Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters, and thereby guarantee the protection of aquatic life and human health. Wastewater quality evaluation is conventionally based on physicochemical parameters, but increasing attention has been paid to integrate physicochemical and biological data. Nevertheless, the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns, and in vitro cell-based assays have thus become an important topic of interest. Hence, the present study intends: (a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells, and (b) maximizing the use of the effluent sample collected, to estimate the environmental condition of the receiving environment. The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity, and the best results were attained by lyophilising the sample as pre-treatment. Due to its response, the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents. The physicochemical status of the sample suggests a high potential for eutrophication, and iron exceeded the permissible level for wastewater discharge, possibly due to the addition of ferric chloride for wastewater treatment. In general, the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries, and both surpassed the aquatic protective values for long-term exposure.     

Tetrabromobisphenol A (TBBPA): A controversial environmental pollutant

Tetrabromobisphenol A (TBBPA) is one of the most widely used brominated flame retardants and is extensively used in electronic equipment, furniture, plastics, and textiles. It is frequently detected in water, soil, air, and organisms, including humans, and has raised concerns in the scientific community regarding its potential adverse health effects. Human exposure to TBBPA is mainly via diet, respiration, and skin contact. Various in vivo and in vitro studies based on animal and cell models have demonstrated that TBBPA can induce multifaceted effects in cells and animals, and potentially exert hepatic, renal, neural, cardiac, and reproductive toxicities. Nevertheless, other reports have claimed that TBBPA might be a safe chemical. In this review, we re-evaluated most of the published TBBPA toxicological assessments with the goal of reaching a conclusion about its potential toxicity. We concluded that, although low TBBPA exposure levels and rapid metabolism in humans may signify that TBBPA is a safe chemical for the general population, particular attention should be paid to the potential effects of TBBPA on early developmental stages.     

长江南京段水体中有机污染物的遗传毒性研究

运用人外周血淋巴细胞彗星试验和蚕豆根尖细胞微核试验对长江南京段水体中有机污染物的遗传毒性进行了研究. 结果表明:长江南京段水体中的有机污染物对人外周血淋巴细胞和蚕豆根尖细胞均产生了不同程度的损伤,存在明显的遗传毒性,有机物是导致水体遗传毒性的主要因素. 试验结果与水体的有机污染状况基本一致. 彗星试验结果及趋势与微核试验相吻合,但前者更为敏感. 彗星试验和微核试验的结合使用在水环境的遗传毒性监测方面具有较大的应用价值.      

Arsenic methylation by an arsenite S-adenosylmethionine methyltransferase from Spirulina platensis

Arsenic-contaminated water is a serious hazard for human health. Plankton plays a critical role in the fate and toxicity of arsenic in water by accumulation and biotransformation.Spirulina platensis(S. platensis), a typical plankton, is often used as a supplement or feed for pharmacy and aquiculture, and may introduce arsenic into the food chain, resulting in a risk to human health. However, there are few studies about how S. platensis biotransforms arsenic. In this study, we investigated arsenic biotransformation by S. platensis. When exposed to arsenite(As(Ⅲ)), S. platensis accumulated arsenic up to 4.1 mg/kg dry weight.After exposure to As(Ⅲ), arsenate(As(Ⅴ)) was the predominant species making up 64% to86% of the total arsenic. Monomethylarsenate(MMA(Ⅴ)) and dimethylarsenate(DMA(Ⅴ))were also detected. An arsenite S-adenosylmethionine methyltransferase from S. platensis(Sp Ars M) was identified and characterized. Sp Ars M showed low identity with other reported Ars M enzymes. The Escherichia coli AW3110 bearing Spars M gene resulted in As(Ⅲ) methylation and conferring resistance to As(Ⅲ). The in vitro assay showed that Sp Ars M exhibited As(Ⅲ) methylation activity. DMA(Ⅴ) and a small amount of MMA(Ⅴ) were detected in the reaction system within 0.5 hr. A truncated Sp Ars M derivative lacking the last 34 residues still had the ability to methylate As(Ⅲ). The three single mutants of Sp Ars M(C59S, C186 S, and C238S) abolished the capability of As(Ⅲ) methylation, suggesting the three cysteine residues are involved in catalysis. We propose that Sp Ars M is responsible for As methylation and detoxification of As(Ⅲ) and may contribute to As biogeochemistry.     

Analysis of the genotoxic potential of low concentrations of Malathion on the Allium cepa cells and rat hepatoma tissue culture

Based on the concentration of Malathion used in the field, we evaluated the genotoxic potential of low concentrations of this insecticide on meristematic and F₁ cells of Allium cepa and on rat hepatoma tissue culture (HTC cells). In the A. cepa, chromosomal aberrations (CAs), micronuclei (MN), and mitotic index (MI) were evaluated by exposing the cells at 1.5, 0.75, 0.37, and 0.18 mg/mL of Malathion for 24 and 48 hr of exposure and 48 hr of recovery time. The results showed that all concentrations were genotoxic to A. cepa cells. However, the analysis of the MI has showed non-relevant effects. Chromosomal bridges were the CA more frequently induced, indicating the clastogenic action of Malathion. After the recovery period, the higher concentrations continued to induce genotoxic effects, unlike the observed for the lowest concentrations tested. In HTC cells, the genotoxicity of Malathion was evaluated by the MN test and the comet assay by exposing the cells at 0.09, 0.009, and 0.0009 mg/5 mL culture medium, for 24 hr of exposure. In the comet assay, all the concentrations induced genotoxicity in the HTC cells. In the MN test, no significant induction of MN was observed. The genotoxicity induced by the low concentrations of Malathion presented in this work highlights the importance of studying the effects of low concentrations of this pesticide and demonstrates the efficiency of these two test systems for the detection of genetic damage promoted by Malathion.     

Assessment of Bisphenol A (BPA) neurotoxicity in vitro with mouse embryonic stem cells

The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell (ESC) properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A (BPA), a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells.     

Role of nitric oxide in the genotoxic response to chronic microcystin-LR exposure in human–hamster hybrid cells

Microcystin-LR (MC-LR) is the most abundant and toxic microcystin congener and has been classified as a potential human carcinogen (Group 2B) by the International Agency for Research on Cancer. However, the mechanisms underlying the genotoxic effects of MC-LR during chronic exposure are still poorly understood. In the present study, human–hamster hybrid (A_L) cells were exposed to MC-LR for varying lengths of time to investigate the role of nitrogen radicals in MC-LR-induced genotoxicity. The mutagenic potential at the CD59 locus was more than 2-fold higher (p < 0.01) in A_L cells exposed to a cytotoxic concentration (1 μmol/L) of MC-LR for 30 days than in untreated control cells, which was consistent with the formation of micronucleus. MC-LR caused a dose-dependent increase in nitric oxide (NO) production in treated cells. Moreover, this was blocked by concurrent treatment with the NO synthase inhibitor N^G-methyl-l-arginine (l-NMMA), which suppressed MC-LR-induced mutations as well. The survival of mitochondrial DNA-depleted (ρ⁰) A_L cells was markedly decreased by MC-LR treatment compared to that in A_L cells, while the CD59 mutant fraction was unaltered. These results provided clear evidence that the genotoxicity associated with chronic MC-LR exposure in mammalian cells was mediated by NO and might be considered as a basis for the development of therapeutics that prevent carcinogenesis.     

Thiolated arsenicals in arsenic metabolism: Occurrence, formation, and biological implications

Arsenic (As) is a notoriously toxic pollutant of health concern worldwide with potential risk of cancer induction, but meanwhile it is used as medicines for the treatment of different conditions including hematological cancers. Arsenic can undergo extensive metabolism in biological systems, and both toxicological and therapeutic effects of arsenic compounds are closely related to their metabolism. Recent studies have identified methylated thioarsenicals as a new class of arsenic metabolites in biological systems after exposure of inorganic and organic arsenicals, including arsenite, dimethylarsinic acid (DMAV), dimethylarsinous glutathione (DMAIIIGS), and arsenosugars. The increasing detection of thiolated arsenicals, including monomethylmonothioarsonic acid (MMMTAV), dimethylmonothioarsinic acid (DMMTAV) and its glutathione conjugate (DMMTAVGS), and dimethyldithioarsinic acid (DMDTAV) suggests that thioarsenicals may be important metabolites and play important roles in arsenic toxicity and therapeutic effects. Here we summarized the reported occurrence of thioarsenicals in biological systems, the possible formation pathways of thioarsenicals, and their toxicity, and discussed the biological implications of thioarsenicals on arsenic metabolism, toxicity, and therapeutic effects.     

The dynamic changes of arsenic biotransformation and bioaccumulation in muscle of freshwater food fish crucian carp during chronic dietborne exposure

Dietary uptake is the major way that inorganic arsenic (iAs) enters into benthic fish; however, the metabolic process of dietborne iAs in fish muscle following chronic exposure remains unclear. This was a 40-day study on chronic dietborne iAs [arsenite (AsIII) and arsenate (AsV)] exposure in the benthic freshwater food fish, the crucian carp (Carassius auratus), which determined the temporal profiles of iAs metabolism and toxicokinetics during exposure. We found that an adaptive response occurred in the fish body after iAs dietary exposure, which was associated with decreased As accumulation and increased As transformation into a non-toxic As form (arsenobetaine). The bioavailability of dietary AsIII was lower than that of AsV, probably because AsIII has a lower ability to pass through fish tissues. Dietary AsV exhibited a high potential for transformation into AsIII species, which then accumulated in fish muscle. The largely produced AsIII considered more toxic at the earlier stage of AsV exposure should attract sufficient attention to human exposure assessment. Therefore, the pristine As species and exposure duration had significant effects on As bioaccumulation and biotransformation in fish. The behavior determined for dietborne arsenic in food fish is crucial for not only arsenic ecotoxicology but also food safety.     

Evaluation of drinking water treatment combined filter backwash water recycling technology based on comet and micronucleus assay

Based on the fact that recycling of combined filter backwash water（CFBW）directly to drinking water treatment plants（WTP）is considered to be a feasible method to enhance pollutant removal efficiency,we were motivated to evaluate the genotoxicity of water samples from two pilot-scale drinking water treatment systems,one with recycling of combined backwash water,the other one with a conventional process.An integrated approach of the comet and micronucleus（MN）assays was used with zebrafish（Danio rerio）to investigate the water genotoxicity in this study.The total organic carbon（TOC）,dissolved organic carbon（DOC）,and trihalomethane formation potential（THMFP）,of the recycling process were lower than that of the conventional process.All the results showed that there was no statistically significant difference（P>0.05）between the conventional and recycling processes,and indicated that the genotoxicity of water samples from the recycling process did not accumulate in 15 day continuous recycling trial.It was worth noting that there was correlation between the concentrations of TOC,DOC,UV₂₅₄,and THMFPs in water and the DNA damage score,with corresponding R² values of 0.68,0.63,0.28,and 0.64.Nevertheless,both DNA strand breaks and MN frequency of all water samples after disinfection were higher than that of water samples from the two treatment units,which meant that the disinfection by-products（DBPs）formed by disinfection could increase the DNA damage.Both the comet and MN tests suggest that the recycling process did not increase the genotoxicity risk,compared to the traditional process.     

The ex vivo and in vivo biological performances of graphene oxide and the impact of surfactant on graphene oxide's biocompatibility

Graphene oxide(GO)displays promising properties for biomedical applications including drug delivery and cancer therapeutics.However,GO exposure also raises safety concerns such as potential side effects on health.Here,the biological effects of GO suspended in phosphate buffered saline(PBS)with or without 1% nonionic surfactant Tween 80 were investigated.Based on the ex vivo experiments,Tween 80 significantly affected the interaction between GO and peripheral blood from mice.GO suspension in PBS tended to provoke the aggregation of diluted blood cells,which could be prevented by the addition of Tween 80.After intravenous administration,GO suspension with or without 1% Tween 80 was quickly eliminated by the mononuclear phagocyte system.Nevertheless,GO suspension without Tween 80 showed greater accumulation in lungs than that containing 1% Tween 80.In contrast,less GO was found in livers for GO suspension compared to Tween 80 assisted GO suspension.Organs including hearts,livers,lungs,spleens,kidneys,brains,and testes did not reveal histological alterations.The indexes of peripheral blood showed no change upon GO exposure.Our results together demonstrated that Tween 80 could greatly alter GO’s biological performance and determine the pattern of its biodistribution in mice.     

铅染毒导致小鼠DNA损伤与氧化损伤

为了研究亚急性铅暴露对小鼠的遗传毒性及氧化损伤的诱导,将不同剂量的醋酸铅对小鼠隔日灌胃4周,用彗星实验检测其外周血淋巴细胞DNA损伤,并测定肝组织中活性氧自由基(ROS)的水平和脂质过氧化主要终产物丙二醛(MDA)的含量.结果显示,随染铅剂量的增加,小鼠肝线粒体中的ROS水平明显升高,染铅剂量为500 mg·kg-1组与对照组相比有显著性差异;肝MDA含量以及淋巴细胞尾长和尾相显著增加,染铅剂量为50、100、500 mg·kg-1组与对照组相比均有显著性差异;MDA与尾长和尾相的变化趋势一致.诱导产生自由基并导致脂质过氧化作用增强及DNA损伤是铅引起机体损伤的主要机制之一.     

基于细菌毒性测试与小鼠肺基因转录分析的PM2.5健康效应

尽管大量流行病学和毒理学研究表明,PM_(2.5)暴露会导致一系列肺部疾病,但是其毒性机制尚不明确.本研究选取不同浓度梯度PM_(2.5)颗粒物样品进行细菌毒性评价,结果显示颗粒物的发光细菌急性毒性、遗传毒性分别为低毒和阴性.此外,采用气管灌注方法模拟小鼠呼吸暴露,研究了肺脏病理改变及差异基因表达.肺脏病理切片分析显示,PM_(2.5)暴露造成肺组织不同程度炎症反应和纤维化损伤,并呈现浓度越高、损伤程度越明显的现象.通路分析发现PM_(2.5)暴露影响到核糖体蛋白功能、脂肪酸与胆固醇代谢功能的正常表达,提示肺部炎症反应源于基因损害,其造成的损害后果可能是不可逆的.GO聚类分析发现免疫功能发生聚类富集,相关基因功能异常表达可能是造成肺部炎症的具体路径.这些发现有助于了解PM_(2.5)暴露危害路径和机制.     

Genotoxicity of drinking water treated with different disinfectants and effects of disinfection conditions detected by umu-test

The genotoxicity of drinking water treated with 6 disinfection methods and the effects of disinfection conditions were investigated using the umu-test. The pretreatment procedure of samples for the umu-test was optimized for drinking water analysis. The results of the umu-test were in good correlation with those of the Ames-test. The genotoxicity and production of haloacetic acids (HAAs) were the highest for chlorinated samples. UV + chloramination is the safest disinfection method from the aspects of genotoxicity, HAA production and inactivation effects. For chloramination, the effects of the mass ratio of Cl₂ to N of chloramine on genotoxicity were also studied. The changes of genotoxicity were different from those of HAA production, which implied that HAA production cannot represent the genotoxic potential of water. The genotoxicity per chlorine decay of chlorination and chloramination had similar trends, indicating that the reaction of organic matters and chlorine made a great contribution to the genotoxicity. The results of this study are of engineering significance for optimizing the operation of waterworks.     

An E. coli SOS-EGFP biosensor for fast and sensitive detection of DNA damaging agents

An E. coli SOS-EGFP biosensor which expresses enhanced green fluorescent protein as a reporter protein under the control of recA gene promoter in SOS response was constructed for detection of DNA damage and evaluation of DNA damaging chemicals. The chemicals that may cause substantial DNA damage will trigger SOS response in the constructed bacterial biosensor, and then the reporter egfp gene under the control of recA promoter is stimulated to express as a fluorescent protein, allowing fast and sensitive fluorescence detection. Interestingly, this biosensor can be simultaneously applied for evaluation of genotoxicity and cytotoxicity. The SOS-EGFP bacterial biosensor provides a sensitive, specific and simple method for detecting known and potential DNA damaging chemicals.     

甲醛致核酸损伤作用的实验研究

应用单细胞凝胶电泳技术(SCGE)和液相色谱-电化学检测法(HPLC-EC)研究了典型环境污染物甲醛对DNA分子的损伤效应．结果表明：甲醛不仅可诱导人外周血淋巴细胞DNA发生链断裂,还可引起DNA-DNA,DNA-蛋白质交联;甲醛与小牛胸腺DNA的体外作用较弱,但在铁离子介导下对DNA的氧化能力增强,可产生一定量的8-羟基脱氧鸟苷(8-OHdG)加合物：动物实验表明甲醛诱导大鼠肺组织DNA氧化损伤生成8-OHdG,提示甲醛较强的遗传毒性．     

微生物对砷的氧化还原竞争

滤池被广泛运用于饮用水厂中,前期研究发现某水厂生物滤池处理含砷地下水时,一方面三价砷可被生物氧化锰氧化为五价砷,另一方面滤池系统中存在的微生物砷还原酶可促使五价砷还原为三价砷,而滤池表面存在的这种微生物竞争关系会影响滤池的稳定性及处理效率.为探讨其内在机制,本研究选取1株锰氧化模式菌(Pseudomonas sp.QJX-1)和1株砷还原模式菌(Brevibacterium sp.LSJ-9),考察在Mn2+、As(As3+、As5+)共存时,两菌株对空间、营养物质以及对砷氧化/还原的竞争关系.结果表明,不同的反应时间,Mn、As质量浓度/价态不同,三价及五价砷体系中,Pseudomonas sp.QJX-1生成的锰氧化物在砷的氧化还原反应中占主导地位,即能迅速氧化本身存在的As3+(三价砷体系)和砷还原菌产生的As3+(五价砷体系),最终两体系中砷都主要以As5+的形式存在.PCR及RT-PCR结果表明,反应过程中锰氧化菌功能基因(cum A)抑制了砷还原酶(ars C)的表达,锰氧化菌16S rRNA表达量始终比砷还原菌高两个数量级,即锰氧化菌在生长竞争过程中占优势.实验结果表明滤池的水力停留时间是决定出水中砷价态的一个重要因素.     

饮水型砷暴露对小鼠多系统脏器的毒性作用

为揭示饮水型砷暴露对机体的毒性,系统研究了砷摄入对实验动物基础生理和多系统脏器的毒性损伤作用.选雄性ICR小鼠为受试动物,以自由饮用含砷10 mg·L^-1的水溶液进行染毒,连续染毒60 d后检测发现,饮水砷暴露对小鼠一般体征和体重无显著影响,肝脏脏器系数显著降低,心脏、肺脏、肾脏和睾丸脏器系数降低,但无统计学意义.砷染毒组血清谷丙转氨酶（ALT）和谷草转氨酶（AST）活性显著升高,总胆固醇（TC）和甘油三酯（TG）含量显著升高,高密度脂蛋白胆固醇（HDL-C）含量显著降低,低密度脂蛋白胆固醇（LDL-C）含量显著升高;肝脏、小肠、心脏、肺脏、肾脏和睾丸组织中还原型谷胱甘肽（GSH）含量和超氧化物歧化酶（T-SOD）活性显著降低,过氧化氢（H₂O₂）和丙二醛（MDA）含量显著升高,并出现程度不同的组织形态结构损伤.研究结果表明,饮水砷暴露可诱发实验小鼠肝脏功能异常、生理代谢紊乱,导致消化系统、循环系统、呼吸系统、泌尿系统与生殖系统等多系统脏器组织的氧化损伤和结构病变,砷暴露对机体的毒性作用存在组织器官差异性,对肝脏的损伤较严重.     

骨炭修复重金属污染土壤和降低基因毒性的研究

采用分级提取的方法研究了施加骨炭对污染土壤重金属的固定效果,结果表明,土壤施加10 mg·kg^-1骨炭后水溶态、交换态、碳酸盐结合态和铁锰氧化物结合态Pb的浓度都显著下降;而加入骨炭后土壤有机结合态Pb的浓度显著上升,表明骨炭可以吸附、固定土壤中的Pb,改变Pb的化学形态,降低Pb的生物可利用性.加入骨炭后土壤中水溶态和交换态Cd的浓度都显著下降,残渣态Cd的浓度明显上升.骨炭处理对土壤中Cu化学形态的影响和Pb相似,表明骨炭可以吸附固定土壤中的Cu.骨炭处理对Zn也有一定的固定效果,降低了土壤中水溶态的Zn.采用植物彗星实验研究了施加骨炭对土壤基因毒性的影响,表明加入骨炭后减轻了污染土壤对洋葱根尖细胞的DNA的损伤,降低了土壤污染对植物的基因毒性.生物评价和化学评价的结果均表明骨炭是重金属污染土壤的有效修复剂,能有效降低污染土壤中重金属的生物有效性和对植物的毒性.