Benzo(a)pyrene-induced genotoxicity: Attenuation by farnesol in a mouse model

In the present study we have evaluated the antigenotoxic effects of Farnesol (FL) a 15-carbon isoprenoid alcohol against benzo (a) pyrene [B(a)P] (125 mg kg^{? 1}.b.wt oral) induced toxicity. B(a)P administration lead to significant induction in Cytochrome P450 (CYP) content and aryl hydrocarbon hydrolase (AHH) activity (p < 0.001), DNA strand breaks and DNA adducts (p < 0.001) formation. FL was shown to suppress the activities of both CYP and AHH (p < 0.005) in modulator groups. FL pretreatment significantly (p < 0.001) restored depleted levels of reduced glutathione (GSH), quinone reductase (QR) and glutathione –S-transferase (GST). A simultaneous significant and at both the doses reduction was seen in DNA strand breaks and in in-vivo DNA adducts formation (p < 0.005), which gives some insight on restoration of DNA integrity. The results support the protective nature of FL. Hence present data supports FL as a future drug to preclude B (a) P induced toxicity.     

Modulatory effect of Piperine on mitochondrial antioxidant system in Benzo(a)pyrene-induced experimental lung carcinogenesis.

Chemoprevention has emerged as a very effective preventive measure against carcinogenesis. Many bioactive compounds present in edible as well in herbal plants have revealed their cancer chemopreventive potential. In the present study, our goal was to investigate the impact of piperine, a principle ingredient of pepper, on alterations of mitochondrial antioxidant system and lipid peroxidation in Benzo(a)pyrene (B(a)P) induced experimental lung carcinogenesis. Oral supplementation of piperine (50 mg/kg body weight) effectively suppressed lung carcinogenesis in B(a)p induced mice as revealed by the decrease in the extent of mitochondrial lipid peroxidation and concomitant increase in the activities of enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and non enzymatic antioxidant (reduced glutathione, vitamin E and vitamin C) levels when compared to lung carcinogenesis bearing animals. Our data suggests that piperine may extent its chemopreventive effect by modulating lipid peroxidation and augmenting antioxidant defense system.     

Benzo(a)pyrene-induced mitochondrial dysfunction and cell death in p53-null Hep3B cells

Benzo(a)pyrene (BaP) has been shown to induce apoptosis and necrosis in various cell types. However, the effect of BaP on mitochondria function and p73, and their possible roles in BaP-induced cell death have not been well studied. This study focused on mitochondria-mediated cell death and the occurrence of p73 protein accumulation in BaP-treated human hepatoma Hep3B (p53-null) cells. We found that BaP (8, 16, 32 and 64μM) induced early necrosis at 12h and delayed apoptosis at 24h. BaP dramatically induced ethoxyresorufin-O-deethylase activity and led to significant increase in oxidative stress at early time points (6 and 12h). Necrotic cell death was concurrent with loss of mitochondrial membrane potential, decrease in the ATP level and activities of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase. However, these changes were reversed in the process of apoptosis. In addition, after BaP treatment, c-Jun N-terminal kinase (JNK) and Bax were activated during apoptosis and no change in p73 protein level was observed. These results revealed that the cells with mitochondria dysfunction and ATP depletion underwent necrosis at early time point and apoptosis afterward when they recovered from mitochondrial dysfunction and ATP depletion. Activation of JNK and Bax possibly contributed to BaP-induced apoptosis.     

Benzo(alpha)pyrene-induced up-regulation of CYP1A2 gene expression: role of adrenoceptor-linked signaling pathways

CYP1A2, a principal catalyst for metabolism of various therapeutic drugs and carcinogens, among others, is in part regulated by the stress response. This study was designed to assess whether catecholamines and in particular adrenergic receptor-dependent pathways, modulate benzo(alpha)pyrene (B(alpha)P)-induced hepatic CYP1A2. To distinguish between the role of central and peripheral catecholamines in the regulation of CYP1A2 induction, the effect of central and peripheral catecholamine depletion using reserpine was compared to that of peripheral catecholamine depletion using guanethidine. The effects of peripheral adrenaline and L-DOPA administration were also assessed. The results suggest that alterations in central catecholamines modulate 7-methoxyresorufin O-demethylase activity (MROD), CYP1A2 mRNA and protein levels in the B(alpha)P-induced state. In particular, central catecholamine depletion, dexmedetomidine-induced inhibition of noradrenaline release and blockade of alpha(1)-adrenoceptors with prazosin, up-regulated CYP1A2 expression. Phenylephrine and dexmedetomidine-induced up-regulation may be mediated, in part, via peripheral alpha(1)- and alpha(2)-adrenoceptors, respectively. On the other hand, the L-DOPA-induced increase in central dopaminergic activity was not followed by any change in the up-regulation of CYP1A2 expression by B(alpha)P. Central noradrenergic systems appeared to counteract up-regulating factors, most likely via alpha(1)- and alpha(2)-adrenoceptors. In contrast, peripheral alpha- and beta-adrenoceptor-related signaling pathways are linked to up-regulating processes. The findings suggest that drugs that bind to adrenoceptors or affect central noradrenergic neurotransmission, as well as factors that challenge the adrenoceptor-linked signaling pathways may deregulate CYP1A2 induction. This, in turn, may result in drug-therapy and drug-toxicity complications.     

Comparison of the effects of arsenic and cadmium on benzo(a)pyrene-induced micronuclei in mouse bone-marrow

This study was undertaken to investigate the genotoxic interactions between the common environmental pollutants: arsenic (As), cadmium (Cd) and benzo(a)pyrene (BaP), which are known to be human carcinogens. C57BL/6J/Han mice were pre-treated with 100mg cadmium chloride (Cd(2+))/L or 50mg sodium arsenite (As(3+))/L in drinking water for 7 days and then given a single dose of 200mg BaP/kg bw by intra-peritoneal injection. A third group of mice did not receive the pre-treatment and was given BaP alone. Mice were sacrificed before or at 12, 24, 48 or 72h after BaP administration. Chromosome damage in bone-marrow cells was assessed by use of the micronucleus test. The study revealed that BaP induced a statistically significant increase in micronucleus (MN) frequency at 48h after administration. In animals exposed to Cd in drinking water no enhancement of genotoxicity was observed compared with the control group that was given tap water only. In Cd/BaP co-exposed animals, the MN frequency at respective time points did not differ from that for the animals exposed solely to BaP. A statistically higher MN frequency was found in bone marrow of animals exposed to As compared with controls that received tap water (0.92+/-0.29% versus 0.38+/-0.13%, respectively). This effect was even more pronounced after combined exposure to As and BaP. In the co-exposed animals, significantly elevated levels of MN were detected in samples examined at 12, 24 and 48h after BaP administration, compared with animals receiving BaP alone (1.14+/-0.31%, 1.26+/-0.3% and 2.02+/-0.45% versus 0.44+/-0.13%, 0.44+/-0.11% and 1.04+/-0.44%, respectively). These findings imply strong interactions between As and BaP, but not between Cd and BaP, in inducing DNA damage in polychromatic erythrocytes in mouse bone-marrow.     

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.     

Chemoprevention of benzo(a)pyrene-induced colon polyps in ApcMin mice by resveratrol

Human dietary exposure to benzo(a)pyrene (BaP) has generated interest with regard to the association of BaP with gastrointestinal carcinogenesis. Since colon cancer ranks third among cancer-related mortalities, it is necessary to evaluate the effect of phytochemicals on colon cancer initiation and progression. In this study, we investigated the preventive effects of resveratrol (RVT) on BaP-induced colon carcinogenesis in Apc^Min mouse model. For the first group of mice, 100 μg BaP/kg body weight was administered to mice in peanut oil via oral gavage over a 60-day period. For the second group, RVT was coadministered with BaP at a dose of 45 μg/kg. For the third group, RVT was administered for 1 week prior to BaP exposure for 60 days. Jejunum, colon and liver were collected at 60 days post BaP and RVT exposure; adenomas in jejunum and colon were counted and subjected to histopathology. RVT reduced the number of colon adenomas in BaP+RVT-treated mice significantly compared to that in mice that received BaP alone. While dysplasia of varying degrees was noted in colon of BaP-treated mice, the dysplasias were of limited occurrence in RVT-treated mice. To ascertain whether the tumor inhibition is a result of altered BaP-induced toxicity of tumor cells, growth, apoptosis and proliferation of adenocarcinoma cells were assessed posttreatment with RVT and BaP. Cotreatment with RVT increased apoptosis and decreased cell proliferation to a greater extent than with BaP alone. Overall, our observations reveal that RVT inhibits colon tumorigenesis when given together with BaP and holds promise as a therapeutic agent.     

Histogenesis of benzo(a)pyrene-induced lesions in tracheal explants

Cytokinetic and histogenic alterations associated with the development of benzo(a)-pyrene (BP) induced epidermoid metaplasia were studied in tracheal explants derived from normal hamsters. Treatment of the explants with BP induced hyperplasia in both the basal and mucous cells. The hyperplasia of the basal cells persisted throughout the duration of the experiment whereas the hyperplasia of the mucous cells subsided between 7 and 10 days after treatment. This was accompanied by stimulation of ciliated cell differentiation and aberrant ciliogenesis which was not limited to the surface cells since some basal cells were observed differentiating into ciliated cells. Subsequently, the differentiation of basal cells into mucous cells was inhibited. Instead, the basal cells differentiated into metaplastic cells. With the progression of the lesions, the mucociliary surface layer was sloughed into the lumen due to the population pressure from the underlying actively proliferating metaplastic cells and their subsequent epidermoid differentiation. Approximately 50% of the explants exhibited focal areas of squamous metaplasia at 7 days after the treatment and extensive epidermoid metaplasia was present in approximately 90% of the explants at 10 days. These results support the hypothesis that BP induced epidermoid metaplasia of tracheal explants originates from the basal cells.     

Gastroenteritis caused by human rotaviruses (serotype three) in a suckling mouse model

The pathogenic potential of human rotaviruses of serotypes 1 through 4 was evaluated in suckling mice. Oral inoculation of three different human rotaviruses of serotype 3 into 5-6 day old CD-1 mice caused disease characterized by diarrhea and dehydration. The mean 50% diarrhea inducing dose (DD50) was 5 X 10(5) pfu. Histopathological examination of small intestines revealed villus epithelial cell vacuolization localized to the distal one-third of the villus. Only Serotype 3 rotaviruses exhibited a rapid phase of viral growth in the intestine between 7 and 12 hours post-inoculation. Larger inocula of rotavirus serotypes 1, 2, and 4 did not cause disease or typical histopathologic changes. However, immunoperoxidase staining for rotavirus antigen was positive in all serotypes tested indicating that infection can occur without apparent disease and is not serotype specific. This convenient in-vivo model can be used to evaluate attenuation of human origin vaccine candidates of serotype 3.     

Benzo[a]pyrene-induced cytochrome P450 1A and DNA binding in cultured trout hepatocytes - inhibition by plant polyphenols

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP) mainly induce lung cancer in humans, but induce liver cancer in fishes. The chemoprevention of cancers through inhibition of molecular events via phytochemicals is a potentially beneficial area of research, and has been carried out in human cell cultures in the past. Carcinogenesis initiation events are thought to occur in similar ways in fish and humans. Our study investigated the feasibility of using cultured rainbow trout CRL-2301 liver cells as a model for BaP-induced carcinogenesis and its prevention by dietary phytochemicals. Treatment with 1 microM BaP resulted in extensive time-dependent covalent binding to cellular DNA and marked cytochrome P450 (CYP) 1A induction, for both about a 20-fold increase, which is similar to what has been observed in cultured human cells. A surprisingly high expression of epoxide hydrolase (EH) activity in these cells likely contributed substantially to the bioactivation of BaP. Two methoxylated flavones and the stilbene resveratrol were effective inhibitors of both the BaP-DNA binding and CYP 1A induction, in particular 5,7-dimethoxyflavone (5,7-DMF), supporting a role for these dietary compounds as cancer chemopreventive agents. Unlike in human liver or bronchial cells, the main mechanism of inhibition of BaP-induced CYP 1A activity in trout liver cells appears to be direct competition at the protein level. Different cellular responses in any particular model used can be expected and the effect of cell context on the biological responses to xenobiotics, including carcinogens as well as polyphenols, must be considered. The trout CRL-2301 cells' sensitivity to BaP treatment is a clear advantage when contemplating a model system for studies of PAH-induced carcinogenesis and cancer chemoprevention. However, extrapolation to human organs should be done cautiously.     

Environmental monitoring for genotoxicity: in vivo sister chromatid exchange in the house mouse (Mus musculus)

Sister chromatid exchange (SCE) values were determined in bone marrow cells isolated from mouse (Mus musculus) femurs after injections of 5-bromo-2'-deoxyuridine (BrdU) and 5-fluorodeoxyuridine (FrdU). Male mice of C3H/J, C57BL/6J, and DBA/2 strains maintained in the laboratory gave mean SCE values of 3.42 +/- 0.07, 3.62 +/- 0.08, and 3.97 +/- 0.13, respectively. Males obtained from natural populations of southwestern Ontario had a higher mean SCE value (6.02 +/- 0.16), as did inbred males maintained in outdoor enclosures for at least 3 weeks (5.07 +/- 0.22). Wild mice housed in the laboratory for 9 months or longer had SCE values similar to laboratory bred mice (3.46 +/- 0.05). The SCE values in wild-caught mice were inversely proportional (r = -0.49) to the distance between the sites where these animals were collected and the nearest major industrial center. Based on these results, SCE analysis in mice is proposed as a possible first-line monitoring procedure for the detection of general changes in environmental genotoxicity.     

Benzo(a) pyrene metabolism in vitamin A deficient rats

Hepatic microsomal metabolism of benzo(a)pyrene, a representative carcinogenic polycyclic hydrocarbon and an ubiquitous environmental pollutant was studied in control and vitamin A deprived (10–12 weeks) male rats. Hydroxylation of benzo(a)pyrene to fluorescent phenols was found to be significantly depressed in the deficient animals. The decreased hepatic metabolism may lead to delayed clearance of the carcinogenic chemicals in this condition and thus may explain at least in part the enhanced susceptibility to carcinogenesis in hypovitaminosis A.     

Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration

Insertional mutagenesis represents a major hurdle to gene therapy and necessitates sensitive preclinical genotoxicity assays. Cdkn2a-/- mice are susceptible to a broad range of cancer-triggering genetic lesions. We exploited hematopoietic stem cells from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. We transduced hematopoietic stem cells in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. Our results validate a much-needed platform to assess vector safety and provide direct evidence that prototypical lentiviral vectors have low oncogenic potential, highlighting a major rationale for application to gene therapy.     

Benzo(a)pyrene hydroxylase activity of immunocompetent cells

A study was made of aryl hydrocarbon hydroxylase activity in immunocompetent cells of varying origin and in hepatocytes from CBA mice. The cells from intact animals may be arranged in the following way with regard to the activity of the enzyme: macrophages greater than hepatocytes much greater than thymocytes greater than splenocytes. The immunostimulants (tilorone and its analogs) altered benzo(a)pyrene hydroxylase activity depending on the cell type.     

Benzo(a)pyrene quinone metabolism in tracheal organ cultures.

The C^? methyl group of methionine-29 of RNAase was enriched with ¹³C. The synthesis involved the reaction of RNAase with ¹³CH₃I at pH 4. S-Methylmethionine-29 RNAase was recovered in 80% yield. This sulfonium derivative was subsequently demethylated with 0.1 M mercaptoethanol at pH 8.5, 25°C for 4 days. These conditions allowed the demethylation reaction to successfully compete with the reaction of the thiol with the four disulfide bridges in RNAase. After dialysis, concentration and chromatography, native RNAase with approx. 50% of its Met²⁹ methyl groups enriched in ¹³C was recovered as was unreversed S-Methylmethionine-29 RNAase. Both proteins showed full enzymatic activity toward cytidine 2′:3′-cyclic monophosphate. ¹³C-methyl signals from enriched RNAase and the sulfonium derivative were observed at 13.8 and 26.7 ppm from TMS respectively. Preliminary denaturation studies with the methylated protein suggest that ¹³C enrichment of methionine methyl groups in RNAase will be a useful technique for following the unfolding transition at these sites of the protein.     

Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry

BACKGROUND: The evaluation of the safety of drugs and other chemicals is an important aspect of toxicology work. The mouse micronucleus assay is a standard in vivo genotoxicity assay. Chromosomal damage is an indicator of genotoxicity, which manifests in the formation of micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. The assay is laborious to perform by manual counting. The laser scanning cytometer allows automated and rapid quantitation of cellular and subcellular fluorescence in monodisperse cell samples on a microscope slide. The object of this study was to evaluate the application of this new technology in the mouse micronucleus genotoxicity assay. Materials and Methods One hundred forty-four mice of various strains were dosed with combinations of carcinogens and antioxidants. Duplicate blood films were prepared 3 days later. One set of slides was stained with acridine orange, and the proportion of micronucleated erythrocytes was counted in 5,000 cells per slide. The duplicates were stained with propidium iodide (40 microg/ml). Five thousand cells per sample were examined using a laser scanning cytometer. The proportion of micronucleated erythrocytes was measured. RESULTS: A coefficient of correlation of 0.96 was found between the data from the two assays. The automation of the assay on the LSC produced a considerable time saving and efficiency gain. CONCLUSIONS: We conclude that with further development, laser scanning cytometry is likely to become the preferred modality for the performance of standard genotoxicity assays.     

Fisetin modulates mitochondrial enzymes and apoptotic signals in benzo(a)pyrene-induced lung cancer

The present study was aimed to delineate in vivo mechanisms of orally administered fisetin with special reference to mitochondrial dysfunction in lung tissues employing benzo(a)pyrene (B(a)P) as the model lung carcinogen. The recent revival of interest in the study of mitochondria has been stimulated by the evidence that genetic and/or metabolic alterations in this organelle lead to a variety of human diseases including cancer. These alterations could be either causative or contributing factors. Hence, the activities of mitochondrial-specific enzymes of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and tumor marker, carcinogenic embryonic antigen were analyzed in control and experimental groups of mice. The induction of apoptotic and anti-apoptotic proteins such as Bcl-2/Bax, cytochrome c, caspase-9 and caspase-3 was confirmed by the immunohistochemistry and Western blot analyses. Furthermore, transmission electron microscopy study of lung sections of B(a)P-induced mice showed the presence of phaemorphic cells with dense granules and increased mitochondria. All the aberrations were alleviated when the mice were treated with fisetin (25 mg/kg body weight). The results proved fisetin to be a very successful drug in combating the mitochondrial dysfunction in an experimental model of lung carcinogenesis induced by B(a)P.     

Poly(ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Benzo(a)pyrene Induced Cell Transformation

Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 μM). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer.