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.     

Effects of quercetin on beta-apo-8'-carotenal-induced DNA damage and cytochrome P1A2 expression in A549 cells

We compared the effects of beta-carotene with those of beta-apo-8'-carotenal (AC, an oxidative product of beta-carotene) on DNA damage and the expression of cytochrome P450 (CYP)1A2 in A549 cells exposed or not to benzo[a]pyrene (BaP), a cigarette-associated carcinogen. Furthermore, we investigated whether quercetin, a flavonoid, modulates these effects. A549 cells were first preincubated with various concentrations of beta-carotene or AC for 1h, followed by incubation with 20 microM BaP for 24h. Next, DNA strand breaks, measured by use of the comet assay, and the expression of CYP1A2, measured by use of western blotting, were assessed. Both beta-carotene and AC at 20 microM significantly enhanced DNA strand breaks and CYP1A2 expression induced by BaP. However, beta-carotene at 2 microM significantly suppressed BaP-induced DNA strand breaks. AC alone induced DNA strand breaks, lipid peroxidation, and the expression of CYP1A2 in A549 cells. The harmful effects of beta-carotene and AC on intracellular DNA were associated with the expression of CYP, because 1-aminobenzotriazole, a CYP inhibitor, partly suppressed these effects. Quercetin significantly inhibited the DNA strand breaks and the increase in CYP1A2 protein induced by AC or beta-carotene in combination with BaP or by AC alone. These findings indicate that the harmful effect of beta-carotene induced by BaP may be through the formation of oxidative products such as AC. Quercetin increased the safety of high doses of beta-carotene, possibly through interaction with beta-carotene's oxidative products or through inhibition of CYP1A2 expression.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.     

In Vitro and In Vivo Studies on Oxygen Free Radical and DNA Adduct Formation in Rat Lung and Liver during Benzo[a]pyrene Metabolism

Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.     

Flavonoids suppresses the enhancing effect of beta-carotene on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A549 cells

This study investigated the individual and combined effects of beta-carotene with a common flavonoid (naringin, quercetin or rutin) on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-related carcinogen in human. A human lung cancer cell line, A549, was pre-incubated with beta-carotene, a flavonoid, or both for 1h followed by incubation with NNK for 4 h. Then, we determined DNA strand breaks and the level of 7-methylguanine (7-mGua), a product of NNK metabolism by cytochrome P450 (CYP). We showed that beta-carotene at 20 microM significantly enhanced NNK-induced DNA strand breaks and 7-mGua levels by 90% (p < 0.05) and 70% (p < 0.05), respectively, and that the effect of beta-carotene was associated with an increased metabolism of NNK by CYP because the concomitant addition of 1-aminobenzotriazole, a CYP inhibitor, with beta-carotene to cells strongly inhibited NNK-induced DNA strand breaks. In contrast to beta-carotene, incubation of cells with naringin, quercetin or rutin added at 23 microM led to significant inhibition of NNK-induced DNA strand breaks, and the effect was in the order of quercetin > naringin > rutin. However, these flavonoids did not significantly affect the level of 7-mGua induced by NNK. Co-incubation of beta-carotene with any of these flavonoids significantly inhibited the enhancing effect of beta-carotene on NNK-induced DNA strand breaks; the effects of flavonoids were dose-dependent and were also in the order of quercetin > naringin > rutin. Co-incubation of beta-carotene with any of these flavonoids also significantly inhibited the loss of beta-carotene incorporated into the cells, and the effects of the flavonoids were also in the order of quercetin > naringin > rutin. The protective effects of these flavonoids may be attributed to their antioxidant activities because they significantly decreased intracellular ROS, and the effects were also in the order of quercetin > naringin > rutin. These in vitro results suggest that a combination of beta-carotene with naringin, rutin, or quercetin may increase the safety of beta-carotene.     

Effect of genetic polymorphism for metabolic enzymes on the relationship between smoking dose and DNA adducts in lymphocytes

The genetic polymorphism of metabolic enzymes on the relationship between smoking dose and DNA adduct levels in lymphocytes were evaluated in 51 smokers. The genetic polymorphisms of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) were analysed by a PCR method. Lymphocyte DNA adducts were measured by two analytical versions of a 32P-postlabelling method; nuclease P1 digested method and butanol extracted method. Mean adduct levels obtained with the nuclease P1 method (1.21 +/- 0.74 per 108 nucleotides) were higher than those obtained with the butanol extracted method (0.82 +/- 0.47, p &lt; 0.01). There was a significant correlation between adduct levels by the nuclease P1 method and those by the butanol extracted method (r = 0.49, p &lt; 0.01). A significant correlation was not found between smoking dose and DNA adduct levels obtained using both methods in lymphocytes of all subjects. When subjects were divided into two groups by CYP1A1 genotypes, significant correlations between smoking indices, such as number of cigarettes per day years or tar intake per day years, and DNA adduct levels measured by the butanol extracted method was found in heterozygous or miner homozygous for CYP1A1 exon 7 polymorphism. We could not get a significant effect of GSTM1 on the relationship between smoking dose and DNA adducts.     

Induction of aryl hydrocarbon hydroxylase and DNA adduct formation in parental and carcinogen transformed C3H/10T1/2 clone 8 cells by benzo[a]pyrene

C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.     

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67 ± 19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations ≥100 μM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations ≥300 μM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by DL-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (≥1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors

It has been reported that the phosphorylated form of histone variant H2AX (gammaH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using gammaH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce gammaH2AX foci in a 24h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce gammaH2AX foci formation in a time- and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of gammaH2AX foci in a time- and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce gammaH2AX foci formation in FL cells; however, AAF can induce gammaH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce gammaH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce gammaH2AX foci. Cell survival analyses indicated that the induction of gammaH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that gammaH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining gammaH2AX foci formation induced by a specific agent.     

Vanadium and 1, 25 (OH)2 vitamin D3 combination in inhibitions of 1,2, dimethylhydrazine-induced rat colon carcinogenesis

The current investigation demonstrates the antitumor effects of combined supplementations of vanadium (V) (4.27 µmol/L drinking water ad libitum) and1α, 25-dihydroxy vitamin D₃ (Vitamin D₃) (0.3 μg/100 μL propylene glycol per os twice a week) on 1, 2 dimethylhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. There was a significant reduction in incidence (70%), multiplicity (P < 0.0001) and volume (P < 0.01) of colon tumors. HPLC-fluorescence assay detected the combinatorial actions of V and Vitamin D₃ against DMH-induced colonic O⁶-methylguanine DNA adducts formation (at four sequential time points; ANOVA, F = 13.56, P < 0.01). Simultaneous inhibition of DNA single strand breaks (P < 0.001) indicates the potency of the combination regimen in limiting the initiation event of colon carcinogenesis. Immunohistochemical analysis revealed that the effect of V and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.001) along with an induction of apoptosis (TUNEL-LI: P < 0.01). The immunoexpression of tumor suppressor p53 and downregulation of antiapoptotic protein BCl-2 in subsequent immunofluorescence assay further provide strong evidence for the combinatorial inhibitory actions of vanadium and vitamin D₃ against DMH-induced rat colon carcinogenesis.     

In vitro and in vivo studies on oxygen free radical and DNA adduct formation in rat lung and liver during benzo[a]pyrene metabolism

Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.     

An alternatively spliced cytochrome P4501A1 in human brain fails to bioactivate polycyclic aromatic hydrocarbons to DNA-reactive metabolites

CYP1A1, a cytochrome P450 enzyme, metabolizes polycyclic aromatic hydrocarbons to genotoxic metabolite(s) that bind to DNA and initiate carcinogenesis. RT-PCR amplification of the complete open reading frame of CYP1A1 generated an amplicon of 1593 bp having deletion of 87 bp of exon-6 that translated into functional P450 enzyme. Unlike wild type CYP1A1, exon 6 del CYP1A1 did not metabolize polycyclic aromatic hydrocarbons such as, benzo(a)pyrene to genotoxic, ultimate carcinogens that form DNA adducts. Exon 6 del CYP1A1 metabolized ethoxyresorufin (the classical substrate for CYP1A1) less efficiently compared with wild type CYP1A1 while pentoxy and benzyloxyresorufin (classical substrates for CYP2B) were dealkylated more efficiently. In silico docking showed alteration of the substrate access channel in exon 6 del CYP1A1 such that benzo(a)pyrene does not bind in any orientation that would permit the formation of carcinogenic metabolites. Genotyping revealed that the splice variant was not generated due to differences in genomic DNA sequence and the variant was present only in brain but not in liver, kidney, lung, or heart from the same individual. We provide evidence that unique P450 enzymes, generated by alternate splicing in a histiospecific manner can modify genotoxic potential of carcinogens such as benzo(a)pyrene by altering their biotransformation pathway.     

Astaxanthin Modulates Autophagy,Apoptosis, and Neuronal Oxidative Stress in a Rat Model of Compression Spinal Cord Injury

The effects of astaxanthin (AST) were evaluated on oxidative mediators, neuronal apoptosis, and autophagy in functional motor recovery after spinal cord injury (SCI). Rats were divided into three groups of sham, SCI?+?DMSO (dimethyl sulfoxide), and SCI?+?AST. Rats in the sham group only underwent a laminectomy at thoracic 8–9. While, the SCI?+?DMSO and SCI?+?AST groups had a compression SCI with an aneurysm clip. Then, this groups received an intrathecal (i.t.) injection of 5% DMSO and AST (10 μl of 0.005 mg/kg), respectively. The rat motor functions were assessed weekly until the 28th day using a combined behavioral score (CBS). Total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were measured in spinal tissue to evaluate oxidative stress-related parameters. Besides, autophagy-related proteins (P62, LC3B, and Beclin1) and apoptosis-associated proteins (Bax and Bcl2) were determined using western blotting on the 1st and 7th days after surgery. Hematoxylin–eosin and Fluoro-Jade B staining were performed to detect the histological alterations and neuronal degeneration. As the result, treatment with AST potentially attenuated rat CBS scores (p?<?0.001) towards a better motor performance. AST significantly reduced the spinal level of oxidative stress by increasing TAC, SOD, and GPx, while decreasing MDA (p?<?0.001). Furthermore, AST treatment remarkably upregulated expression of LC3B (p?<?0.001), and Beclin1 (p?<?0.05) in the spinal cord, but downregulated P62 (p?<?0.05) and the Bax/Bcl2 ratio (p?<?0.001). Consequently, AST reduced SCI-induced histological alterations and neuronal degeneration (p?<?0.001). In conclusion, AST can improve motor function after SCI by reducing oxidative stress/apoptosis and increasing neuronal autophagy.

     

Ethyl Pyruvate Inhibits Peroxynitrite-induced DNA Damage and Hydroxyl Radical Generation: Implications for Neuroprotection

Ethyl pyruvate (EP) has recently been reported to afford protection against neurodegenerative disorders. However, the mechanism underlying EP-mediated neuroprotection remains to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of EP is associated with inhibition of peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite elicited cytotoxicity. Incubation of φX-174 plasmid DNA with 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time- dependent manner. The presence of EP (0.5–10 mM) was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent fashion. The consumption of oxygen induced by 250 μM SIN-1 was found to be decreased in the presence of EP (0.5–10 mM), indicating that EP might affect the auto-oxidation of SIN-1. It was observed that incubation of the plasmid DNA with authentic peroxynitrite caused significant DNA strand breaks, which could also be dramatically inhibited by EP (0.5–10 mM). EPR spectroscopy in combination with spin-trapping technique using 5,5-dimethylpyrroline-N- oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adducts (DMPO-OH) from authentic peroxynitrite, and that EP at 0.5–10 mM inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that EP can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation.     

Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco-2 cells

Protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide (tert-BOOH)- and menadione-induced DNA single strand breaks was investigated in Caco-2 cells. Both tert-BOOH and menadione induced DNA single strand breaks in a concentration-dependent manner. Pre-incubation of Caco-2 cells with either quercetin or rutin for 24 h significantly decreased the formation of DNA single strand breaks evoked by tert-BOOH (P <.05). Iron chelators, 1,10-phenanthroline (o-Phen) and deferoxamine mesylate (DFO), also protected against tert-BOOH-induced DNA damage, whereas butylated hydroxytoluene (BHT) had no effect. Quercetin, and not rutin, decreased the extent of menadione-induced DNA single strand breaks. DFO and BHT, and not o-Phen, protected against menadione-induced DNA strand break formation (P <.05). From the results of this study, iron ions were involved in tert-BOOH-induced DNA single strand break formation in Caco-2 cells, whereas DNA damage evoked by menadione was far more complex. We demonstrated that the flavonoids, quercetin and rutin, protected against tert-BOOH-induced DNA strand breaks by way of their metal ion chelating mechanism. However, quercetin, and not rutin, protected against menadione-induced DNA single strand breaks by acting as both a metal chelator and radical scavenger.     

Differential response to benzo[A]pyrene in human lung adenocarcinoma cell lines: the absence of aryl hydrocarbon receptor activation.

Benzo[a]pyrene (B[a]P) has been shown to produce DNA adducts and to initiate pulmonary carcinogenesis in animals. We observed differential susceptibility to B[a]P in two human lung adenocarcinoma cell lines, A427 and CL3. DNA adducts were induced by B[a]P treatment in CL3 cells, however, A427 cells were much less responsive to B[a]P treatment. Cytochrome P450 1A1 (CYP1A1) is involved in bioactivation of B[a]P in nonhepatic tissues. Cotreatment with alpha-naphthoflavone, a CYP1A1 inhibitor, abolished DNA adduct formation by B[a]P in CL3 cells. Nevertheless, CYP1A1 inducer beta-naphthoflavone, enhanced DNA adduct formation by B[a]P in both A427 and CL3 cells. Both enzyme activity and mRNA levels of CYP1A1 were highly induced by 1 or 10 microM B[a]P treatment for 24 hr in CL3 cells but not in A427 cells. Protein levels of AhR and aryl hydrocarbon receptor nuclear translocator (Arnt) were similar in A427 and CL3 cells before B[a]P treatment. However, B[a]P induced a retarded band with the [32P]-dioxin responsive element in CL3 cells, but not in A427 cells. This study demonstrated that variation in AhR-mediated CYP1A1 induction contributes to differential susceptibility to B[a]P-DNA adduct formation in human lung cells. Since AhR and/or Arnt function is impaired in A427 cells, this cell line offers a model for investigating the repression mechanisms of CYP1A1 induction by B[a]P in lung cells.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24h exposure, 0.01 microM for both PAHs). The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 0.1 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively). The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 (R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21(WAF1) levels (R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (> or = 1.5-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses. The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 0.1 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 microM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.     

H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides

Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs), we have studied the phosphorylation of H2AX (H2AXγ). A549 cells were exposed to benzo[a]pyrene diol epoxide [(+)-anti-BPDE] (a bay-region PAH) and dibenzo[a,l]pyrene diol epoxide [(−)-anti-DBPDE] (a fjord-region PAH) and H2AXγ was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H₂O₂) was used to induce oxidative DNA damage and strand breaks. As showed with single cell gel electrophoresis, neither of the diol epoxides resulted in DNA strand breaks relative to H₂O₂. Visualisation of H2AXγ formation demonstrated that the proportion of cells exhibiting H2AXγ staining at 1 h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H₂O₂ treatment, almost all cells demonstrated H2AXγ at 1 h. Western blot analysis of the H2AXγ formation also showed concentration and time-dependent response patterns. The kinetics of H2AXγ formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AXγ formation and persistence was related to both the number of adducts and their structural features.     

Protective effect of vanillic acid against benzo(a)pyrene induced lung cancer in Swiss albino mice

Vanillic acid (VA) is found in high concentrations in various plants and used as traditional medicine for various diseases. The aim of the existing study is to illustrate the protective effects of VA against benzo(a)pyrene (B(a)P)‐induced lung cancer in Swiss albino mice. B(a)P (50 mg/kg b.wt.) was given orally to induce lung cancer in mice. The body weight, tumor incidence, carcinoembryonic antigen (CEA), neuron‐specific enolase (NSE), and enzymatic/nonenzymatic antioxidants (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and glutathione) were estimated. Further histochemical investigation through hematoxylin and eosin staining was also carried out. B(a)P administered groups showed increased levels of serum pathological markers CEA, NSE along with reduced final body weight as well as decreased tissue enzymatic and nonenzymatic antioxidants activities, whereas VA treatment (200mg/kg/b.wt) along with B(a)P showed significantly reverted the above changes, which proves as prominent anticancer effects in experimentally induced lung cancer. Overall, these results suggest that VA has an efficient preventive action against B(a)P‐induced lung cancer, and this is attributed to its free‐radical scavenging antioxidant activities.