Effects of in utero- lactational- and direct exposure to selected endocrine disrupting chemicals on the rat male reproductive system

Abstract

Endocrine disrupting chemicals (EDCs) are ubiquitous natural or synthetic substances, present in the environment, that possess hormonal activity. EDCs have the ability to disrupt hormonally dependent processes and potentially elicit adverse health effects in both animals and humans. Possible adverse effects on fertility and reproductive parameters following acute and chronic exposure to these chemicals have been reported in the scientific literature. However, the association between exposure to EDCs present in a malaria area and impaired male reproductive health remains inconsistent. In South Africa (SA), malaria remains a public health threat and various programs are in place in an effort to prevent malaria transmission. EDCs in a malaria endemic area in the Limpopo Province, SA were identified as: (i) the organochlorine pesticide, 1,1,1- trichloro-2,2-bis(p-chlorophenyl)ethane (DDT); (ii) the persistent metabolite of DDT, 1,1- dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE); (iii) the active substance in insecticidetreated nets (ITNs) and used for indoor residual spraying (IRS), deltamethrin (DM); (iv) the anti-oxidant used in the preparation of lubricating oil additives, resins, plasticizers and found in polyvinyl chloride (PVC), para-nonylphenol (p-NP); (v) and phytoestrogens (coumestrol, genistein, zearalenone), which form part of a normal diet. The aim of the study was to investigate the effects of in utero-, lactational- and direct exposure to selected concentrations of previously studied EDCs found in the environment on male reproductive health using the rat model. The objectives were to (1) determine the impact of in utero-, lactational- and direct exposure to EDCs on malespecific endocrine sensitive endpoints (anogenital distance and gonadosomatic index), male accessory glands (prostate, seminal vesicles), epididymis and liver (2) determine the effects of EDC exposure on epididymal sperm count and testosterone levels, (3) assess and compare the testicular histology and spermatogenesis cycle using the spermatogenesis staging program STAGES and the Johnsen Scoring system, (4) determine the possible relationship between exposure to selected EDCs and the increase incidence of testicular apoptosis. We adapted the Organization for Economic Cooperation and Development (OECD) 415 reproductive toxicity protocol to include one control and three experimental groups, a longer prenatal exposure period, and several additional endpoints. Male Sprague- Dawley rats were exposed in utero- for 2 weeks, indirectly during lactation for 3 weeks and directly for 10 weeks to cottonseed oil [control - group 1; n=24]; DDT (35mg/kg) [group 2; n=11]; DDE (35mg/kg) [group 3; n=27]; and a mixture of DDT (35mg/kg), DM (0.5mg/kg), p-NP (2.5?g/kg), genistein (2.5?g/kg), coumestrol (2.5?g/kg) and zearalenone (2.5?g/kg) [group 4; n=15]. Following exposure and at 13 weeks of age, changes in organ weights, epididymal sperm counts, histological assessments, staging of the spermatogenic cycle and testicular apoptosis were assessed. Treatment effects were found for male reproductive tract development as evidenced by anogenital distance (AGD) in newborns and in liver characteristics. Compared with AGD in the control group (group 1; 17.54 +/- 0.65mm), AGD was significantly shorter in the mixture group (group 4; 15.20 +/- 0.16mm; P = 0.005), indicating possible feminization. In comparison with the control group mean liver mass (group 1; 17.36 +/- 2.16 g), was significantly higher in all three experimental groups: DDT (group 2; 21.16 +/- 1.29g; P <0.001), DDE (group 3; 20.65 +/- 5.06g; P = 0.003) and the mixture (group 4; 19.45 +/- 2.00g; P = 0.031). Since enlargement of the liver is a marker of liver toxicity, the increase in liver mass observed in this study indicates that exposure to these selected EDCs had a significant effect on the liver of male rats. Lipid droplet formation and hepatic disorganization were present in the liver of the DDT, DDE and mixture groups suggesting that the liver may be a primary target. The changes in liver function may therefore be involved in the reproductive effects observed in this study. When animals had reached adulthood at the end of the study, the effects of EDC exposures were found for a number of endpoints. Prostate mass in the control group (group 1; 0.83 +/-0.24g) was significantly higher in the DDT group (group 2; 1.02 +/- 0.19g; P = 0.018). Prostate mass was not, however, correlated with testosterone levels which were significantly higher in the DDE and mixture groups. Testicular histology revealed marked effects in all groups including dilated tubular lumens, detachment of the seminiferous epithelium, necrosis in the interstitium, disorganization of the seminiferous epithelium with few germ cells present, reduced seminiferous tubule diameter with no lumen, absent seminiferous tubules and decreased layers of germ cells. Although these changes were not seen in all tubules, treatment was associated with decreased mean seminiferous tubule diameters, decreased epithelial thickness, and smaller luminal diameters. Application of the Johnsen scoring system showed that the treatment effects manifested primarily as a Johnsen Score of 9 tubules (a Johnsen Score of 9 is defined as a seminiferous tubules with many spermatozoa present, but germinal epithelium disorganization with marked sloughing or obliteration of the lumen). Specifically, controls had, on average, 19% abnormal tubules, compared 46%, 25% and 56% in the DDT, DDE and mixture groups, respectively. Surprisingly, however, the lesions in histology did not translate into changes in epididymal sperm counts. This suggests that spermatogenesis proceeded normally in a proportion of tubules, resulting in sperm production sufficient to maintain apparently normal epididymal sperm stores. The results of this study indicate that in utero-, lactational- and direct exposure to mixture of EDCs found in a malaria area, at the levels used here, has negative impacts on normal genital development after in utero exposure and on spermatogenesis in adulthood after combined prenatal, lactational and postnatal exposure. These findings raise concerns to EDC exposures to mothers living in malaria-areas and the reproductive health of their male offspring. Significant differences were found in the endocrine-sensitive endpoints: AGD, testosterone, testicular STAGES and Johnsen score. This study shows that in utero-, lactational- and direct exposure to EDCs present in a malaria-area negatively affects male reproductive parameters in rats. These findings raise concerns to EDC-exposures to mothers living in malaria-areas and the reproductive health of their male offspring. Since this reproductive toxicology study constitutes in utero-, lactational and direct lifespan exposure to environmentally relevant concentrations of EDCs present in a currently malaria-vector control area, these results might be considered indicative of the effects following similar human exposures. Safer alternatives should be sought particularly in malaria vector-control programs where adverse reproductive health effects have been reported following chronic exposure to these potentially harmful chemicals.