氯化饮水中有机提取物对大鼠和人肝肿瘤细胞（HepG2）的遗传毒性及脂？…

应用体内,体外微核分析和单细胞凝胶电泳技术（慧星分析,ｃｏｍｅｔ分析）探讨氯化饮水的有机提取物对人肝肿瘤细胞和大鼠肝细胞,骨髓嗜多染红细胞的遗传损伤作用,并观察其对大鼠组织脂质过氧化物作用的影响。结果表明,氯化饮水低浓度提限物能诱导大鼠ＰＣＥｓ微核的增加,而高浓度提取物则能增加ＨｅｐＧ２微核的发生率。     

Vinclozolin, a widely used fungizide, enhanced BaP-induced micronucleus formation in human derived hepatoma cells by increasing CYP1A1 expression

Vinclozolin, a widely used fungicide, can be identified as a residue in numerous vegetable and fruit samples. To get insight in its genetic toxicity, we investigated the genotoxic effect of vinclozolin in the human derived hepatoma cell line HepG2 using the micronucleus (MN) assay. Additionally, to evaluate the co- or anti-mutagenic potency of vinclozolin, we treated HepG2 cells with different concentrations of vinclozolin for 24 h. Subsequently, the cells were exposed to benzo[a]pyrene (BaP) for 1h. Exposure of HepG2 cells to 50-400 microM vinclozolin alone did not cause any induction of micronuclei. However, a pronounced co-mutagenic effect was observed. MN frequencies caused by BaP increased by 30.6%, 52.8% and 65.3% after pretreatment of the cell cultures with 50, 100 and 200 microM vinclozolin, respectively. The highest concentration (400 microM) of vinclozolin tested caused cytotoxicity. Therefore, micronuclei were not considered for that concentration. To clarify the mechanism of cogenotoxicity, we assayed cytochrome P450 1A1 (CYP1A1), which plays a pivotal role in activation of BaP. Cells exposed to vinclozolin led to significant increase of CYP1A1 expression in Western blot. The result suggested that induction of CYP1A1 by vinclozolin account for its enhancing effect on genotoxicity caused by BaP.     

Ethanol enhanced the genotoxicity of acrylamide in human, metabolically competent HepG2 cells by CYP2E1 induction and glutathione depletion

In the present study, the genotoxicity of acrylamide (AA) was investigated in HepG2 cells using SCGE. Additionally, the influence of ethanol on the modulation of AA-induced DNA-migration caused by CYP2E1-upregulation and/or GSH-depletion was examined in the same cell line. For the ethanol/AA combination assays, the cells were treated with ethanol for 24 h prior to exposure to 5 mM AA for another 24 h. 1.25 to 10 mM AA-induced DNA migration (OTM) in HepG2 cells in a concentration-dependent manner, e.g., exposure to 10 mM AA, resulted in an 8-fold increase of DNA migration compared to the negative control. Treatment with 120 mM ethanol prior to exposure to 5 mM AA increased the level of DNA migration more than 2-fold as compared to cells treated with 5 mM AA alone. Immunoblotting showed a clear ethanol-induced increase of CYP2E1, which plays a pivotal role in AA toxification. Additionally, intracellular GSH levels were significantly reduced after ethanol or AA treatment. In the ethanol/AA combination experiments, GSH depletion was comparable to the additive effect of the single compounds. No induction of apoptosis (ssDNA assay), but necrosis was identified as responsible for the reduction of viability with increasing compound concentration. The data clearly show a higher genotoxic potential of ethanol/AA combination treatment compared to AA treatment alone. In conclusion, both the ethanol-mediated induction of CYP2E1 and the depletion of GSH provide a mechanistic explanation for the over-additive effects of ethanol and AA. Even though the concentrations used in this study were rather high, consequences for the dietary intake of AA-containing food and alcoholic beverages should be discussed.     

Cytotoxicity and genotoxicity in human lung epithelial A549 cells caused by airborne volatile organic compounds emitted from pine wood and oriented strand boards

Due to the massive reduction of air-change rates in modern, energy-saving houses and dwellings, the contribution of volatile organic compound (VOCs) emissions from wood-based materials to indoor air quality has become increasingly important. To evaluate toxicity of VOC mixtures typically emitted from pine wood and oriented strand boards (OSB) and their main constituents (selected terpenes and aldehydes), cytotoxicity and genotoxicity were investigated in human A549 lung cells. To facilitate exposure directly via gas phase, a 250 L emission chamber was combined with a Vitrocell^® exposure system. VOC exposure concentrations were measured by GC/MSD. Biological effects were determined after an exposure time of 1 h by measuring cytotoxicity (erythrosine B staining) and genotoxicity (comet assay). Neither cytotoxic nor genotoxic effects were observed for VOC mixtures emitted from pine wood or OSB at loading factors of approximately 13 m²/m³ (worst case conditions) of the panels (with maximum VOC levels of about 80 mg/m³) in comparison to clean air. While α-pinene and Δ³-carene did not induce toxic effects even at exposure concentrations of up to 1800 mg/m³ and 600 mg/m³, respectively, hexanal showed a cytotoxic effect at 2000 mg/m³. The α,β-unsaturated aldehydes 2-heptenal and 2-octenal caused genotoxic effects in concentrations exceeding 100 mg/m³ and 40 mg/m³, respectively. In conclusion, high concentrations of VOCs and VOC mixtures emitted from pine wood and OSB did not lead to adverse effects in A549 human lung cells even at concentrations 10² to 10⁵-fold higher than those found in normal indoor air. Attention must be paid to mutagenic and possibly carcinogenic α,β-unsaturated aldehydes.     

DNA damage caused by extracts of chlorinated drinking water in human derived liver cells (HepG2)

Dong (D) lake and the Yangtze (Y) river are the main water supplies of the city of Wuhan, PR China. In the present study, the genotoxic effect of chlorinated drinking water (CDW) processed from raw water of D lake and Y river was evaluated in human HepG2 cells using the Comet assay and the micronucleus test. For that, HepG2 cells were exposed to XAD extracts of CDW corresponding to 0.167, 1.67, 16.7 and 167 ml CDW/ml cell culture. All CDW extracts caused a significant and dose-dependent increase of DNA migration in HepG2 cells. The level of DNA damage varied depending on the sampling time (season) and sampling site. The lowest concentration which caused a significant increase of DNA migration was 1.67 ml CDW/ml culture for water samples collected in August. Water samples collected in March showed their lowest observable effect levels in 167 ml and 16.7 ml CDW/ml culture for Y river and D lake, respectively. Additionally, significant increases of micronuclei (MN) frequencies were found in HepG2 cells after CDW treatment. However, in the MN assay the CDW samples collected in March exhibited higher genotoxicity than the August samples. In conclusion, HepG2 cells provide a useful tool for the detection of genotoxic effects of environmental mixtures.     

3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells

3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.     

Protective effects of Brussels sprouts towards B[a]P-induced DNA damage: a model study with the single-cell gel electrophoresis (SCGE)/Hep G2 assay.

The aim of this study was to investigate the chemoprotective effects of Brussels sprouts juice towards benzo[a]pyrene (B(a)P)-induced DNA damage in the single-cell gel electrophoresis (SCGE)/Hep G2 test system. This assay combines the advantages of the SCGE assay with that of the use of human-derived cells possessing inducible phase I and phase II enzymes. Co-treatment of Hep G2 cells with small amounts of Brussels sprouts juice (0.25-2.0 microl/ml) and B(a)P reduced the genotoxic effect of the latter in a dose-dependent manner. Contrary to the results with the crude juice, unexpected synergistic effects were observed with allyl isothiocyanate (AITC, 1.0-6.0 microM), a breakdown product of sinigrin, which is the most abundant glucosinolate in Brussels sprouts. Although these concentrations of AITC did not cause DNA damage per se, at higher concentrations (> or =25 microM), the compound caused a pronounced dose-dependent DNA damage by itself. Mechanistic studies showed that Brussels sprouts juice causes induction of activities of ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) at dose levels which were protective towards B(a)P. In combined treatment experiments with (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE, 5.0 microM), the main genotoxic metabolite of B(a)P, and Brussels sprouts juice, only weak protection was found indicating that the mechanism of chemoprotection of Brussels sprouts is not mediated through inactivation of this metabolite. In conclusion, our findings show that Brussels sprouts are highly protective against B(a)P-induced DNA damage in human-derived cells.     

Use of primary blood cells for the assessment of exposure to occupational genotoxicants in human biomonitoring studies

The Comet assay is an often used approach for the assessment of genetic damage in primary cells of exposed populations. In the majority of these studies lymphocytes are used. Therefore, we reviewed human biomonitoring studies of occupational exposure using the Comet assay with lymphocytes. We also tried to elucidate the strengths of the studies, which were that (i) data could be obtained in a fast and cost-effective manner, (ii) the ease at which these cells can be collected and (iii) a remarkable concordance between Comet assay and cytogenetic assays. However, the analysis also revealed some shortcomings: (i) the low number of study participants, (ii) the bias in the distribution of gender, (iii) lack of qualitative and quantitative exposure data, (iv) omission to consider differences in physical activity and diet between control and exposed groups, (v) lack of uniformity in the Comet assay procedures, and (vi) controversy in the sensitivity of Comet assay since it picked up DNA damage caused by agents which were found to be weak genotoxicants or non-genotoxicants in other tests, but gave inconsistent results with known mutagens/carcinogens such as cigarette smoke.     

Benzo(a)pyrene induced micronucleus formation was modulated by persistent organic pollutants (POPs) in metabolically competent human HepG2 cells

Due to their bioaccumulation and their biological properties persistent organic pollutants (POPs) attract wide attention. In the present study we investigated the genotoxicity, cogenotoxicity and antigenotoxicity of three selected POPs (DDT, aroclor-1254 and toxaphene) in the HepG2 micronucleus assay. Exposure of HepG2 cells to DDT (17.8-60 microM) and aroclor-1254 (23-184 microM) alone did not increase the micronucleus-frequencies. A slight genotoxic effect could be observed after exposure to toxaphene (20-40 microM). Additionally, the ability of POPs to enhance/decrease the benzo(a)pyrene (BaP)-induced micronucleus formation was investigated. Exposure of HepG2 cells to 50 microM BaP alone led to a more than 2-fold increase of micronuclei (MN) compared with the background frequency. But when the cells were pretreated with 23-181 microM aroclor-1254 or 10-20 microM toxaphene, BaP exposure caused significantly more MN than BaP alone. In contrast, DDT (17.8-60 microM) reduced BaP-induced micronucleus induction by 6-38%. Mechanisms of action are discussed.