MUTAGENICITY OF BENZENE METABOLITES

苯代谢物的致突变性

• 批准号: 6178665
• 负责人: WILLIAM J BODELL
• 金额: $ 15.48万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178665
• 关键词: bacteriophage lambda; benzene; carcinogen testing; chemical carcinogen; chemical carcinogenesis; enzyme activity; gene mutation; hydroquinones; mutagen testing; mutagens; nucleic acid sequence; peroxidases; tissue /cell culture; transfection /expression vector

DESCRIPTION: (Adapted from the investigator's abstract) Benzene is one of the few environmental agents which is unequivocally established as initiating huma cancer. This is a important public health issue due to the fact that virtually everyone is exposed to varying levels of benzene. In contrast to its biologica importance, relatively little is known about the molecular mechanisms by which benzene induces its genotoxic effects. To address this, they proposed the development of a human cell line which can be used to investigate the mutageni properties of benzene's metabolites and related environmental agents. They proposed: (1) To isolate HL-60 cell line containing stably integrated copies o a lambda/lacI shuttle vector. This cell line will be developed by transfection of HL-60 cells with a lambda/lacI shuttle vector and a plasmid encoding resistance to neomycin. The clone selected for this study will have the following properties: (a) a high rescue efficiency for lambda/lacI shuttle vector, (b) contain significant levels of peroxidase enzyme and activate hydroquinone to form DNA adducts, and (c) shows a low spontaneous and a significant increase in lacI mutation frequency in treated cells. (2) To measure the frequency of lacI mutations induced by treatment of HL-60/lacI cells with varying concentrations of either p-benzouione or hydroquinone. They will determine if treatment of HL-60/lacI cells with these metabolites significantly increases the lacI mutation frequency compared to treatment with solvent alone. The formation of DNA adducts in treated cells will be determine by 32P-postlabeling; allowing us to estimate the levels of DNA adducts formed under the conditions used for analysis of lacI mutations. (3) To sequence individual lacI mutants from the control and the treatment groups. This analysis will allow them to determine if the mutation spectrum in the treatmen groups are different than the mutation spectrum in the control group. They wil determine if the spectrum of mutations observed in the para-benzoquione and hydroquione treatment groups are consistent with being derived from N2-(4-hydroxy phenyl) deoxyguanosine the DNA adduct identified as being formed by these two metabolites. These studies will provide insights as to the nature of mutations induced by these benzene metabolites. Although this proposal uses established procedures, it is innovative for the following reasons: (1) it wil use cellular peroxidase enzymes for activation of the metabolites, (2) it will allow for comparison of mutation induction with adduct formation, (3) the cell line developed can be applied to the analysis of the mutagenic potential of a wide range of environmental, occupational and medicinal agents.

描述：（改编自研究者摘要）苯是 少数几个明确确定为 引发人类癌症 这是一个重要的公共卫生问题， 事实上，几乎每个人都暴露在不同程度的苯中。 在 与其生物学上的重要性相比， 苯诱导其遗传毒性作用的分子机制。 到 为了解决这个问题，他们提出了一种人类细胞系的发展， 用于研究苯代谢产物的致突变性， 相关环境因素。 本研究提出：（1）分离HL-60细胞， 含有稳定整合的λ/lacI穿梭载体拷贝的细胞系。 该细胞系将通过用重组质粒转染HL-60细胞来开发。 λ/lacI穿梭载体和编码新霉素抗性的质粒。 本研究选择的克隆将具有以下特性： λ/lacI穿梭载体高拯救效率，（B）含有 显著水平过氧化物酶和活化氢醌形成 DNA加合物，和（c）显示低自发和显着增加， 处理细胞中lacI突变频率。 (2)测量的频率 用不同浓度的抗肿瘤药物处理HL-60/lacI细胞诱导的lacI突变 浓度的对苯甲酮或对苯二酚。 他们将决定 如果用这些代谢物处理HL-60/lacI细胞， 与溶剂处理相比，增加了lacI突变频率 一个人 在处理的细胞中DNA加合物的形成将通过 32 P-后标记;使我们能够估计形成的DNA加合物的水平 在用于分析lacI突变的条件下。 (3)测序 来自对照组和处理组的单个lacI突变体。 这 分析将使他们能够确定基因组中的突变谱是否存在 治疗组与对照组的突变谱不同 组 他们将确定是否观察到的突变谱， 对苯苯醌和对苯二酚治疗组与 衍生自N2-（4-羟基苯基）脱氧鸟苷，DNA加合物经鉴定 是由这两种代谢物形成的 这些研究将提供 这些苯代谢物引起的突变的性质的见解。 虽然这项建议使用了既定的程序，但它是创新的， 原因如下：（1）它将使用细胞过氧化物酶， （2）它将允许比较突变 加合物形成的诱导，（3）开发的细胞系可以应用于 分析广泛的环境， 职业和药物制剂。