HPLC法研究苯乙烯-DNA的加合特性

目的：研究苯乙烯的DNA加合特性。方法：采用高效液相色谱法研究苯乙烯-7，8-氧化物（S0）、苯乙烯、苯乙醇酸（MA）、苯乙醛酸（PGA）和DNA的加合反应。结果：苯乙烯、MA、PGA不与DNA发生加合反应；SO可与脱氧鸟苷酸及脱氧腺苷酸发生加合反应，生成以共价键结合的加合物。结论：苯乙烯进入机体后，通过其活性中间代谢物SO与DNA起加合作用，SO攻击DNA脱氧鸟苷酸及脱氧腺苷酸形成加合物，如果在细胞复制前所形成的DNA加合物没有被修复或者被错误修复的话，就有可能导致基因突变。苯乙烯的其他代谢物未见此效应。     

职业接触苯乙烯的生物限值研究

目的：研究职业接触苯乙烯的生物限值。方法：高效液相色谱法测定苯乙烯作业工人班前、班后尿中苯乙醛酸（PGA）和苯乙醇酸（MA）的含量，监测工人8h苯乙烯的接触水平，同时研究两者的相关性。结果：尿中的PGA和MA浓度与苯乙烯的接触量有明显的剂量－反应关系。根据作业场所空气中苯乙烯的国家卫生标准，按回归方程推导出职业接触苯乙烯的生物限值。结论：对职业接触苯乙烯的生物限值提出的推荐值：工作班末MA为220mmol/mol肌酐或300mg/g肌酐，下一班前为88mmol/mol肌酐或120mg/g肌酐；PGA班末为7mmol/mol肌酐或100mg/g肌酐，下一班前为30mmol/mol肌酐或40mg/g肌酐。     

苯乙烯作业工人染色体畸变率与尿中代谢物相关性研究

目的　研究苯乙烯作业工人染色体畸变率与尿中代谢物苯乙醛酸 (PGA)、苯乙醇酸 (MA )浓度的关系。方法　对 10 0名接触苯乙烯的工人和 10 0名非苯乙烯作业的工人进行流行病学调查 ,测其班后尿中PGA、MA浓度 ,检测外周血淋巴细胞的染色体畸变率。结果　苯乙烯在一定浓度下可造成作业工人染色体畸变率增加 ,且经单因素分析 ,班后尿中MA +PGA浓度、工龄等因素与工人的染色体畸变率相关。结论　苯乙烯对接触者的染色体具有损伤作用 ,且与班后尿中MA +PGA浓度有很好的相关性。建议班后尿中MA +PGA浓度的卫生标准应低于 (0 4 4 2± 0 0 5 6 )g/L。     

苯乙烯生物标志物的研究

目的 研究苯乙烯的生物标志物，为苯乙烯的生物监测提供理论依据。方法 采用高效液相色谱法对苯乙醇酸(MA)、苯乙醛酸(PGA)、苯乙烯巯基尿酸(MUA)进行监浏。结果 晨尿中MA、PGA、MUA和苯乙烯接触浓度间相关关系分别为y＝2．58x 70．82，y＝1．66. 37．42，y＝0．05x 0．555;班末尿中MA、PGA、MUA和苯乙烯接触浓度间相关关系分别为y＝1．85x 89．02，y=1．33x 4．32，y＝0．04x |0．68，均呈良好的相关性。结论 晨尿及班末尿中的MA、PGA、MUA测定均可作为苯乙烯的生物监测指标。     

苯乙烯遗传损伤研究进展

苯乙烯常温下为无色透明、具有芳香气味的液体,是合成橡胶和塑料的重要单体之一.因其主要中间代谢产物苯乙烯-7,8-氧化物(Styrene-7,8 -oxide,SO)为直接致癌物,国际癌症研究机构(IARC)将其列为G2B组.苯乙烯进入人体后,在肝混合氧化酶的作用下主要被氧化为SO,SO主要代谢过程为:①经P450酶系作用,在谷胱甘肽-S-转移酶的作用下,形成谷胱甘肽结合物,随后在肾脏及胰腺中γ-谷氨酰转肽酶作用下,断裂其谷氨酰基,继而在肝脏及肾脏中的半胱氨酰甘氨酸酶及氨肽酶的共同催化下,成为半胱氨酸结合物,随后在N-乙酰转移酶的催化下,生成苯乙烯巯基尿酸从尿中排出;②经P450酶系作用,在环氧化物水解酶作用下转变为苯乙烯乙二醇,并继续氧化为苯乙醇酸(Mandelic acid,MA)和苯乙醛酸,随尿排出,当接触高浓度苯乙烯时,部分MA还会转化成非特异性马尿酸,随尿排出;③经P488酶系作用,与DNA发生反应,生成DNA加合物或引起DNA链断裂,从而引发生物学效应[1].     

苯醌-脱氧鸟苷酸加合物的结构及化学特性初步研究

目的 测定苯醌(BQ)与脱氧鸟苷酸(dGMP)反应形成的加合物结构与化学特性。方法 高效液相色谱—质谱联机技术及紫外分光光度法。结果 苯醌与脱氧鸟苷酸反应形成两种可检测的加台物Ad1和Ad2，质谱显示Ad1的分子量为437，在不同pH条件下的特征紫外吸收光谱及结合键类型测定结果表明，Ad1为BQ与dGMP共价结合而成，推测结合位点在dGMP的N—1和N^2位，分子式为C16H16O8N5P。BQ与小牛胸腺DNA反应水解产物经高效液相色谱(HPLC)分离同样检出了与Ad1具有相同保留时间的化合物。Ad2分子量为241，其分子式为C11H702N5，可能是由BQ进攻dGMP的N—9位并脱掉糖基所得。结论 苯醌可在体外试管反应条件下形成DNA加合物，其中一种主要的加合物结构为(3’-经基)—1，N^2—苯乙烯基—2’—脱氧鸟苷—5’—磷酸。     

不同膳食硒水平对甲基苄基亚硝胺（NMBzA）诱发大鼠食道上皮细胞DNA烷基化和

本文报道了硒对NMBzA诱发大鼠食道上皮细胞DNA加合物的形成，DNA加合物修复酶（甲基转移酶）的活性以及-cmyc的基因表达的影响，一次腹腔注射NMBzA 2.5mg/Kg体重，6小时后测定产食上皮细胞中O6－甲基脱氧鸟嘌呤核甙酸（O6－MedGua)和N7－甲基脱氧鸟嘌呤核甙酸（N7－MedGua)的含量，结果表明饲料中不同硒含量对DNA加合物无明显影响；对O6－MedGua DNA甲基转移酶活性也无明显作用，给大鼠每周一次灌胃NMBzA 3mg/kg体重，共8周诱发大鼠食道上皮细胞c-myc基因表达，结果膳食缺硒（＜0．02ppm)和补硒（2ppm)对 c-myc基因表达亦无影响.     

苯乙烯对大鼠不同脑区多巴胺递质含量及单胺氧化酶活性的影响

目的观察苯乙烯在不同染毒期限和染毒剂量下对大鼠神经系统多巴胺递质含量及单胺氧化酶活性的影响。方法大鼠随机分为5组,每组12只动物,雌雄各半。苯乙烯急性染毒剂量为600mg/kg,亚急性染毒剂量为150~600mg/kg,恢复组在苯乙烯染毒后正常饲养3周,L-dopa组在苯乙烯染毒同时腹腔注射600mg/kgbw的L-dopa。方法监测动物尿中苯乙烯代谢物苯乙醇酸(MA)和苯乙醛酸(PGA)的含量作为苯乙烯染毒的内剂量,测定苯乙烯染毒大鼠不同脑区多巴胺(DA)含量及参与多巴胺代谢的单胺氧化酶(MAO)活性的变化。结果尿中的PGA和MA含量与染毒剂量均呈正相关,由于存在一定的环境本底,MA比PGA在作为苯乙烯染毒内剂量的指标上更有代表性。视网膜、垂体和纹状体中的DA含量在苯乙烯染毒下显著降低,垂体中的MAO活性增加,而纹状体和视网膜中的MAO活性减小。结论苯乙烯可以通过多巴胺通路产生对机体的神经损伤。     

香烟侧流烟雾引起的DNA分子氧化损伤

目的：通过研究环境烟草烟雾的侧流烟雾（ETSS）对DNA分子的氧化损伤，探讨环境烟草烟雾（ETS）的遗传毒性效应及其分子机制。方法：以DNA加合物8－羟基脱氧鸟苷（8－OHdG)作为DNA氧化损伤的生物学标志物，用高压液相色谱－电化学检测（HPLC－EC）法对ETSS染毒后的DNA中8－OHdG进行定量检测，通过气质联用法（GC－MS）对香烟烟雾进行有机成分分析和原子吸收法（AAS）对其进行无机元素分析，并从化学组成成分的角度探讨DNA氧化损伤的分子机理。结果：ETSS的颗粒物和挥发性有机物污染物123种和84种，有机元素7种，其中醌类，多酚等化合物具有自氧化作用，不需要任何生物活性系统，在体外就可产生大量的活性氧自由基，并在金属的催化作用下进攻DNA的碱基，形成加合物8－OHdC ,结论：ETSS对DNA具有氧化能力，体现了直接的遗传毒性效应，8－OHdG是DNA氧化损伤较好的效应标志物。     

香烟烟雾成分分析及其对DNA生物氧化能力研究

目的 从分子生物学水平上探讨香烟烟雾对DNA的生物氧化能力。方法 以DNA加合物8－羟基脱氧鸟苷（8－OHdG）作为DNA氧化损伤的生物学标志物,用高压液相色谱－电化学检测法对香烟烟雾染毒后的DNA中8－OHdG进行定量。通过气质联用法（GC－MS）对香烟烟雾进行有机分成分析,用原子吸收法（AAS）对其进行无机元素分析。结果 香烟烟雾颗粒物和挥发性有机物可不同程度地引起DNA基损伤产生8－OHdG,并分别检出有机污染物157种和78种,无机元素5种。香烟烟雾对DNA的生物氧化能力主要是由于存在大量有机污染物,其中的醌类、多酚等化合物具有自氧化作用,在体外可产生大量的活性氧自由基,并在金属的催化作用下进攻DNA的碱基,形成加合物8－OHdG。结论 香烟烟雾对DNA具有氧化能力,香烟烟雾具有直接的遗传毒性效应。     

体外甲基丙烯酸环氧丙酯-DNA加合物的研究

目的 探讨新诱变剂甲基丙烯酸环氧丙酯（ＧＭＡ）与ＤＮＡ形成加合物的类型和特性。方法 在体外条件下,dＡＭＰ、dＣＭＰ、dＧＭＰ、dＴＭＰ及小牛胸腺ＤＮＡ与ＧＭＡ发生反应,反应产物经紫外光谱、反相高效液相色谱（ＲＰ－ＨＰＬＣ）和质谱先进方法分析。结果 ＧＭＡ能与dＡＭＰ、dＣＭＰ、dＧＭＰ及小牛胸腺ＤＮＡ形成专一性共价结合。已 被证实１捉ＧＭＡ－ＤＮＡ加合物为Ｎ３－甲基丙烯酸－２羟丙－基－脱氧胞嘧啶核苷一磷     

Hemoglobin adducts as a marker of exposure to chemical substances, especially PRTR class I designated chemical substances

Many new biomarkers are being studied, in addition to classical biomarkers, such as chemical substances and their metabolites in blood and urine and modified enzymes. Among these new biomarkers, hemoglobin adducts are thought to be especially useful for the estimation of chemical exposures. We review here the use of biomarkers for monitoring exposures to nine substances, mainly focusing on PRTR class I designated chemical substances, styrene, phenyloxirane (styrene oxide), 4,4'-methylendiphenyl diisocyanate (MDI), 4,4'-methylendianiline (MDA), 1,3-butadiene, ethylene oxide, propylene oxide, acrylamide and acrylonitrile. Hemoglobin adduct levels were elevated after exposures to styrene, MDI, MDA, 1, 3-butadiene, ethylene oxide, acrylamide and acrylonitrile. Moreover, hemoglobin adducts of butadiene, ethylene oxide, acrylamide and acrylonitrile have several useful advantages. For example, the hemoglobin adduct of 1,3-butadiene is an even more useful biomarker of exposure than urinary metabolites, and in the case of ethylene oxide, even though the concentration of ethylene oxide-Hb in the blood of workers did not exceed the value of the German exposure equivalent, a significant difference in it was found between workers and a control group. Also hemoglobin adducts of acrylamide and acrylonitrile can reflect their exposures because there are no urinary metabolites of acrylamide and acrylonitrile that are useful for exposure assessment. In addition to these advantages, hemoglobin adducts are superior to DNA adducts with respect to the availability of large amounts, availability of methods for chemical identification, and well-defined life spans due to the absence of repair. Hemoglobin adducts can be effective biomarkers for assessing exposure to and the effects of chemicals.     

Albumin and hemoglobin adducts as biomarkers of exposure to styrene in fiberglass-reinforced-plastics workers

Objective: The purpose of this work was to compare levels of styrene-7,8-oxide (SO) adducts of albumin (Alb) and hemoglobin (Hb) with those of two urinary metabolites of styrene, mandelic acid (MA) and phenylglyoxylic acid (PGA), among workers exposed to styrene in the reinforced-plastics industry and in unexposed subjects. We also wished to determine whether cigarette smoking influenced adduct levels among these subjects. Methods: A group of 22 male workers was selected on basis of an expectedly high level of exposure to styrene, and a group of 15 controls was selected from hospital blood donors and hospital staff. In the exposed group, MA and PGA were quantified by high-performance liquid chromatography (HPLC) analysis of urine samples collected prior to the work shift. The SO adducts were cleaved from cysteine residues by reaction with Raney nickel to give 1-phenylethanol (1-PE) and 2-phenylethanol (2-PE), which, after derivatization, were measured using gas chromatography-mass spectrometry (GC-MS) in the negative-chemical-ionization (NCI) mode. Results: The estimated mean levels of MA and MA+PGA were 74 and 159 mg/g creatinine, respectively. Using the levels of urinary metabolites, an average styrene concentration of about 100 mg/m³ in the workplace air was estimated. The mean levels of 2-PE and 1-PE adducts in exposed workers were 2.84 and 0.60 nmol/g Alb and 5.44 and 0.43 nmol/g Hb, respectively. When subjects were stratified by level of urinary metabolites [zero (controls), low-level exposure (MA+PGA ≤159 mg/g creatinine), and high-level exposure (MA+PGA> 159 mg/g creatinine)] and smoking status (smokers versus nonsmokers), a difference in Alb adduct levels was found among the groups (2-PE P=0.002, 1-PE P=0.052). The difference in 2-PE-Alb levels was related to exposure category, to smoking status, and to their interaction. Correlations at or near a 0.05 level of significance were observed among the workers (n=22) between individual levels of SO-protein adducts and MA+PGA (2-PE Alb, r=0.54, 2-PE Hb, r=0.40). Conclusion: Our data suggest that only exposure to relatively high levels of styrene allows a clear relationship to be detected between styrene exposure and SO adducts, due in part to the effects of cigarette consumption and to the high background levels of these adducts observed in unexposed subjects. Received: 3 February 1997 / Accepted: 9 June 1997     

Fast repair activities towards dGMP hydroxyl radical adducts by silybin and its analogues

The repair activities of silybin (SLB) and its analogues towards the oxidizing deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts are investigated by pulse radiolytic techniques. On pulse irradiation of nitrous oxide saturated 2.0 mM dGMP aqueous solution containing 0.1 mM silybin at neutral pH, the transient absorption spectrum of the dGMP hydroxyl radical adducts decreases with the formation of the phenoxyl radical of silybin within tens of microseconds, indicating that there is a repair reaction between the dGMP hydroxyl radical adduct and silybin. The rate constant of the repair reaction is calculated to be 1.0 x 10(9) M(-1)s(-1) for silybin. The repair activity of hesperetin (HESP), naringin (NAN) and naringenin (NAR) towards hydroxyl radical adducts of dGMP are also studied.     

Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA

Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 10⁸ dm³ mol⁻¹ s⁻¹ and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10–1000 μmol dm⁻³) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid.     

Urinary excretion of specific mercapturic acids in workers exposed to styrene

Styrene is an important chemical of wide industrial use, particularly in the manufacture of polymers and reinforced plastics. Environmental and occupational exposures to styrene occur predominantly via inhalation. Styrene undergoes biotransformation mainly by side chain oxidation catalyzed by cytochrome P-450 enzymes to its reactive metabolite, styrene oxide. The (R)- and (S)-enantiomers of styrene oxide can be conjugated with glutathione to both (R)- and (S)-diastereoisomers of specific mercapturic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (MI) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). We conducted this biomonitoring study with the aim of evaluating the association between excretion of specific mercapturic acids (M1 and M2) and level of exposure to styrene among occupationally exposed people. The mean time-weighted average (TWA) exposure was about one-half the current threshold limit value, the range of the values varied from 44 to 228 mg/m³. Geometric mean (GM) concentrations of 650. 1,084, and 31.8 μg/g creatinine were measured, respectively, for M1-S, M2, and M1-R. The environmental styrene concentration exhibited a significant correlation with total specific mercapturic acid (Mtot = sum of M1-R, M1-S, and M2), making it possible for the first time to calculate the approximate relationship between styrene uptake and excretion of these substances. The M2 mercapturic acid had a better correlation (r = 0.56) with respect to M1-R and M1-S. Significant correlations were found also between the excretion of specific mercapturic acids and biological exposure indices (i.e., mandelic and phenylglyoxylic acids and urinary styrene). Am. J. Ind. Med. 31:636–644, 1997. © 1997 Wiley-Liss, Inc.     

紫外光谱法测定混配农药的DNA加合作用

目的 研究混配农药的DNA加合作用。方法 采用紫外光谱移动法研究农药的DNA加合作用。结果 农药马拉硫磷、呋喃丹、氯氰菊本及其两两混配农药均可引起小牛胸腺DNA紫外光谱的改变。结论 农药马拉硫磷、呋喃丹、氯氰菊酯及其两两混配 经进入机体后，有与DNA形成加 的，如果在细胞复制前所形成的DNA加合物没有被修复或者被错误修复的活，则有可能导致基因突变，从而产生化学损伤。