In vitro and in vivo assessment of lead toxicity on mammalian female reproduction and effect of antioxidants

Abstract

Heavy metals are among the environmental toxicants incurring great concern for human and animals because of their toxicity even at low concentrations. Lead (Pb) has been shown to induce severe and long lasting effects on female fertility and pregnancy outcomes in many animal species. The aim of this work was to investigate the effect of Pb on bovine granulosa cells (GCs) and preimplantation embryos and its association with dysregulation of Nrf2 and NF-κB and their downstream genes. We further aimed to study the role of <em>in utero</em> Pb exposure and the possibility of using antioxidant as prophylactic agent against Pb toxicity using rat model. For this, three experiments were conducted. First, <em>in vitro</em> cultured GCs were exposed to Pb toxicity which subsequently attenuated GCs proliferation and altered the cell cycle progression. Lead exposure suppressed the expression of both Nrf2 and NF-κB and their downstream genes. Additionally, Pb challenge on GCs increased the expression of endoplasmic reticulum stress marker genes (GRP78 and CHOP) and the pro-apoptotic gene (caspase-3), while the anti-apoptotic gene (BCL-2) was reduced.<br /> Furthermore, treatment of bovine preimplantation embryos with Pb in a stage specific manner resulted in similar phenotypes. Blastocysts derived from different treatment groups exhibited aberrant developmental phenotypes regardless of the exposure stage. Exposure to Pb caused higher accumulation of ROS and reduced blastocyst cell number. Besides, the mRNA and protein levels of NF-κB were elevated with Pb treatment along with TNF-α level. On the contrary, the expression of Nrf2 protein showed significant reduction in all treatment groups. Apoptosis under Pb exposure was manifested by the higher ratio of BAX/BCL-2 and the number of TUNEL positive nuclei as compared to the control blastocysts. Moreover, Pb significantly upregulated DNMT1, a gene involved in maintenance of DNA methylation.<br /> In order to investigate the effect of Pb toxicity <em>in vivo</em>, pregnant rats were orally ingested by Pb during the period of organogenesis, while the natural antioxidant, taurine (TA) was given throughout the gestation period. The dams and their fetuses were checked for morphological, biochemical and histopatholgical parameters. Results showed that, Pb caused a significant decline in the maternal body weight gain and an increase in the rate of abortion. Fetuses maternally-received Pb showed growth retardation and malformations in their skeleton. Additionally, Pb induced hematological and biochemical impairments in both dams and fetuses. Histopathological examination of the placenta and hepatic DNA fragmentation revealed the toxicity of Pb. However, these events have been alleviated by TA pretreatment without affecting the normal course of pregnancy.<br /> The present work demonstrates that Pb-induced oxidative stress displayed direct deleterious effect on bovine GCs proliferation and preimplantation embryo development. This effect may be in part through disrupting the Nrf2/NF-κB interaction and could vary according to the dose, the period of exposure and the type of cells. It is quite evident that even small doses of Pb are reprotoxic where the soundest approach is to minimize Pb exposure <em>in vivo</em> rather than treatment. Administration of antioxidants such as taurine could be promising approach to be used as a prophylactic agent against environmental heavy metals.