Effects of two DDT metabolites and one synthetic DDE-analogue on testicular steroidogenesis in primary porcine Leydig cells in vitro during neonatal development

Abstract

Although the production and use of the insecticide DDT have been restricted or banned since the 1970s, DDT and its metabolites are persistent in the environment and may still pose a hazard of toxic effects in wildlife and humans. One area, which has been given increasing attention in the past years, is the possible link between exposure to pesticides and disturbance of endocrine functions including reproductive functions. The present study investigated the direct effect of two DDT metabolites and one synthetic DDE-analogue on basal testicular steroidogenesis in primary neonatal porcine Leydig cells in vitro. The two DDT metabolites, 3-MeSO2-DDE and o,p’-DDD, as well as the synthetic DDE-analogue, 3,3’-(bis)MeSO2-DDE, do all have known endocrine effects as they exert toxic effects towards the adrenal cortex in humans and several species. Because of these properties, o,p’-DDD is in fact the main drug for adrenocortical carcinoma and Cushing’s syndrome. Neonatal Leydig cells were obtained from castrations, purified by a discontinuous Percoll gradient and the purity of Leydig cells was determined by staining for 3β-hydroxysteroid dehydrogenase. The primary cultures of purified Leydig cells were then exposed to six different concentrations of each compound up to 20 μM and solvent control (0.1% DMSO) for 48 hours. The aim was to assess the compounds’ effect on cell viability, hormone production (testosterone, estradiol, progesterone and cortisol) and expression of 16 genes involved in testicular steroidogenesis. Only samples exposed to the next highest concentration (10 μM) were used in the gene expression analysis. The expression in the 10 μM samples was then compared against the solvent controls. The results for cell viability showed that only o,p’-DDD was cytotoxic at the highest concentration. For hormone production, all three compounds stimulated testosterone secretion with most effect for 3-MeSO2-DDE and o,p’-DDD exposure. 3,3’-(bis)MeSO2-DDE caused only an increase at the highest concentration. Estradiol production was not much altered in comparison, but a stimulatory effect was seen for 3-MeSO2-DDE and o,p’-DDD, and a decrease was seen with 3,3’-(bis)MeSO2-DDE. The Leydig cells did not produce progesterone and cortisol. The results for gene expression showed that the three compounds were able to alter the effect of some genes. All genes altered were down-regulated with the exception of one gene and o,p’-DDD exerted most effect with down-regulation of eight genes in total. Since 3-MeSO2-DDE and o,p’-DDD caused an increase in hormone secretion at 10 μM, especially for testosterone, the reduction seen in mRNA levels does not explain the 4 mechanism behind the stimulatory effect. This might indicate that the compounds interact at the protein level. Taken together, the results suggest that the compounds are able to exert effect on basal testicular steroidogenesis and the possible endocrine effects on the male reproductive tract are thus concerning.