In-utero Exposure to Low Doses of Genistein and Di-(2-ethylhexyl) Phthalate (DEHP) Alters Innate Immune Cells in Neonatal and Adult Rat Testis.

BACKGROUND 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. OBJECTIVES 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. MATERIALS AND METHODS 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. RESULTS 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. DISCUSSION AND CONCLUSION 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 suggest 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.