Dere Edward, Wilson Shelby K, Anderson Linnea M, Boekelheide Kim
*Division of Urology, Rhode Island Hospital, Providence, Rhode Island 02903 Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912.
Toxicol Sci. 2016 Oct;153(2):327-40. doi: 10.1093/toxsci/kfw137. Epub 2016 Jul 27.
Traditional testis histopathology endpoints remain the gold standard for evaluating testicular insult and injury in a non-clinical setting, but are invasive and unfeasible for monitoring these effects clinically in humans. Assessing testicular injury in humans relies on semen and serum hormone analyses, both of which are insensitive and poor indicators of effect. Therefore, we hypothesized that sperm messenger RNA (mRNA) transcripts and DNA methylation marks can be used as translatable and sensitive indicators or testicular injury. Dose-response studies using adult male Fischer 344 rats subchronically exposed to model Sertoli cell toxicants (0.14, 0.21, and 0.33% 2,5-hexanedione, and 30, 50, and 70 mg/kg/day carbendazim), and a model germ cell toxicant (1.4, 3.4, and 5.1 mg/kg/day cyclophosphamide) for 3 months were evaluated for testicular injury by traditional histopathological endpoints, changes in sperm mRNA transcript levels using custom PCR arrays, and alterations in sperm DNA methylation via reduced representation bisulfite sequencing. Testis histopathological evaluation and PCR array analysis of the sperm transcriptome identified dose-dependent changes elicited by toxicant exposure (P < 0.05). Global sperm DNA methylation analysis of subchronic 0.33% 2,5-hexandione and 5.1 mg/kg/day cyclophosphamide exposure using a Monte Carlo approach did not identify differentially methylated regions (methylation difference > 10% and q < 0.05) with robust signatures. Overall, these results suggest that sperm mRNA transcripts are sensitive indicators of low dose toxicant-induced testicular injury in the rat, while sperm DNA methylation changes are not. Additionally, the Monte Carlo analysis is a powerful approach that can be used to assess the robustness of signals resulting from -omic studies.
传统的睾丸组织病理学指标仍是非临床环境下评估睾丸损伤的金标准，但具有侵入性，在临床上监测人类的这些影响并不可行。评估人类睾丸损伤依赖于精液和血清激素分析，而这两者对于损伤的指示既不敏感，效果也不佳。因此，我们推测精子信使核糖核酸（mRNA）转录本和DNA甲基化标记可作为可转化的敏感指标用于指示睾丸损伤。我们使用成年雄性Fischer 344大鼠进行剂量反应研究，将其亚慢性暴露于模型支持细胞毒物（0.14%、0.21%和0.33% 的2,5 -己二酮以及30、50和70毫克/千克/天的多菌灵）和一种模型生殖细胞毒物（1.4、3.4和5.1毫克/千克/天的环磷酰胺）3个月，通过传统组织病理学指标评估睾丸损伤情况，利用定制的PCR芯片检测精子mRNA转录水平变化，并通过简化代表性亚硫酸氢盐测序分析精子DNA甲基化的改变情况。睾丸组织病理学评估以及精子转录组的PCR芯片分析确定了毒物暴露引发的剂量依赖性变化（P < 0.05）。使用蒙特卡罗方法对亚慢性暴露于0.33% 的2,5 -己二酮和5.1毫克/千克/天环磷酰胺的大鼠进行精子全基因组DNA甲基化分析，未发现具有显著特征的差异甲基化区域（甲基化差异> 10%且q < 0.05）。总体而言，这些结果表明精子mRNA转录本是大鼠低剂量毒物诱导睾丸损伤的敏感指标，而精子DNA甲基化变化并非如此。此外，蒙特卡罗分析是一种强大的方法，可用于评估组学研究结果信号强度。
