Effects of bisphenol A exposure during pregnancy on expression and activity of inducible nitric oxide synthase in hippocampus of rat offspring at different developmental stages

Background Available studies have shown that bisphenol A (BPA) could induce neurotoxicity. Nitric oxide (NO) is an important retrograde messenger in the nervous system, and the alternations of its level play an important role in the mechanism underlying neurotoxicity. When exogenous chemicals are applied to the body, changes in the expression and activity of inducible nitric oxide synthase (iNOS) may have an impact on the production of NO.

Objective This experiment investigates the effects of BPA exposure during pregnancy on the expression and activity of iNOS in the hippocampus of rat offspring at different developmental stages.

Methods Sixty SPF-grade healthy SD pregnant rats were randomly divided into control group and 0.05, 0.5, 5, and 50 mg·kg^-1·d^-1 BPA exposure groups, with 12 rats in each group. The pregnant rats in the exposure groups were exposed to BPA (dissolved in corn oil) via oral gavage daily from gestational day (GD) 5 to GD19, and the rats in the control group were fed with corn oil. Six pregnant rats were sacrificed in each group on GD20, the placenta was harvested and the hippocampus was separated from brain tissues. The remaining six pregnant rats of each group were fed continuously, and the infant rats were weaned on postnatal day (PND) 21. Six rat offspring of each group were sacrificed on PND21 and PND56 respectively after ether anesthesia, and the hippocampus was also taken. The organ coefficients of brain tissues of rat offspring at different developmental stages (GD20, PND21, and PND56) were calculated. The levels of NO and the activity of iNOS in the hippocampus were detected using biochemical kits. The expression levels of iNOS mRNA and protein in the hippocampus was detected by real-time quantitative polymerase chain reaction and Western Blotting respectively.

Results The hippocampal NO levels of GD20 rat offspring in the BPA exposure groups were lower than that in the control group (Ps < 0.05); the hippocampal NO levels of PND21 and PND56 rat offspring in the BPA exposure groups (except PND21 rat offspring in the 0.05 mg·kg^-1 BAP exposure group) were significantly higher than those in the control group; however, the hippocampal NO levels of PND56 rat offspring in the 0.5, 5, and 50 mg·kg^-1 BPA exposure groups were lower than that in the 0.05 mg·kg^-1 BPA exposure group (Ps < 0.05). Besides, the iNOS mRNA expression levels in the hippocampus of rat offspring in the BPA exposure groups at all developmental stages were higher than that in the control group (Ps < 0.05). The iNOS mRNA expression level in the hippocampus of GD20 rat offspring in the 50 mg·kg^-1 BPA exposure group was lower than that in the 0.5 mg·kg^-1 BPA exposure group (P < 0.05). Similar to the results on GD20, the iNOS mRNA expression levels in the hippocampus of PND21 rat offspring exposed to 0.5, 5 and 50 mg·kg^-1 BPA were lower than that in the 0.05 mg·kg^-1 BAP exposure group, and the iNOS mRNA expression level of rat offspring in the 5 and 50 mg·kg^-1 BPA exposure groups were lower than that in the 0.5 mg·kg^-1 BAP exposure group (Ps < 0.05). The iNOS mRNA level in the hippocampus of PND56 rat offspring in the 5 mg·kg^-1 BPA exposure group was higher than those in the 0.05 mg·kg^-1 and 0.5 mg·kg^-1 BPA exposure groups, and the level in the 50 mg·kg^-1 BPA exposure group was lower than that in the 5 mg·kg^-1 BPA exposure group (Ps < 0.05). The relative expression levels of iNOS protein in the hippocampus of rat offspring exposed to 0.5, 5, and 50 mg·kg^-1 BPA at different developmental stages were higher than those in the control group and the 0.05 mg·kg^-1 BPA exposure group (Ps < 0.05). The relative expression level of iNOS protein in the hippocampus of GD20 rat offspring exposed to 50 mg·kg^-1 BPA was also higher than that in the 5 mg·kg^-1 BPA exposure group (P < 0.05). There was no significant difference in iNOS protein relative expression level between rat offspring in the control group and the 0.05 mg·kg^-1 BPA exposure group at different developmental stages (Ps>0.05). The activity of iNOS in the hippocampus of GD20 rat offspring in the 50mg·kg^-1 BPA exposure group was higher than those in the control group and the other BPA exposure groups (Ps < 0.05). The iNOS activities in the hippocampus of PND21 and PND56 rat offspring in the BPA exposure groups were higher than that in the control group (Ps < 0.05). The iNOS activity in the hippocampus of PND21 rat offspring in the 0.5 mg·kg^-1 BPA exposure group was also higher than that in the 0.05 mg·kg^-1 BPA exposure group (Ps < 0.05).

Conclusion BPA exposure during pregnancy can promote the mRNA and protein expressions and the activity of iNOS in the hippocampus of rat offspring at different developmental stages, which may play roles in the increase of NO level after birth and the neurotoxicity caused by the NO upregulation.