Effects of prenatal and postnatal exposure to a low dose of bisphenol A on behavior and memory in rats

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Abstract

Endocrine disruptors (EDs) are increasingly common chemicals in the environment. Bisphenol A (BPA), used to manufacture polycarbonate plastics, is an ED recognized for its estrogenic, anti-estrogenic, and anti-androgenic effects. Behavior is considered a vital characteristic for an animal's life cycle. This study evaluated the effect of exposure to low doses of BPA during pregnancy and/or lactation on several aspects of rat behavior, including memory, locomotion, and the exploratory instinct. Pups at 16 weeks of age (females and males) were divided into groups according to the mother's exposure to BPA (40 μg/kg/day): CON (vehicle only); PRE (during pregnancy); LAC (during lactation); PRE-LAC (during both pregnancy and lactation). In the PRE-LAC group, exposure to BPA impaired both short-term (STM) and long-term memory (LTM) in inhibitory avoidance and the object recognition task, and also affected locomotor activity and spatial memory. Some sex-specific behavioral characteristics disappeared in the LAC group. Sex-specific memory and behavior impairment were caused by BPA exposure during brain organogenesis and differentiation.

Introduction

Exposure to gonadal steroid hormones during intrauterine and neonatal life plays a pivotal role in the development of brain circuits, and this differentiation persists, determining behavior patterns throughout life (Kubo et al., 2001, Patisaul and Polston, 2008). Estrogens cause proliferation and differentiation of nervous system cells and synaptic plasticity. Estrogens could act through genomic (nuclear-receptor) or non-genomic (membrane receptors) pathways. Estrogen receptors (ERs), ERα and ERβ, are estrogen-regulated, are expressed in various regions of the central nervous system (CNS) including the hippocampus, and have an important direct and indirect influence on higher cognitive functions (Prange-Kiel and Rune, 2006).

Endocrine disruptors (EDs) are synthetic or natural substances that are able to affect the action of endogenous hormones. EDs mimic or block endogenous hormones and may modify the synthesis, transport, metabolism, or elimination of natural hormones (Mlynarcıkova et al., 2005, Hanet et al., 2008). These chemicals influence the development and function of the CNS and the reproductive system (Kubo et al., 2001, Kubo et al., 2003, Fujimoto et al., 2007). Bisphenol A (BPA) is a well-known ED, and is recognized as having both estrogenic and anti-estrogenic effects, in addition to anti-androgenic and anti-thyroid effects (Zoeller et al., 2005, Fujimoto et al., 2007; Gonçalves et al., unpublished). About 3.0 million tons of BPA are produced annually, and it is used in the manufacture of polycarbonate, epoxy resins, polysulfone, polyester, and polyether ketones, and also as an antioxidant and flame retardant. BPA has been shown to leach from these materials, and in this way has become ubiquitous in the environment. Suspected of being hazardous to humans, BPA has been found in human urine (Calafat et al., 2005), milk, and plasma samples, and also in placental tissues (Schönfelder et al., 2002). Despite hepatic BPA metabolism (mainly glucurodation) be fast, BPA presence in human fluids indicates a constant exposure.

Low dose of BPA increases ERα mRNA and ERβ mRNA brain levels (see review in Vom Saal and Hughes, 2005). Therefore, BPA has been linked as a potential cause of alteration in sexual dimorphisms of the CNS and behavioral impairment in rats (Kubo et al., 2001).

For BPA, the lowest-observed-adverse-effect-level (LOAEL) was 50 mg/kg/day, and was used to calculate a reference dose of 50 μg/kg/day based on experiments conducted in the 1980s (IRIS, 1988). Exposure to a low dose (low compared to the reference dose) of BPA during the fetal and suckling periods in mice affected their exploratory emotional behavior, decreasing the normal sexual differences in behavior (Kubo et al., 2003). Rats exposed to a low dose of BPA showed behavioral defeminization in females and demasculinization in males (Kubo et al., 2001, Gioiosa et al., 2007).

Taking into consideration the widespread presence of BPA in the environment, and considering its weak estrogen activity and experimental limitations, some authors have affirmed (Vom Saal and Hughes, 2005) and others have doubted (Dekant and Völkel, 2008) the real risk of human exposure to BPA.

Concerning experimental condition, via of administration deserves some consideration: while the oral route is the main pathway in BPA exposure, parenteral administration avoids BPA first-pass in the liver (glucuronide conjugation). On the other hand, the toxicological core “the dose makes the poison” is confronted with hormesis mechanisms (i.e., low dose effect) or non-monotonic dose response curves (Vandenberg et al., 2009). Current human BPA exposure occurs in low dosage. Thus, low dose BPA effect in a single dose is interesting to observe, under adequate experimental conditions.

Independently of dosage, exposure period is crucial. Organisms exposed to EDs such as BPA during embryonic, fetal (free BPA increase for deconjugation β-glucuronidase-mediated in placental and fetal tissue) or neonatal (hepatic immaturity in glucuronidation capacity) life would present predisposition to dysfunctions observed in puberty or adulthood. Therefore, an important experimental approach is to study the effect of different BPA exposure times, at the same dosage, on memory and behavioral tasks.

This study evaluated the effects of a low dose of BPA (40 μg/kg/day, administered orally) on litters of female and male Wistar rats during the pregnancy and/or lactation periods, with respect to conditioning and spatial memory, locomotion, exploratory activity, and emotional state.

Section snippets

Materials and methods

All the procedures involving animal subjects were reviewed and approved by the Institutional Research Ethics Committee of the Universidade Federal do Rio Grande, Rio Grande, Rio Grande do Sul (CEPAS-Approval: 15/2008).

Female and male Wistar rats (Rattus norvegicus) at 16 weeks of age obtained from the Universidade Federal do Rio Grande breeding colony were housed in standard cages. After confirmed copulation, females were exposed to one of the following treatments: corn oil-treated, with BPA,

Statistical analysis

Data were expressed as mean ± SEM. When the assumptions of homogeneity and normality of variance were verified, one- or two-way ANOVA was carried out to determine the main effect of sex and treatment and possible sex × treatment interactions. One-way ANOVA was performed first between litters for each treatment and sex, to exclude litter effects. Once litter effect was excluded, statistical tests were performed. The ANOVA was followed by a Student–Newman–Keuls post hoc test. If the assumption of

Step-down inhibitory avoidance task (IAT)

The training session step-down latency among groups did not differ significantly in a Kruskal–Wallis analysis of variance (p > 0.05).

Latency time in the test session showed no significant differences between sexes, in both STM (p = 0.239) and LTM (p = 0.625). The test results for STM (Fig. 1A) and LTM (Fig. 1B), expressed as mean and interquartile range (25/75), are shown for each sex and treatment. PRE-LAC-treated females and males showed impairment in conditioning memory as measured in the

Discussion

Memory formation is a process that involves many biochemical and molecular events in specific brain regions (Izquierdo and Medina, 1997). Soon after learning, memory is very susceptible to external and internal interferences (Gioiosa et al., 2007). We used the IAT test to evaluate the possible interference of BPA exposure during pregnancy and/or lactation with STM and LTM, in a paradigm of aversive learning. The aversive learning model is normally adaptive, and it contributes to remembering

Conflicts of interest

All authors declare they have no potential competing interest.

Acknowledgements

This study was supported by a grant, Proc. No. 0613848-FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul), to the Physiological Sciences Post-graduate Program of the Universidade Federal do Rio Grande. D.M. Barros is a research fellow of the Brazilian agency CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). C.R. Gonçalves received a graduate fellowship from the Brazilian agency CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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