and vitamin C. ATRA supplementation (0.585 mg/kg body weight) attenuated all these alterations, which indicates the anti-cancer effect that was further confirmed by histopathological analysis. Overall, the above data show that the anti-cancer effect of ATRA is more pronounced when used as an chemopreventive agent against B(a)P-induced lung carcinogenesis.

Acute and prolonged stress responses of brain monoaminergic activity and plasma cortisol levels in rainbow trout are modified by PAHs (naphthalene, beta-naphthoflavone and benzo(a)pyrene) treatment.

We have investigated if treatment with two different PAHs such as naphthalene (NAP) and benzo(a)pyrene (BaP), and the PAH-like compound beta-naphthoflavone (BNF), may modify the stress responses elicited in rainbow trout by acute or prolonged stress stimuli, and the possible involvement of brain monoamines in those responses. Two experiments (acute and prolonged stress) were performed. In the acute stress experiment, fish were i.p. injected with vegetable oil alone (control) or oil containing NAP, BNF or BaP (10 mg kg(-1)), and 72 h after injection fish were acutely stressed by chasing for 15 min. In the prolonged stress experiment, a similar group-design and injection protocol were followed, but fish were submitted to severe confinement stress by maintaining fish under high stock density (70 kg fish mass m(-3)) for 72 h. The levels of cortisol, glucose and lactate were assayed in plasma. In addition, the contents of dopamine (DA), noradrenaline (NA) and serotonin (5HT), as well as their oxidized amine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy-3-indoleacetic acid (5HIAA) were assayed in telencephalon, hypothalamus, preoptic region, optic tectum and brain stem, as well as the pituitary. Both acute and prolonged stress stimuli increased plasma levels of cortisol, which further increase with NAP and BNF treatments after acute stress. In contrast, cortisol levels of fish exposed to prolonged stress showed a clear tendency to decrease after the treatment with BNF and BaP. Stress stimuli also increased plasma glucose levels, which were not affected by PAHs in acute stressed fish but decreased in fish exposed to prolonged stress. Increased plasma levels of lactate in fish exposed to stress decreased after PAHs treatment in acute stress but not in prolonged stress. With respect to monoaminergic systems, major changes induced by both acute and prolonged stress were increases of the metabolites DOPAC and 5HIAA and DOPAC/DA or 5HIAA/5HT ratios in several brain regions. PAHs induced alterations in the normal responses of monoaminergic systems to stress, with dopaminergic system being the most affected after acute stress, and serotonergic system after prolonged stress. Those alterations, especially after prolonged stress, showed certain parallelism with alterations of plasma cortisol levels. Thus, results suggest that in stressed fish PAH effects on plasma cortisol levels (and its derived metabolic actions) could be in part mediated by alterations on the monoaminergic systems at the CNS of rainbow trout.

The potent protective effect of wild ginseng (Panax ginseng C.A. Meyer) against benzo[alpha]pyrene-induced toxicity through metabolic regulation of CYP1A1 and GSTs.

Wild Panax ginseng C.A. Meyer (WG) is a well-known medicinal herb. In this study, the protective effects of a water extract from the root of WG on benzo[alpha]pyrene (BP)-induced hepatotoxicity and the mechanism of these effects were investigated for the first time. The effects of WG on liver toxicities induced by BP were assessed by blood biochemical and histopathological analyses. BP caused severe liver injury in rats, as indicated by elevated plasma ALT, AST and LPO levels. Pretreatment with WG for 4 weeks completely abrogated increases in the ALT, AST and LPO levels when challenged with BP. Reductions in GSH content and GST activity by BP were reversed by WG. These protective effects of WG against BP-induced toxicity were consistent with the results of histopathological examinations. We next examined the effects of WG on the gene expression of the enzymes that metabolize BP in H4IIE cells. CYP1A1 mRNA and protein expression were increased by BP. WG moderately inhibited BP-induced CYP1A1 gene expression. Moreover, GSTA2, GSTA3 and GSTM2 gene expressions were significantly increased by WG through the Nrf2/antioxidant responsive element pathway for enzyme induction. In summary, WG is efficacious in protecting against BP-induced hepatotoxicity as results of metabolic regulations through both the inhibition of metabolic enzyme activation and the enhancement of electrophilic detoxification, implying that WG should be considered a potential chemopreventive agent.

Effects of aloe, aloesin, or propolis on the pharmacokinetics of benzo[a]pyrene and 3-OH-benzo[a]pyrene in rats.

This study was conducted to examine the effects of aloe and aloesin on the weight gain and blood chemistry as well as the pharmacokinetics of benzo[a]pyrene (BaP) and 3-OH-BaP in rats. The rats treated with multiple doses of aloe and aloesin (100 mg/kg every 12 h for 14-19 d) did not show any significant changes in the weight gain and blood biochemical parameters. In addition, the effects of oral treatment with aloe, aloesin, and propolis on the absorption and pharmacokinetics of benzo[a]pyrene (BaP) and its metabolite, 3-OH-BaP, were studied in rats. The treatment with a single oral dose (200 mg/kg) of aloe, aloesin, and propolis did not alter the concentration-time profiles of BaP and 3-OH-BaP after iv and oral administration of BaP. At higher oral doses (500 mg/kg), the biliary excretion of BaP and the urinary excretion of 3-OH-BaP were significantly increased, but the urinary excretion of BaP and the fecal excretion of 3-OH-BaP remained unaltered. Whether high doses of aloe increase the overall elimination of BaP deserves further investigation.

Inhibition of clastogenicity of benzo[a]pyrene and of its trans-7,8-dihydrodiol in mice in vivo by fruits, vegetables, and flavonoids.

In the in vivo mouse bone marrow micronucleus assay, homogenates of spinach, artichoke, peaches, and blue grapes as well as commercial concentrates of these vegetables and fruits reduced induction of micronuclei by benzo[a]pyrene (BaP) by 43-50%. Concentrates of strawberries (31% reduction) and of cauliflower (20% reduction) were less potent. Inhibition of genotoxicity by spinach and peaches was not caused by any delay in maturation of micronucleated erythrocytes as shown by experiments with sampling times of 24, 48, and 72 h after dosing of BaP. Pre-treatment of the mice with spinach 48, 24, and 12h before application of BaP resulted in a 44% reduction of micronuclei while peaches generated only a marginal effect. A post-treatment procedure administering spinach or peaches 6h after dosing of BaP did not indicate any protective effects. When trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-OH) was applied for induction of micronuclei spinach and peaches reduced the number of micronuclei by 55 and 48%, respectively. Pre-treatment of mice with spinach 96, 72, and 60 h before sacrifice caused a decline of hepatic 7-ethoxyresorufin-O-dealkylase (EROD) and of 7-pentoxyresorufin-O-dealkylase (PROD) activities by factors of 2.2 and 1.4, respectively. However, statistical significance was not reached. On the other hand, peaches had no influence on hepatic EROD or PROD activities. The flavonoids quercetin and its glucoside isoquercitrin, administered orally in doses of 0.03 mmol/kg body weight simultaneously with intraperitoneally given BaP, reduced the number of micronuclei in polychromatic erythrocytes of the bone marrow of mice by 73 and 33%. Ten-fold higher concentrations, however, reversed the effects with a particular strong increase observed with isoquercitrin (+109%; quercetin: +16%).

Effect of dietary vitamin A on forestomach tumorigenesis during the total and postinitiation stages in mice treated with high- or low-dose benzo(a)pyrene.

The effects of dietary vitamin A on forestomach tumorigenesis during the total stage of the initiation and postinitiation periods and during the postinitiation stage were evaluated in ICR/Jcl mice treated with either high or low doses of benzo(a)pyrene (B(a)P). In experiment 1, the animals were initiated with a high carcinogenic dose of B(a)P to a total of 20 mg, while in experiment 2 the animals were treated with a low dose of B(a)P to a total of 2 mg. A control group of animals received no carcinogens. Five different dietary levels of vitamin A supplements were used in each experiment and in the control study. In experiment 1, a high incidence of tumorigenesis was observed in every group, with 74% to 96% developing papilloma and 19% to 46% developing carcinoma. In experiment 2, the incidence of tumorigenesis in the high-dose vitamin A groups, including those given during the total and postinitiation stages, was found to be significantly reduced at 7.4%, compared with that in the low-dose vitamin A group of 57.7% (P < 0.05). These results suggest that a high dietary level of vitamin A can reduce the incidence of tumorigenesis when low carcinogenic dose levels of B(a)P are given in both the total and postinitiation stages.

Southern flounder hepatic and intestinal metabolism and DNA binding of benzo[a]pyrene (BaP) metabolites following dietary administration of low doses of BaP, BaP-7,8-dihydrodiol or a BaP metabolite mixture.

Certain finfish species living in chemically polluted environments exhibit a high incidence of gastrointestinal tract tumors. Carnivorous fish in such environments are likely to consume invertebrates which contain chemical procarcinogens and the invertebrate biotransformation products of these compounds. The retention in tissues, extent of DNA adduct formation in liver and intestine, and metabolite composition of bile was investigated in southern flounder following gavage with pure [3H]- or [14C]benzo[a]pyrene (BaP), pure [14C]benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8D), or hepatopancreas from spiny lobsters previously dosed with [3H]- or [14C]BaP (Metab.HP). Metab.HP contained mainly polar conjugates of BaP diols, triols and tetraols. BaP-7,8D was retained in fish tissues and bile at 24 h to a greater extent (33.6% of the dose), than either BaP (19.00%) or Metab.HP (6.6%). Hepatic and intestinal DNA isolated from all dosed fish contained covalently bound radioactivity, but exposure to BaP-7,8D or BaP resulted in significantly higher binding in both tissues than exposure to Metab.HP. Hepatic DNA from BaP and BaP-7,8D-dosed flounder contained 0.24 +/- 0.07 and 0.33 +/- 0.06 pmol BaP equivalents/mg DNA respectively (mean +/- S.E.), while hepatic DNA isolated from Metab.HP-dosed flounder contained 0.006 +/- 0.002 pmol BaP equivalents/mg DNA. Binding of radioactivity to intestinal DNA was significantly higher than to hepatic DNA for flounder dosed with Metab.HP (0.026 +/- 0.003) or with BaP (0.76 +/- 0.27) but not for flounder dosed with BaP-7,8D (0.44 +/- 0.09). These studies show that dietary BaP, and metabolites likely to be present in invertebrates, can be absorbed by the southern flounder and form DNA adducts in target organs.

[Modifying effect of the chemical components of dust on the induction of lung tumors by physical and chemical carcinogens (experimental study]. / O modifitsiruiushchem vliianii khimicheskikh komponentov pyli na induktsiiu opukholei legkogo fizicheskim i khimicheskim kantserogenom (éksperimenta'lnoe issledovanie).

Chronic inhalation intake of benzo(a)pyrene (BP) and polonium-210 (210Po) together with aluminum oxide caused increase of tumor formation in the lungs of mice. Synergy of BP and 210Po carcinogenic effect was pointed out, it was characterized by summation and possible effect involution by tumor development rates and the duration of the latent period. BP and 210Po carcinogenic effectiveness depended on the type of dust carrier and probably on the presence of silicon dioxide and also carcinogenic metals. It was pointed out that further studies were necessary to determine an etiologic role of mineral dust chemical components in carcinogenic activity of the above substances and also carcinogenic effectiveness of dusts as carriers of chemical carcinogens and alpha-active radionuclides.