DNA damage in mouse organs and in human sperm cells by bisphenol A

Abstract

The in vivo genotoxic potential of bisphenol A using the comet assay in mice and in human sperm cells in vitro without metabolizing enzymes was studied. Male mice were exposed by oral gavage to the following doses of bisphenol A (0 125, 250 and 500?mg/kg body weight). DNA damage was investigated in liver, kidney, testes, urinary bladder, colon and lungs cells. In testicular cells, a significant increase in DNA strand breaks was observed in the lowest, but not in the medium or highest dose groups. Histopathological investigation of the testicular samples did not show any treatment dose-related effects. No DNA strand breaks were observed in any of the other investigated tissues. In human sperm cells in vitro, bisphenol A did not induce DNA strand breaks.     

Genotoxic effects of two different classes of insecticide in developing chick embryos

Pesticides provide considerable protection against pest population; however, rampant accumulation of these chemicals into varied habitats across the globe necessitates the need for a careful screening of each chemical due to toxic manifestations. In the current study, the genotoxic potential of two different classes of commercial insecticides – chlorpyrifos and cypermethrin combination and Spinosad, a naturalyte were compared. Rhode Island Red chick embryos were exposed to different doses of either of these insecticides individually, by in ovo treatment. Genotoxicity was then evaluated through micronucleus (MN) test and Comet assay. The combination insecticide exposure at low doses of 0.05 and 0.1 μg/egg induced DNA damage as evidenced by an increased tail moment in the Comet assay. Further, the presence of micronucleated erythrocytes and also various abnormal cells including dacryocytes, microcytes, erythroplastids, squashed/notched nuclei, and spindle-shaped erythrocytes in the blood smear consolidates indicate the presence of insecticide-induced genotoxicity. Spinosad, however, was found only mildly genotoxic but at a high dose of 1.5 mg/egg. The results indicate that usage of naturalyte insecticide may be a better option to minimize the harmful effects of chemical insecticides.     

人血淋巴细胞检测浊漳河地表水的遗传毒性

为评价浊漳河水体中有机污染物的致突变性,采用单细胞凝胶电泳(SCGE)技术,研究了浊漳河水体3个不同样点的有机提取物对人外周血淋巴细胞DNA损伤效应。结果显示,各水样中的有机提取物均能引起DNA损伤,且随剂量的增高,有机物对DNA的损伤加重;细胞DNA损伤与对照组相比均有显著性差异(p<0.01)。这表明人外周血淋巴细胞对水体有机污染物的毒作用非常敏感。细胞彗星尾部DNA含量(%DNAT)和染毒剂量呈极显著正相关(R2>0.8547,p<0.01)。以上结果表明,浊漳河水体受到一定程度的有机物污染,这些有机污染物可诱导淋巴细胞DNA损伤,具有遗传毒性。SCGE技术作为一种简便、快速和灵敏的检测方法在水环境遗传毒性监测方面具有较大的应用价值。     

Effect of latex material on antioxidant enzymes,lipid peroxidation,DNA damage,and chromosomal aberration

Cell integrity is affected by oxidative stress when the production of active oxidants overwhelms antioxidant defense mechanisms. Latex, a natural polymer obtained from Hevea brasiliensis, is used in medical industry for manufacturing surgical gloves, urinary catheters, and dental dams. The aim of this study was to evaluate the effects of latex material on oxidative stress by in vivo and in vitro methods. In addition, the material was screened for its ability to induce any chromosomal aberrations (CAs) by in vitro method. In vivo studies were carried out with implanted latex material onto subcutaneous tissue of various batches of experimental Wistar rats. At the end of experimental period, animals were anesthetized, blood was collected for serum analysis, and sacrificed. Liver was excised for the determination of antioxidant enzymes and lipid peroxidation (LPO). Subcutaneous tissues were obtained for the extraction of genomic DNA from implanted animals and checked for the presence of 8-hydroxy-2-deoxyguanosine (8-OHdG), considered an indicator of DNA damage. Simultaneously, in vitro studies were carried out using fresh liver and subcutaneous tissue obtained from Swiss albino mice treated with physiological saline extract of latex material. For the estimation of both in vitro and in vivo oxidative stress, 10% liver homogenate was assessed for stress indicators like reduced glutathione, glutathione reductase, glutathione peroxidase, LPO and protein content. The results of both in vivo and in vitro studies indicated that the chemical leachents from the latex material did not significantly affect LPO and the levels of antioxidant enzymes. There was also no significant increase in 8-OHdG content due to the presence of implanted latex material. Finally, the results of in vitro CA test and G banding indicated that extracts of test material did not induce any chromosomal abnormalities.     

Assessment of micronuclei and chromosomal anomalies of five biocompatible materials in mice

In vivo genetic toxicology tests measure direct DNA damage or the formation of gene or chromosomal mutations, and are used to predict mutagenic and carcinogenic potential of compounds for regulatory purposes. These adverse genotoxic effects may be manifested in the form of gene mutations, structural chromosomal aberrations (CA), recombination, and numerical changes. The present investigation was carried to assess genotoxic effects of five different implantable biomaterials developed in different laborataries of Sree Chitra Tirunal Institute of Medical Sciences and Technology. All biomaterials were developed for clinical applications. CA and micronuclei (MN) studies are biomarkers of genotoxicity testing. Leachants from the extract of biomaterials are capable of inducing structural and numerical chromosomal changes. The studies were conducted in Swiss albino mice with the physiological saline extract of materials together with cyclophosphamide and physiological saline as positive and negative controls. Animals were administered intraperitoneally (ip) with a single injection of test, positive (cyclophosphamide), and negative (physiological saline) control and sacrificed after 24 or 48?h. Bone marrow cells were collected for CA and MN assays. Data showed that all five biomaterials did not significantly exert genotoxic effects. Hence, the study indicates that these biomaterials do not induce any chromosomal anomalies.     

Induction of micronuclei in tadpoles of Odontophrynus americanus (Amphibia: Leptodactylidae) by the pyrethroid insecticide cypermethrin

Cypermethrin (CY) is an active cyano pyrethroid effective against a wide range of pests encountered in agriculture and forestry. Although CY is not mutagenic in in vitro assays for gene mutation, in vivo assays showed conflicting results. In vivo genotoxicity of the synthetic pyrethroid CY in erythrocytes of Odontophrynus americanus tadpoles was examined. The frequency of micronuclei (MN) was recorded in blood smears obtained from tadpoles exposed in vivo to four different nominal concentrations 5, 10, 20 or 40?µg?L^?1 of the compound and fixed at two sampling times 48 and 96?h. As a positive control larvae were exposed to 40?mg?L^?1 of cyclophosphamide (CP). Tadpoles exposed to all CY treatments showed a significant increase in single small MN compared to the negative control group after 48?h and at 5 and 10?µg?L^?1 of CY at 96?h. Results obtained here demonstrated the genotoxic effects of the commercial formulation CY in the anuran larvae analyzed. Thus, data suggest that measurements of MN and other erythrocytes morphological aberrations performed in circulating blood samples of O. americanus tadpoles is a method for detecting cytogenetic damage in other native species.     

Genotoxic effects of Ag2S and CdS nanoparticles in blue mussel (Mytilus edulis) haemocytes

The use of functionalised metal sulphide nanoparticles (NPs) for nanoremediation and biomedical application is rapidly increasing, which could lead to significant inputs into the marine environment. The potential impact of some NPs on marine organisms is still poorly understood. In the present paper the genotoxic potential of Ag₂S and CdS NPs on Mytilus edulis haemocytes was assessed. MPEG-SH (thiol-terminated methyl polyethylene glycol), was used as capping agent to avoid NPs agglomeration. TEM analysis showed that the Ag₂S NPs size was 13±7 nm, whereas CdS quantum dots had an average diameter of 4±1 nm. DNA integrity was evaluated by Comet assay following exposure to increasing concentration series (0.01–10 mg/L). Both silver and cadmium NPs showed genotoxic effects at the highest dose. MPEG-SH was also found to exert a weak genotoxic activity, suggesting that at least part of the genotoxic potential of functionalised NPs on mussel cells might be attributable to the capping agent. These results confirm the genotoxic potential of Ag₂S NPs for mussel cells and demonstrated, for the first time, that CdS NPs is genotoxic in a marine organism.     

Toxicity of 4-methylimidazole on isolated brain mitochondria: using both in vivo and in vitro methods

4-methylimidazole (4MI) is a compound widely used in various industrial and consumer applications. The most important sources of exposure include chemical caramel coloring, ammoniated molasses, dyes and pigments, rube, cleaning and agricultural chemicals. Toxicity attributed to 4MI in foods has recently become a focus of research. Recent studies showed that 4MI induced adverse changes in various target tissues. Brain is known to be a target organ for 4MI-induced toxicity but its cytotoxic mechanisms have not yet been elucidated. In this study, experiments were divided into two parts: (1) using in vivo methodology, doses of 4MI at 100, 200, or 300 mg/kg were administered orally to mice daily for 14 to obtain brain mitochondria; and (2) utilizing in vitro methodology, brain mitochondria were incubated with 4MI at 400, 800, or 1600 μM concentrations. Subsequently, the neurotoxicity of 4MI was assessed using mitochondrial dysfunction tests, including reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, and cytochrome c release. Our results from both in vivo and in vitro experiments on isolated brain mitochondria showed a significant decrease in complex II activity and also marked elevation in the ROS formation, MMP collapse, mitochondrial swelling, and enhanced release of cytochrome c. Data indicated that 4MI induced neurotoxicity through the impairment of electron transfer chain especially at complex II and elevated ROS formation leading to subsequent oxidative stress events including mitochondrial membrane depolarization, mitochondrial swelling, and release of cytochrome c, which is the starting point of mitochondrial-mediated apoptosis signaling and neurodegeneration.     

Genotoxicity study of high aspect ratio silver nanowires

The genotoxicity potential of silver nanowires synthesized via the solution-based polyol method has been investigated. They were found to be non-mutagenic in three Salmonella strains and were not genotoxic in a clastogenicity assay in mice. Residual surfactant was found to have an effect on the toxicological properties of the nanowires by increasing the rate of Ag⁺ release. Residual surfactant can be easily degraded via a UV treatment.     

利用彗星实验检测丙烯酰胺对小鼠两种细胞的DNA损伤和修复

为了研究丙烯酰胺致小鼠睾丸细胞和外周血淋巴细胞DNA的损伤及修复情况,同时比较这两种细胞对丙烯酰胺的敏感性,将雄性昆明种小鼠一次性腹腔注射丙烯酰胺(50mg·kg-1(bw)),在暴露后第1、2、4、6、8、10、12d,分别对其睾丸组织细胞和外周血淋巴细胞DNA损伤进行彗星实验分析.结果表明,暴露结束后每个时间点小鼠睾丸组织细胞、外周血淋巴细胞DNA的迁移率均显著高于阴性对照组,随时间推移两种细胞DNA迁移距离逐渐降低,同一时间点睾丸组织细胞DNA损伤较外周血淋巴细胞DNA损伤更为严重,两者差异显著(p<0.05).以上结果表明,睾丸组织和外周血淋巴细胞可能是丙烯酰胺的作用位点;机体对丙烯酰胺造成的遗传损伤有一定的修复能力;与淋巴细胞相比,睾丸细胞对丙烯酰胺导致的遗传损伤更为敏感.     

The microcystins-induced DNA damage in the liver and the heart of zebrafish,Danio rerio

Microcystins (MCYST) are the freshwater cyanobacterial toxins, known to induce hepatocellular carcinoma, necrosis, intrahepatic bleeding, as well as human and livestock mortality. Within hepatocytes, MCYST selectively bind to protein phosphatases 1 and 2A, resulting in severe liver damage. The toxicology of MCYST in mice and rats has been well studied, but little is known regarding genotoxicity in aquatic animals. In this study, the zebrafish, Danio rerio was exposed to crude extract of Microcystis aeruginosa bloom. Liver and heart were examined for MCYST-induced toxicity. Light microscopy at 36?h revealed severe, widespread apoptotic necrosis of the majority of hepatocytes, and cytoskeletal deformation in myocardiocytes. Hepatocytes were dissociated with cell shrinkage and margination of nuclear chromatin. Laddering of genomic DNA from the liver and heart of the exposed fish in an increment of 180–200?bp was consistent with apoptosis. Fluorimetric analysis of DNA unwinding was carried out to determine the DNA strand breakage. After 36?h exposure, the % double-stranded DNA was significantly reduced in hepatocytes and myocardiocytes. In conclusion, the results obtained in this study indicate that, the extract of M. aeruginosa bloom is genotoxic to fish. The DNA damage observed in this study may be attributed to the activation of DNA endonucleases. This model of DNA damage may contribute for identifying novel molecular mechanisms of interest for therapeutic application.     

Genotoxic assessment of anthracene and benzo [a] pyrene to milkfish Chanos chanos

Mutagenic and genotoxic effects of polycyclic aromatic hydrocarbons, anthracene and benzo [a] pyrene (BaP), in milkfish Chanos chanos were determined using micronucleus (MN) test and comet assay (CA). Distinct mean frequencies of nuclear abnormalities such as MNs; binucleated micronuclei, nuclear bud, and fragmented apoptotic cells were measured. Significant increase in DNA damage with five classes of damage level was observed and expressed in terms of arbitrary unit (AU). Mean frequencies of total nuclear abnormalities were 0.5?±?0.25 cells in control; 0.67?±?0.33 cells in solvent control; 70?±?9.60 cells in 0.176?mg?L^?1 anthracene, and 91.83?±?6.25 cells in 0.031?mg?L^?1 BaP. The greatest DNA damage of 170AU was observed in 0.176?mg?L^?1 anthracene-exposed group and 182AU was observed in 0.031?mg?L^?1 BaP-treated fish. This study confirmed that the CA and MN assays are useful tools in determining potential genotoxicity of water pollutants and might be appropriate as a part of monitoring program.     

In situ evaluation of the genotoxic potential of the river Nile: I. Micronucleus and nuclear lesion tests of erythrocytes of Oreochromis niloticus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822)

This study aimed to investigate the genotoxic potential of chemicals present along the course of the river Nile using frequencies of micronuclei (MN) and nuclear lesions (NL) in erythrocytes of Nile tilapia Oreochromis niloticus niloticus and African catfish Clarias gariepinus, as biomarkers. Results showed that most of the physicochemical parameters detected and heavy metal concentrations were significantly higher in the water collected from the estuaries of the river Nile compared to other sites of the upper Nile. The frequencies of MN and NL in peripheral blood erythrocytes of Nile tilapia and African catfish were significantly higher in estuary sites in Damietta and Rosetta compared to upper sites. The lowest level of genotoxicity was observed at two sites (Aswan and Kena), considered to be less contaminated. Our results suggested that higher frequencies of MN and NL determined at Damietta and Rosetta sites may be indicative of damage produced by pollutants in these areas. The most remarkable result was that MN and NL frequencies appear to be strongly related to water quality at different sites examined, indicating that MN frequencies may serve as a reliable biomarker for testing genotoxicity in situ. The positive correlation between MN and NL induction suggested that NL may be a useful complementary assay for genotoxicity analyses when fish are used as experimental animals. It was also found that seasonal variations in MN and NL frequencies might contribute to a better understanding of genotoxic responses in the field. The use of fish as indicator organisms for monitoring the presence of genotoxic-inducing contaminants in the environment seemed justified because the effects of exposure to a “complex mixture” such as river water were obtained. Nile tilapia appears to be a more suitable bioindicator species than African catfish in studying genotoxic chemical pollution in the river Nile attributed to a higher sensitivity.     

Effects of benzo(k)fluoranthene,a polycyclic aromatic hydrocarbon on DNA damage,lipid peroxidation and oxidative stress in marine gastropod Morula granulata

Benzo(k)fluoranthene [B(k)F] is one of the widespread priority pollutants of polycyclic aromatic hydrocarbons that has been scarcely studied for exposure assessment. With studies reporting a high amount B(k)F in sediments and water samples around the world, it has become vital to study its effects on aquatic organisms. In this connection, this study is conducted to study the effect of different concentrations of B(k)F (1, 10, 25 and 50?µg/L) in marine gastropod Morula granulata exposed in vivo for 96?h. A concentration-dependent increase in percentage tail DNA (TDNA) as measured by comet assay was observed in snails exposed to B(k)F. Exposure concentrations above 1?µg/L B(k)F showed significant increase in superoxide dismutase (SOD) activity and lipid peroxidation value in snails. After 96?h, SOD activity was found to be doubled for 50?µg/L B(k)F in comparison to control. A significant increase in catalase and glutathione S-transferase activity was observed at all exposure conditions at the end of the exposure time. Our study showed that B(k)F induces oxidative stress in snails which further lead to genotoxic damage. To our knowledge, this is the first study on oxidative stress and genotoxic damage in gastropods exposed to B(k)F.     

Cytogenotoxicity and histopathological assessment of Lekki Lagoon and Ogun River in Synodontis clarias (Linnaeus, 1758)

The cytogenotoxicity and histopathological alterations induced by xenobiotics in Lekki Lagoon and Ogun River on Synodontis clarias were investigated. Fish from these water bodies and a fish farm (control) were examined for micronucleated, binucleated, and immature erythrocytes in both gill and peripheral blood. Also gill, liver, kidney, and ovary were processed for histopathology using hematoxylin-eosin staining. Concentrations of cadmium, zinc, lead and copper in the water were determined. There was significant (p < 0.05) increase in micronucleated, binucleated, and immature erythrocytes in both gill and peripheral blood of S. clarias from the lagoon and river compared to the reference site. Loss and disorganization of the primary and secondary lamellae, multifocal degeneration, hemorrhages, cellular infiltration, congestions, vacuolations, atresia, and necrosis were common lesions in the examined tissues of fish from the lagoon and river. Cd, Zn, Pb, and Cu in water samples from the lagoon and river were higher than the reference site. Xenobiotics in Lekki Lagoon and Ogun River, mostly metals, induced deoxyribonucleic acid (DNA) and pathological damage in S. clarias.     

Effects of sublethal concentrations of permethrin on bioaccumulation of cadmium in zebra cichlid (Cichlasoma nigrofasciatum)

The aim of this study was to evaluate the synergistic and antagonistic effects of permethrin on the bioaccumulation of cadmium in zebra cichlid (Cichlasoma nigrofasciatum). Zebra cichlid was exposed to various sublethal concentrations of permethrin combined with cadmium chloride for 15 days. Although at the lowest concentrations, permethrin (0.40 µg L^?1) decreased the bioconcentration potential of cadmium in fish, bioaccumulation of cadmium increased along with increasing concentrations of permethrin (0.80–1.20 µg L^?1). However, the magnitude of changes in the accumulation of cadmium in the fishes simultaneously exposed to 1.60 µg L^?1 permetrin and cadmium chloride was similar to the cadmium levels observed in fishes after exposure to the cadmium chloride alone. The results of this study suggest that the synergistic effects of permethrin on the bioaccumulation of cadmium highly depend on the concentrations of this pesticide in water.     

Effects of nanopesticide chlorfenapyr on mice

The effects of chlorfenapyr nano preparation on mice were examined. The animals were administered with different doses of 4.84, 9.68, or 19.36?mg?kg^?1 of chlorfenapyr common preparation or nano preparation intraperitoneally (IP). Micronucleus (MN) test and comet assay (CA) experiments revealed that at the same dose, similar DNA damage in the peripheral blood lymphocytes of the mouse and chromosome damage in bone marrow cells of the mouse occurred with chlorfenapyr nano preparation; however, the severity of these effects was less than that found in common preparation. It is noteworthy that there were no differences in apoptotic ratio and live cell percentage in the liver cells of the mouse between these two pesticide formulations using flow cytometry. Data indicate that chlorfenapyr nanoformulation is less toxic to mouse cells than the common formulation.     

Blood concentrations of lead,cadmium, mercury and their association with biomarkers of DNA oxidative damage in preschool children living in an e-waste recycling area

Reactive oxygen species (ROS)-induced DNA damage occurs in heavy metal exposure, but the simultaneous effect on DNA repair is unknown. We investigated the influence of co-exposure of lead (Pb), cadmium (Cd), and mercury (Hg) on 8-hydroxydeoxyguanosine (8-OHdG) and human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) mRNA levels in exposed children to evaluate the imbalance of DNA damage and repair. Children within the age range of 3–6 years from a primitive electronic waste (e-waste) recycling town were chosen as participants to represent a heavy metal-exposed population. 8-OHdG in the children’s urine was assessed for heavy metal-induced oxidative effects, and the hOGG1 mRNA level in their blood represented the DNA repair ability of the children. Among the children surveyed, 88.14% (104/118) had a blood Pb level >5 μg/dL, 22.03% (26/118) had a blood Cd level >1 μg/dL, and 62.11% (59/95) had a blood Hg level >10 μg/dL. Having an e-waste workshop near the house was a risk factor contributing to high blood Pb (r _s  = 0.273, p < 0.01), while Cd and Hg exposure could have come from other contaminant sources. Preschool children of fathers who had a college or university education had significantly lower 8-OHdG levels (median 242.76 ng/g creatinine, range 154.62–407.79 ng/g creatinine) than did children of fathers who had less education (p = 0.035). However, we did not observe a significant difference in the mRNA expression levels of hOGG1 between the different variables. Compared with children having low lead exposure (quartile 1), the children with high Pb exposure (quartiles 2, 3, and 4) had significantly higher 8-OHdG levels (β _Q2 = 0.362, 95% CI 0.111–0.542; β _Q3 = 0.347, 95% CI 0.103–0.531; β _Q4 = 0.314, 95% CI 0.087–0.557). Associations between blood Hg levels and 8-OHdG were less apparent. Compared with low levels of blood Hg (quartile 1), elevated blood Hg levels (quartile 2) were associated with higher 8-OHdG levels (β _Q2 = 0.236, 95% CI 0.039–0.406). Compared with children having low lead exposure (quartile 1), the children with high Pb exposure (quartiles 2, 3, and 4) had significantly higher 8-OHdG levels.     

茶多酚对三丁基锡诱导的小鼠氧化损伤的保护作用

为研究茶多酚对三丁基锡诱导的小鼠氧化损伤是否具有保护作用,采用灌胃方法,对雄性小鼠进行TBT染毒, 然后分别用不同剂量的茶多酚进行保护．结果表明,茶多酚保护组小鼠肝组织活性氧(ROS)活性和丙二醛(MDA)含量均明显低于TBT对照组;彗星实验发现茶多酚保护组小鼠淋巴细胞尾长较正常,而尾相与TBT对照组相比没有显著改变．电镜观察结果表明茶多酚保护组胸腺细胞核和线粒体损伤明显减轻．因此茶多酚对TBT诱导的氧化损伤具有一定的预防作用,并且对细胞核损伤也有一定的保护作用．茶多酚对TBT所引起的细胞核损伤的保护作用机制可能是抑制脂质过氧化反应,防止细胞氧化损伤,从而保护DNA．     

Tissue-specific stress and hepatic DNA damage in Pelteobagrus fulvidraco caused by low concentrations of cadmium

Physiological stress and DNA damage in Pelteobagrus fulvidraco induced by continuous exposure to cadmium at concentrations of 170 and 1700 µg/L for up to 28 days was evaluated. The activities of acetylcholinesterase and monoamine oxidase in brain tissue, Na⁺-K⁺-ATPase and glutathione in gill tissue, and superoxide dismutase and catalase in liver tissue were measured. In studying random amplified polymorphic DNA to evaluate cadmium-induced hepatic genotoxicity, both the appearance of new bands and the disappearance of existing bands were observed, as well as increased levels of monoamine oxidase and Na⁺-K⁺-ATPase and decreased activities of antioxidant enzymes. The results suggest that continuous exposure to cadmium at the studied levels can induce biochemical and physiological changes and DNA damage in P. fulvidraco.