Department of Chemical Toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway.
Mutagenesis. 2010 Jul;25(4):417-25. doi: 10.1093/mutage/geq024. Epub 2010 May 20.
Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 microM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 microM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE-DNA adducts as measured with (32)P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.
暴露于遗传毒物可能会损害男性生殖细胞的 DNA 完整性，使未来的后代面临发育缺陷和疾病的风险。为了研究精子 DNA 损伤作为遗传毒物暴露的生物标志物的有用性，我们已经研究了苯并[a]芘（B[a]P）在体外对人精子的细胞和分子变化，并对吸烟者和非吸烟者进行了比较。通过碱性彗星试验评估，来自 5 名吸烟者的精子 DNA 确实比 6 名非吸烟者的精子 DNA 更碎片化（分别为 26.5%和 48.8%的尾 DNA，P<0.05）。通过免疫染色检测到吸烟者体内 B[a]P 相关 DNA 加合物水平升高。成熟精子细胞直接暴露于 B[a]P（10 或 25 microM）会导致 DNA 片段化适度增加，而无需添加人肝 S9 混合物以激活 B[a]P 的酶促反应，这表明精液中存在一些未知的 B[a]P 代谢。体外将样本暴露于不同剂量的 B[a]P（有或没有 S9），并未显示吸烟者和非吸烟者对 DNA 碎片化的敏感性有任何显著差异。用 B[a]P 的近位代谢物苯并[a]芘-r-7,t-8-二氢二醇-t9,10-环氧化物（BPDE）孵育会产生剂量依赖性的 DNA 片段化（20 或 50 microM），但仅当彗星试验中包含 formamidopyrimidine DNA 糖基化酶处理时才会发生。然而，与用（32）P 后标记法测量的非常高的 BPDE-DNA 加合物水平相比，这些 DNA 片段化水平较低。我们得出结论，精子 DNA 损伤可用作适应于精子的彗星试验中精子直接暴露的生物标志物，因此该方法可适用于队列研究。尽管敏感性相对较低，但可以以更高的效率检测到早期精母细胞发生的 DNA 损伤。
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