Spatio-temporal characteristics of genotoxicity in the Yangtze River under the background of COVID-19 pandemic

The global spread of viruses can lead to the release of large amounts of disinfectants or antiviral drugs into the water environment.The resulting disinfection byproducts(DBPs)and residual antiviral drugs,acting as genotoxic substances or their precursors,may pose risks to aquatic animals and drinking water sources;however,to date,no studies have analyzed the changes in genotoxicity in the Yangtze River before and after the epidemic.In the present study,water and sediment samples from the Yangtze River were collected during different seasons,just before and after the outbreak of COVID-19,and were assessed using the SOS/umu test(with and without liver S9).The results indicated that water samples exhibited more pronounced genotoxicity than did sediments,with direct genotoxicity being the primary factor.Additionally,there were significant regional differences,with notably greater genotoxicity observed in the upper Yangtze River than in the lower reaches before the COVID-19 epidemic.However,this trend was reversed six to ten months later,suggesting the accumulation of DBPs or antiviral drugs after the COVID-19 pandemic.Moreover,the risk quotient indicated that 65%of the water samples posed a high risk for Paramecium caudatum,whereas 71%of the samples posed a medium risk for Danio rerio,thereby representing a potential threat to the ecological security of the Yangtze River.In conclusion,this study,at the basin scale,revealed the impacts of COVID-19 on the Yangtze River,highlighting the need to prevent DBPs and pharmaceutical pollution during similar events in the future.

Genotoxicity of substituted nitrobenzenes and the quantitative structure－activity relationship

The genotoxicity of 22 substituted nitrobenzenes were evaluated by the chromosome aberrations test in in vitro human peripheral lymphocytes.18 of 22 compounds exhibit genotoxic activities.Quantitative structure-activity relationship model was established to correlate the genotoxicity of substituted nitrobenzenes with the characteristics of the substituents on benzene ring.

磷酸西格列汀中基因毒性杂质及其检测方法的研究进展

目的磷酸西格列汀作为广泛应用于2型糖尿病治疗的口服二肽基肽酶-Ⅳ抑制剂,其安全性直接关系到众多糖尿病患者的健康。该文旨在为研究人员和监管机构提供关于磷酸西格列汀生产中可能产生的基因毒性杂质的综合信息,并为未来新合成路线中的基因毒性杂质筛查提供依据。方法检索了中国知网、PubMed、万方等数据库中与磷酸西格列汀合成相关的基因毒性杂质及其检测技术。结果总结了磷酸西格列汀常用合成途径中可能产生的10种潜在基因毒性杂质及相关检测手段。探讨了近年来在提高这些杂质检测灵敏度、特异性和准确性方面的进展。结论严格控制磷酸西格列汀在生产中产生的基因毒性杂质对于保障糖尿病患者的健康安全至关重要。

Evaluation of the Genotoxicity of Polymeric Microparticles Containing Ametryn Herbicide

Agrochemicals have been used throughout the ages to eradicate weeds and pests. Use of agrochemicals is now commonplace, serving as an aid in farming procedures and contributing to greater agricultural production. However, the indiscriminate use of agrochemicals is a cause for concern because they can exert toxic effects on the environment and hence on living beings. This work involved a genotoxic analysis of controlled release formulations of the herbicide ametryn encapsulated in microspheres of poly（hydroxybutyrate） and poly（hydroxybutyrate-co^valerate）. Genotoxicity was analyzed using cytogenetic, micronucleus, comet and Allium cepa assays, as well as molecular analysis. The results showed that the rate of chromosome breakdown caused by unencapsulated ametryn was much higher than that caused by ametryn encapsulated in the polymer microspheres （p 〈 0.05）. This indicates that controlled release delivery systems employing the polymer formulations should be significantly safer for the environment and for living beings.

HPLC法测定富马酸伏诺拉生片中三种潜在基因毒性杂质的含量

目的建立高效液相色谱(HPLC)法测定富马酸伏诺拉生片中三种潜在基因毒性杂质的含量,并通过方法学验证该方法的可行性。方法采用色谱柱YMC-Pack ODS-AQ,以高氯酸钠溶液(pH 2.5)为流动相A,乙腈为流动相B,进行梯度洗脱,检测波长为265 nm,柱温为30℃,进样量为60μl。结果该方法专属性良好,三种潜在基因毒性杂质分别在0.01483~0.29662μg/ml、0.01479~0.29587μg/ml和0.01618~0.33944μg/ml浓度范围内线性关系良好,平均回收率分别为97.2%、98.3%、97.6%,相对标准偏差(RSD)分别为1.2%、1.6%、4.9%;使用该方法对国产和原研进口的富马酸伏诺拉生片进行检测,均未检出上述潜在基因毒性杂质。结论所建立的HPLC分析法简便、准确、易于操作,可用于富马酸伏诺拉生片潜在基因毒性杂质含量测定,并为其质量控制提供参考依据。

离子色谱法测定维格列汀片中基因毒性杂质氯乙酸含量

目的建立测定维格列汀片中氯乙酸含量的离子色谱法。方法分析柱为Dionex IonPac AS11-HC阴离子分离柱(250 mm×4.6 mm),保护柱为Dionex IonPac AG11-HC阴离子保护柱(50 mm×4.0 mm),淋洗液为10 mmol/L氢氧化钾溶液,流速为1.0 mL/min,辅助气为99.99%氮气,检测器为抑制电导检测器,检测池温度为35℃,进样量为25μL。结果氯乙酸的质量浓度在0.01~2.00μg/mL范围内与峰面积线性关系良好(r=0.9995,n=7);检测限为11.77 ng/g,定量限为62.77 ng/g;精密度、稳定性试验结果的RSD均小于3.0%;平均加样回收率为101.64%,RSD为4.60%(n=12)。10个厂家的10批样品中,有5批检出氯乙酸杂质,其含量为0.0002%~0.0007%,均低于限度规定的0.0015%。结论该方法操作简便、灵敏度高、专属性强、高效快速,可用于维格列汀片中氯乙酸的含量测定。

Genotoxicity evaluation and a primary risk assessment of organic pollutants in the drinking water sources of Nanjing, China

An increasing number of industrial, agricultural, and commercial chemicals in the aquatic environment leads to various deleterious effects on organisms, which is becoming an increasingly serious problem in China. In this study, the comet assay was conducted to investigate the genotoxicity to human body caused by organic concentrates in the drinking water sources of Nanjing City from Yangtze River of China, and health and ecology risk due to expose to these organic pollutants were evaluated with the multimedia environmental assessment system （MEAS）. For all the water samples, they were collected from four different locations in the drinking water sourcr samples, es of Nanjing City. The results of the comet assay showed that all the organic concentrates from the water samples could induce different levels DNA damages on human peripheral blood lymphocytes, and a statistically significant difference （p〈0.01） was observed compared with the solvent control, which demonstrated the genotoxicity was in existence. According to the ambient severity （AS） of individual compound, we had sorted out the main organic pollutants in the drinking water source of the four waterworks, and the results showed that there was some potential hazard to human body for all the source water, namely the total ambient severity （TAS） of health for each water source was more than 1. However, the TAS of ecology for each water source was less than 1, which indicated that it was safe to ecology. The results of this investigation demonstrate the application of the comet assay and the MEAS in aquatic environmental monitoring studies, and the comet assay found to be fast, sensitive, and suitable for genotoxicity monitoring programs of drinking water source.

吉非替尼中SM1、中间体1、中间体2残留研究

鉴于基因毒性杂质对人体产生不可逆转的伤害,制定科学合理的杂质研究控制策略,建立基于风险评估的药物杂质研究方法显得尤其重要。本研究采用UPLC-MS/MS测定吉非替尼中基因毒性杂质SM1[2-氨基-4-甲氧基-5-(3-氯丙氧基)苯甲腈]、中间体1[3-(3-氯丙氧基)-4-甲氧基苯甲腈]和中间体2[2-硝基-4-甲氧基-5-(3-氯丙氧基)苯甲腈]的残留量,采用Agilent SB-C_(18)色谱柱进行分离,梯度洗脱,流动相A为0.2%乙酸溶液,流动相B为甲醇,质谱检测器,流速0.4 mL/min。结果显示,SM1、中间体1、中间体2的线性范围为3.6~36.0 ng/mL,相关系数r为0.9998~1.0000,定量限浓度为3.6 ng/mL,检测限浓度为1.1 ng/mL;精密度试验和精准度试验中3种化合物的平均回收率分别为100.43%~101.78%和99.01%~102.07%,RSD均小于5.0%。本方法可简便、快速、灵敏、准确地定量测定吉非替尼中SM1、中间体1和中间体2杂质含量。

Formation potentials of typical disinfection byproducts and changes of genotoxicity for chlorinated tertiary effluent pretreated by ozone

The effects of ozonation on the formation potential of typical disinfection byproducts （DBPs） and the changes of genotoxicity during post chlorination of tertiary effluent from a sewage treatment plant were investigated. Ozonation enhanced the yields of all detected chlorine DBPs except CHCI3. At a chlorine dose of 5 mg/L, the three brominated THMs and five HAAs increased, while chloroform decreased with the increase of ozone dose from 0 to 10 mg/L （ozone dose in consumption base）. At a chlorine dose of 10 mg/L, the two mixed bromochloro species THMs and two dominant HAAs （DCAA and TCAA） increased firstly and then decreased with the increase of ozone dose, with the turning point approximately occurring at an ozone dose of 5 mg/L. The genotoxicity detected using umu test, on the other hand, was removed from 7 Ixg 4-NQO/L to a negligible level by ozonation under an ozone dose of 5 mg/L. Chlorination could further remove the genotoxicity to some extent. It was found that SUVA （UV absorbance divided by DOC concentration） might be used as an indicative parameter for monitoring the removal of genotoxicity during the oxidation.

Studies of the Genotoxicity of Glycidyl Methacrylate (GMA)

The following experiments were conducted to evaluate the genotoxic effects of GMA (glycidyl methacrylale) on mammalian and human cells.(1) Using the absorption spectrum shift method in vitro, we observed that the maximums of calf thymus DNA and GMA were shifted toward longer wavelengths (a change of more than 15nm) and the absorbance decreased after incubation at room temperature for 15min or more.The result indicates that binding of DNA and GMA had occurred.The binding force is strong, not affected by the addition of concentrated sodium chloride solution, and only slightly decreased by the addition of 8 M urea solution.Therefore the bond between DNA and GMA might be covalent.(2) In cell cultures, unscheduled DNA synthesis (UDS) in human and/or rat lymphocyte was induced and DNA semiconserva-tive replication was inhibited by GMA at concentrations of less than 5.2 mM.(3) Sperm abnormality tests and assays of UDS in germ cells of male mice were conducted to study the in vivo genotoxicity of GMA.The results revealed that GMA could damage DNA, increase sperm abnormality frequency, and reduce the number of sperm cells, 1990 Academic Press.Inc.

Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7cell line model using comet assay

Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta(E.hirta)in MCF-7 cell line model using comet assay.Methods:The cytotoxicity of E.hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E.hirta was assessed by using Comet assay.Results:Both toxicity tests exhibited significant toxicity result.In the comet assay,the E.hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage.The extract of E.hirta showed significant toxicity against brine shrimp with an LC_(50)value of 620.382μg/mL(24 h).Comparison with positive control potassium dichroniate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity.Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E.hirta.However,the observed toxicity of E.hta extracts needs to be confirmed in additional studies.

超高效液相色谱-串联质谱法定量分析羟考酮纳洛酮缓释片中基因毒性杂质7,8-二脱氢纳洛酮

目的建立羟考酮纳洛酮缓释片中潜在基因毒性杂质7,8-二脱氢纳洛酮的超高效液相色谱-串联质谱(UHPLC-MS/MS)检测方法。方法色谱柱为Waters Acquity CSH C 18(2.1 mm×150 mm,1.7μm),以0.77 g·L^(-1)乙酸铵水溶液(冰醋酸调节pH为5.0)为流动相A,以乙腈为流动相B,梯度洗脱,流速为0.5 mL·min-1,柱温为50℃。采用多反应监测(MRM)模式,对羟考酮纳洛酮缓释片中的7,8-二脱氢纳洛酮进行定量检测。结果7,8-二脱氢纳洛酮浓度在1.2~48 ng·mL^(-1)范围内具有良好的线性关系。检测限和定量限分别为0.6 ng·mL^(-1)和1.2 ng·mL^(-1)。低、中、高3个浓度的加样回收率在93%~99%之间(n=9),RSD为2.4%。结论该方法灵敏度高、专属性强,可用于羟考酮纳洛酮缓释片中7,8-二脱氢纳洛酮杂质的含量测定,为羟考酮纳洛酮缓释片杂质控制提供科学依据和定量分析方法。

Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay

The potential harm of organic pollutants in drinking water to human health is widely focused on in the wodd; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference （P 〈 0.01） was observed when compared with the solvent control, The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100x; the degree of DNA damage treated by Hushu waterworks （at town level） was the most serious, the arbitrary units （AU） was 141.62±6.96, however, that of Shangyuanmen waterworks （at city level） was only 109.64±2.97. The analysis also revealed that the genotoxicity of town＇s water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM (Pagrosomus major) FIN CELL LINE RSBF

A continuous marine fish cell line RSBF (i.e. Red Sea Bream Fin) was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine (PEI) and nickel chloride (NiCl 2) in this study on the deleterious effects of aquatic genotoxins on fish. At the 0.01 to 1 μg/ml concentration tested, PEI had acute toxicity to the treated RSBF cells (IC 50 =1.12, 0.92, 0.88 and 0.64 μg/ml PEI for time 0 h, 24 h, 48 h and 72 h after treatment, respectively) and markedly inhibited their proliferation in a dose dependent manner. At the 0.001 to 5 μmol/L concentration tested, NiCl 2 posed no acute toxicity but significantly stimulated their growth (107%-214% of control). Random amplified polymorphic DNA (RAPD) technique was used to detect the genotoxic effects of PEI and NiCl 2 by comparing the RAPD banding patterns of the control and treated cells. RAPD analysis indicated that at the concentrations tested, PEI was more genotoxic than NiCl 2 to RSBF cells; that there was a slight dose dependent response in the genotoxic effect of PEI but not NiCl 2; and that RAPD technique might provide a sensitive, non specific genotoxic endpoint. And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene vector in fish gene transfer and human gene therapy.

Genotoxicity of Pesticide Waste Contaminated Soil and Its Leachate

Objective Improper land disposal of hazardous waste can result in leaching of hazardous constituents which may contaminate ground and surface water leading to adverse impact on human health and environment consequences. The present study utilized mammalian cell culture for the genotoxicity assessment of waste and its leachate. Methods Genotoxic potential and chemical analysis of pesticide derived tarry waste contaminated soil extract and its leachate was assessed using in vitro human lymphocyte cultures and GC-MS. Results The investigation revealed that the soil extract could cause significant to highly significant genotoxicity in the form of DNA strand break at 25 mL (P<0.01), 50 mL, 100 mL and 200 mL (P<0.001) and chromosomal aberration at 25 mL (P<0.01) and 50 mL and 100 mL (P<0.001). The leachate could cause significant DNA strand break and chromosomal aberration only at 100 mL and 200 mL (P<0.01) dose levels. Conclusion The genotoxicity observed is attributed to carbaril and tetra methyl naphthyl carbamate, the major ingredients of the extracts, as revealed by GC-MS.

The Study on Genotoxicity of PM_(2.5) Mineral Dusts to A_(549) Cells

By detecting the influence of six main ingredients of PM2.5 mineral dusts on the A549 cell morphology, proliferation inhibition rate, micronuclei and DNA damage, to explore the genotoxicity of PM2.5 mineral dusts. (1) After exposure to six kinds of dusts of 200 μg/mL concentration for 24 hours, the morphology of A549 cells were observed using Wright-Giemsa staining. (2) After exposure to different concentrations of mineral dusts for 24 hours, the proliferation inhibition rate of A549 cells was detected by MTT assay. (3) Cells were exposed to PM2.5 mineral dusts at a concentration of 200 μg/mL for 24 h. After Wright-Giemsa staining, the rates of micronucleus cells were counted under oil microscope. (4) Observe Comet phenomenon by SCGE electrophoresis, the degree of DNA damage was observed by OTM. (1) Compared to the control group, membrane destruction, nuclear pyknosis and mineral surface adhesion were mainly seen in the Sericite group and Albite group. In the Quartz group and Montmorillonite group, enlarged cell gaps, loosely arranged cells, absorption of a large number of minerals on the cell surface, and cell pyknosis were observed. (2) The proliferation inhibition rate of the six kinds of dusts to A549 cells were (from large to small): KWC-M>Nano-SiO2>KWC-S>KWC-Q>KWC-A>KWC-C.The dust concentration was positively related to the inhibition of cell proliferation rate. (3) With the dusts concentration increased, the incidence of micronuclei gradually increased. The rate was positively correlated to exposure concentration. (4) The six mineral dusts can damage DNA of the A549 cells by dose-response relationship.The higher concentration of the mineral dusts, the more obvious of the DNA damagenation. There’s statistically significant compared with the control group. The six main ingredients of the PM2.5 mineral dusts can change A549 cell morphology from varying degrees, improve proliferation inhibition rate of the cells, increase the number of micronuclei cells, damage DNA.Then we come to the conclusion that PM2.5 mineral dusts can change the genotoxicity of the cells.

Hexabromocyclododecane-induced Genotoxicity in Cultured Human Breast Cells through DNA Damage

To investigate the genotoxicity and reveal the potential toxicological mechanisms of Hexabromocyclododecane （HBCD）, human breast cells HBL-100 were exposed to a sequence of HBCD concentrations （0, 5, 10, and 50 mg/L） for 24 h. With a series of zymology and molecular biology methods, we found that HBCD induced dose-dependent oxidative stress on HBL-100 DNA. As revealed in q RT-PCR, activated prognostic factor ATM down-regulated tumor suppressor gene BRCA1 and prompted DNA repair genes h OGG1 and h MTH1 expression in lower concentrations of HBCD （〈 10 mg/L）. However, DNA repair were inhibited as well as cell proliferation rate by higher concentrations of HBCD （50 mg/L）. The results inferred that the genotoxicity of HBCD was dose-dependent and related to DNA repair pathway.

Evaluation of naproxen-induced oxidative stress, hepatotoxicity and in-vivo genotoxicity in male Wistar rats

Naproxen(NP), a nonsteroidal anti-inflammatory drug(NSAID), is used for the treatment of common pain, inflammation and tissue damage. Genotoxicity testing of NP is of prime importance as it represents the largest group of drugs to which humans are exposed. Not many genotoxic studies are reported on NP;therefore, the present study investigated the detailed genotoxic and oxidative stress properties of NP.Male Wistar rats were administered NP orally at the doses of 38.91 and 65.78 mg/kg body weight for 14 days. Reduced glutathione(GSH), superoxide dismutase(SOD), catalase(CAT) and lipid peroxidation(LPO) activities/levels were measured in the liver, kidney and brain tissues. The aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(ALP) activities, and total bilirubin(TBIL) levels were measured in the liver tissues. Micronucleus frequency(micronucleus test MNT)and DNA damage(comet assay) were performed in the bone marrow cells and leukocytes, respectively.The results showed that NP treatment decreased the GSH levels and increased the SOD, CAT, LPO, ALT,AST, ALP and TBIL activities/levels compared to the control(p o 0.05). Results of MNT showed an increased micronucleus induction and comet assay showed a significant increase in DNA damage in the NP treated animals(p o 0.05). Treatment of NP resulted in the biochemical imbalance and induced oxidative stress that deteriorated the integrity of the cells, which caused significant damage to the genetic material and affected liver function in male Wistar rats. Therefore, NP is a potential genotoxic agent that induces genotoxicity and oxidative stress.

Effect of Buchanania lanzan Spreng.bark extract on cyclophosphamide induced genotoxicity and oxidative stress in mice

Objective:To elucidate the effect of ethanolic extract of Buchanania lanzan Spreng.(B.lanan) bark against cyclophosphamide induced genotoxicity and oxidative stress in mice.Methods: The prevalence of micronuclei in bone marrow,the extent of lipid peroxidation,reduced glutathione and the status of the antioxidant enzymes,superoxide dismutase and catalase in liver of mice were used as intermediate biomarkers for chemoprolection.Lipid peroxidation and associated compromised antioxidant defenses in cyclophosphamide treated mice were observed in the liver.Results:Pre-treatment with B.lanzan 250,500 and 1 000 mg/ kg,p.o.,daily for 7 days significantly reduced the chromosomal damage and lipid peroxidation with concomitant changes in antioxidants and detoxification systems.Conclusions:These results point out the presence of chemopreventive phytoconstituents in the crude extract offering protection against cyclophosphamide induced genotoxicity and oxidative stress in mice.

Evaluation of Cytotoxicity and Genotoxicity of Insecticide Carbaryl to Flounder Gill Cells and Its Teratogenicity to Zebrafish Embryos

In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill （FG） cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was determined with methods including MTT and neutral red uptaking （NRU）, lactate dehydrogenase （LDH） releasing and Hoechst 33342 and propidiurn idodide （PI） double staining. Moderate cytotoxicity in a concentration-dependent manner was observed. The 24h-IC50 value of 53.48 ± 1.21, 59.13 ± 1.19 and 46.21 ± 1.24 mgL 1 carbaryl was obtained through MTT, NRU and LDH assays, respectively. Double fluorescence staining dem- onstrated that carbaryl induced the death of FG cells mainly through necrosis. There was no significant genotoxicity found in the FG cells exposed to the highest testing concentration of carbaryl （20mgL-1, P〉0.05） as was demonstrated by Comet assay. Zebrafish embryos exposed to carbaryl at concentrations 〉10 mg L-1 displayed moderate toxic effects on the survival, spontaneous movement, hatching, heart rates of the embryos and their development, which were evidenced by yolk and pericardial sac edemas, body length reduction and tail flexure in time- and concentration-dependent manners at specific stages. The 24h-, 48h- and 96h-LC50 values of carbaryl to zebra fish embryos were 41.80 ± 1.10, 17.80 ± 1.04 and 14.46 ± 1.05 mg L-1, respectively. These results suggested that car- baryl is moderately toxic to FG ceils cultured in vitro and zebrafish embryos, and the FG cells were similar to zebrafish embryos in their sensitivity to carbaryl as 24h-IC50 and LC50 indicated.