Di (2-ethylhexyl) phthalate-induced reproductive toxicity involved in dna damage-dependent oocyte apoptosis and oxidative stress in Caenorhabditis elegans

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer with a high environmental exposure level. As a persistent organic pollutant, DEHP causes reproductive and developmental toxicity in mammals. In this paper, the reproductive toxicity of DEHP was discussed using the model organism Caenorhabditis elegans to determine the sensitivity indices for evaluating the ecotoxicological effects of DEHP. L4 C. elegans larvae to evaluate the LC50 of DEHP and the changes in brood size and generation time, we found that the LC50 of DEHP to C. elegans exceeded 100 mg/L. And 10 mg/L DEHP exposure significantly reduced the brood sizes but not the generation time. Results of oocyte and distal-tip cell (DTC) counting suggested that the number of oocytes were decreased and apoptotic cells that from the unilateral gonad arm were increased in the 1 mg/L and 10 mg/L DEHP exposed groups. In contrast, there was no significant difference in the fluorescence intensity of DTC. Fluorescence analysis of HUS-1 showed that HUS-1 protein was overexpressed after DEHP exposure. The H₂O₂ level and DNA damage were measured by Bradford protein assay and AP staining respectively. The results showed that there was no significant difference in H₂O₂ level after DEHP exposure, in contrast, DNA damage was increased significantly. Moreover, 10 mg/L concentration DEHP exposure significantly increased the expression levels of apoptosis-related genes cep-1, egl-1, ced-4, and ced-3 and decreased the expression levels of ced-9. It suggested that cep-1, egl-1, ced-4, and ced-3 genes promote apoptosis and the ced-9 gene inhibits apoptosis. Meanwhile, 10 mg/L concentration DEHP exposure decreased the expression of oxidative stress-related genes mev-1 and gas-1. The mev-1 and gas-1 are mainly involved in the inhibition of oxidative stress in nematodes. In short, the decreased oocyte numbers and increased apoptosis oocyte numbers in C. elegans when exposed to DEHP, which may involve in the DNA damage induced by oxidative stress.

Keywords: Caenorhabditis elegans; Ecotoxicological effect; Endocrine disruptor; Germ cells; Oogenesis.