Independent and combined effects of Bisphenol A and Diethylhexyl Phthalate on gestational outcomes and offspring development in Sprague-Dawley rats

Highlights

• Prenatal exposure to combinations of BPA and DEHP increased abortion rates in rats. Exposure to individual chemicals did not induce abortions.

• Relative heart weight increased in male offspring prenatally exposed to the combination of BPA and high dose-DEHP.

• Prenatal exposure to BPA or HD-DEHP reduced the relative thymus weight in males; while exposure to HD-DEHP increased the apoptotic index in both sexes.

Abstract

Bisphenol A (BPA) and Diethylhexyl Phthalate (DEHP) are well-studied endocrine disrupting chemicals (EDCs), however, the effects of mixtures of these EDCs are not. To assess the consequences of prenatal exposure to a mixture of these EDCs, dams were orally administered either saline (control), BPA (5 μg/kg BW/day), high dose DEHP (HD-D; 7.5 mg/kg BW/day), or a combination of BPA with HD-D in experiment 1; saline, BPA (5 μg/kg BW/day), low-dose DEHP (LD-D; 5 μg/kg BW/day) or a combination of BPA with LD-D in experiment 2. Gestational weights, number of abortions, litter size and weights, number of live births and stillbirths were recorded. Morphometric measures were obtained at birth and body weight, food and water intake were monitored weekly from postnatal weeks 3–12. Offspring were sacrificed at 16–24 weeks of age and organ weights were measured. The abortion rate of dams exposed to HD-D and the mixtures, BPA + LD-D and BPA + HD-D were higher at 9, 14 and 27% respectively. Prenatal exposure to BPA or HD-D significantly decreased relative thymus weights in male but not female offspring. Apoptotic cells were detected in thymus sections of both male and female offspring prenatally exposed to DEHP. Relative heart weights increased in BPA + HD-D exposed male offspring compared to the other groups. The results indicate that a mixture of BPA and DEHP, produced a pronounced effect on pregnancy outcomes. Male offspring appear to be more susceptible to the programming effects of these EDCs or their mixture suggesting a need to reconsider the possible additive, antagonistic or synergistic effects of EDC mixtures.

Introduction

Increased prevalence of human health risks has been correlated with environmental contaminants, including the plastic-derived compounds: Bisphenol-A (BPA) and Di-2-ethylhexyl phthalate (DEHP). BPA is a weak estrogen while DEHP has anti-androgenic properties. They interfere with the endocrine system by activating a broad range of signaling pathways (Gould et al., 1998; Grun and Blumberg, 2006; Latini et al., 2004). BPA and DEHP are ubiquitously present in the environment. They are extensively used in a variety of consumer products including plastic bottles, dental fillings and sealants, eyeglass lenses, sports equipment, household electronics and food packaging (Hashimoto and Nakamura, 2000; Yoshida et al., 2001). Exposure to these chemicals occurs through ingestion of contaminated food or water, inhalation of contaminated air and dust, and dermal absorption (Hernandez-Diaz et al., 2009; Erythropel et al., 2014; Diamanti-Kandarakis et al., 2009; Morgan et al., 2018). BPA and DEHP can leach into the environment from plastic products as they degrade over time and increase human exposures (Erythropel et al., 2014; Yoshida et al., 2001; Yamamoto and Yasuhara, 1999).

The prevalence of BPA and DEHP in the environment has raised concerns, in particular because they have been detected in the serum (Schonfelder et al., 2002), amniotic fluid (Jensen et al., 2012; Calafat et al., 2006; Edlow et al., 2012; Ikezuki et al., 2002), placental tissue (Schonfelder et al., 2002; Jimenez-Diaz et al., 2010) and fetal serum (Talsness et al., 2009; Troisi et al., 2014; Schonfelder et al., 2002; Ikezuki et al., 2002). This implies that BPA and DEHP can cross the placenta during the critical periods of development and program the fetus for possible adult-onset disorders (Liu et al., 2013).

Animal and human studies have demonstrated some of the permanent and irreversible in utero effects of low-dose BPA and DEHP on different organ systems (Colborn, vom Saal, and Soto, 1993) (vom Saal et al., 1998; Hatch et al., 2010; Naville et al., 2013). Early life exposure to DEHP is associated with a decrease in pregnancy weight gain (Gray et al., 2000), fetal growth (Ferguson et al., 2016), pup weight at birth (Gray et al., 2000) and anogenital distance in male infants (Swan et al., 2005; Gray et al., 2000) (Parks et al., 2000). In utero exposure to BPA is shown to produce sex-specific reductions in offspring birth weight (Huo et al., 2015), increase in gestational length (Veiga-Lopez et al., 2015), and shortened anogenital distance in male offspring (Sun et al., 2018). Besides, exposure to environmentally relevant low doses BPA and DEHP have been linked to a number of chronic diseases including metabolic, reproductive, cardiovascular, and neurological disorders (De Coster and van Larebeke, 2012; Rochester, 2013; Richter et al., 2007; Vandenberg et al., 2009; Rubin and Soto, 2009; Rubin, 2011; Lyche et al., 2009). However, the effects of prenatal exposures to a mixture of low doses of these chemicals have not been studied.

This study aimed to investigate the effects of prenatal exposure to a mixture of low dose BPA and DEHP on Sprague Dawley rat development, as well as the sex differences in their biological effects. We used a single low dose of BPA (5 μg/kg BW) in combination with either a low dose (5 μg/kg BW) of DEHP to match the BPA dose or a higher dose (7.5 mg/kg BW) based on earlier studies (Rattan et al., 2018; Tinwell et al., 2002). Exposures were limited to the prenatal period (gestational days 6–21). We hypothesized that prenatal exposure to EDCs will “program” the offspring and affect their postnatal morphometric parameters, body weights, and organ weights in a sex-specific manner with the EDC mixture showing a more pronounced effect than individual EDCs.