Bisphenol A and Bisphenol S Oxidative Effects in Sheep Red Blood Cells: An In Vitro Study

Abstract

Bisphenols (BPs) are plastic components widely used worldwide and occurring in the environment. Exposure to these compounds is known to be harmful for animals and humans at different levels. The aim of this study was to evaluate and compare the oxidative effects of bisphenol A (BPA) and bisphenol S (BPS) in sheep. Reactive oxygen species (ROS) production and correlated structural alterations in sheep erythrocytes were investigated in vitro. Blood samples from four ewes were collected at fasting from the jugular vein using vacuum collection tubes containing EDTA. For ROS assay in erythrocytes, blood was properly diluted and BPA or BPS was added to obtain final bisphenol concentrations in the range between 1 and 300 μM. 2 ${}^{\prime }$ ,7 ${}^{\prime }$ -Dichlorodihydrofluorescein diacetate (H2DCF-DA) 3 μM was added to the samples, and fluorescence was read in four replicates using a microplate reader. To evaluate erythrocyte shape, blood smears of blood treated with the different concentrations of BPS and BPA were prepared. A significant increase in ROS production was observed when concentrations of BPS and BPA increased from 1 to 100 μM ( $p<0.05$ ). At the higher concentrations of the two studied BPs (300 μM of BPS and 200-300 μM of BPA), a ROS decrease was observed when compared to the control group ( $p<0.01$ ). Erythrocytes’ shape alterations were observed in cells treated with BPS and BPA 200-300 μM 4 hours after the beginning of the treatment. This study confirms that BPA and BPS exhibit oxidative effects on sheep erythrocytes. At higher concentrations, BPA was able to modify erythrocytes’ shape, while BPS altered their membrane as a sign of a protein clustering that could lead to eryptosis. These BPs’ effects are consequent to intracellular ROS increase.