Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, USA.
The Research Institute of the McGill University Health Centre, McGill University, Montreal, QC, Canada.
Andrology. 2020 Jul;8(4):943-964. doi: 10.1111/andr.12840. Epub 2020 Jul 3.
Although humans are exposed to mixtures of endocrine disruptor chemicals, few studies have examined their toxicity on male reproduction. We previously found that fetal exposure to a mixture of the phytoestrogen genistein (GEN) and the plasticizer di(2-ethylhexyl) phthalate (DEHP) altered gene expression in adult rat testes.
Our goal was to investigate the effects of fetal exposure to GEN-DEHP mixtures at two doses relevant to humans on testicular function and transcriptome in neonatal and adult rats.
Pregnant SD rats were gavaged with vehicle, GEN or DEHP, alone or mixed at 0.1 and 10 mg/kg/day, from gestation day 14 to birth. Fertility, steroid levels, and testis morphology were examined in neonatal and adult rats. Testicular transcriptomes were examined by gene array and functional pathway analyses. Cell-specific genes/proteins were determined by quantitative real-time PCR and immunohistochemistry.
GEN-DEHP mixtures increased the rates of infertility and abnormal testes in adult rats. Gene array analysis identified more genes exclusively altered by the mixtures than individual compounds. Altered top canonical pathways included urogenital/reproductive developmental and inflammatory processes. GEN-DEHP mixtures increased innate immune cells and macrophages markers at both doses and ages, more strongly and consistently than DEHP or GEN alone. Genes exclusively increased by the mixture in adult testis related to innate immune cells and macrophages included Kitlg, Rps6ka3 (Rsk2), Nr3c1, Nqo1, Lif, Fyn, Ptprj (Dep-1), Gpr116, Pfn2, and Ptgr1.
These findings demonstrate that GEN-DEHP mixtures at doses relevant to human induce adverse testicular phenotypes, concurrent with age-dependent and non-monotonic changes in testicular transcriptomes. The involvement of innate immune cells such as macrophages suggests immediate and delayed inflammatory responses which may contribute to testicular dysfunction. Moreover, these effects are complex and likely involve multiple interactions between immune and non-immune testicular cell types that will entail further studies.
尽管人类暴露于内分泌干扰物化学混合物中，但很少有研究探讨其对男性生殖的毒性。我们之前发现，胎儿接触植物雌激素染料木黄酮（GEN）和增塑剂邻苯二甲酸二（2-乙基己基）酯（DEHP）的混合物会改变成年大鼠睾丸中的基因表达。
我们的目标是研究在与人类相关的两个剂量下，胎儿暴露于 GEN-DEHP 混合物对新生和成年大鼠睾丸功能和转录组的影响。
从妊娠第 14 天到出生，SD 孕鼠经口给予载体、GEN 或 DEHP，单独或混合，剂量分别为 0.1 和 10mg/kg/天。检查新生和成年大鼠的生育能力、类固醇水平和睾丸形态。通过基因芯片和功能途径分析检查睾丸转录组。通过实时定量 PCR 和免疫组织化学法测定细胞特异性基因/蛋白。
GEN-DEHP 混合物增加了成年大鼠不育和睾丸异常的发生率。基因芯片分析鉴定出更多仅由混合物改变的基因，而不是单个化合物。改变的主要经典途径包括泌尿生殖/生殖发育和炎症过程。GEN-DEHP 混合物在两个剂量和年龄下均增加了固有免疫细胞和巨噬细胞标志物，比 DEHP 或 GEN 单独作用更强且更一致。混合物在成年睾丸中仅增加的与固有免疫细胞和巨噬细胞相关的基因包括 Kitlg、Rps6ka3（Rsk2）、Nr3c1、Nqo1、Lif、Fyn、Ptprj（Dep-1）、Gpr116、Pfn2 和 Ptgr1。
这些发现表明，与人类相关剂量的 GEN-DEHP 混合物会导致不良的睾丸表型，同时伴有睾丸转录组的年龄依赖性和非单调变化。固有免疫细胞（如巨噬细胞）的参与表明可能导致睾丸功能障碍的即时和延迟炎症反应。此外，这些影响是复杂的，可能涉及免疫和非免疫睾丸细胞类型之间的多种相互作用，需要进一步研究。
