Bisphenol AF exposure reveals concentration and sex dependent mRNA expression of xenobiotics metabolism genes: higher exposures are correlated with wound healing
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Contributing OfficeNational Center for Toxicological Research
Abstract
Bisphenol AF (BPAF), a structural analogue of BPA, is a compound manufactured for its heat-resistant, flame-retardant, and caustic-resistant properties and used in fluoroelastomers and specialty polymers. Research in rodents suggests that it is an endocrine-disrupting compound (EDC), with studies showing that certain exposure concentrations can potentially affect reproductive health, offspring development and neurodevelopment, and result in metabolic and/or endocrine disorders. This experiment studied the effects of BPAF on the enzymes involved in the different phases of xenobiotic (XB) metabolism both trans generational between mother and offspring and between male and female pups. Xenobiotic metabolism facilitates the excretion of exogeneous substances from the body by chemically altering the XBs in two phases. Phase I enzymes render compounds more reactive, introducing groups that can be conjugated with glucuronic acid, sulfate, acetate, glutathione; thus, metabolic reactions convert xenobiotics from inactive to biologically active compounds. Phase II enzymes detoxify the XB through biotransformation reactions that facilitate the excretion of the XB and its biproducts. This process is important for maintaining homeostasis and regulating how various pollutants and other XBs are processed. Banked RNA samples from an experiment in which rats were dietary exposed to BPAF [ 0ppm (control),250ppm, or 1000ppm] in the feed through in utero and postnatal developmental stages were used in this study. These banked RNA samples were from the terminal ileum that was collected on postnatal day (PND) 4, PND 28 and week 13. The RNA was then extracted from the ileum by using TRIzol reagent. The RNA was DNAse treated and converted to cDNA. The mRNA expression for genes involved in the xenobiotic metabolism was assessed using real-time quantitative PCR. The preliminary analysis of the PCR data from the 13-week rats (male and female pups), showed that at 1000ppm BPAF exposure females had lower expressions of metabolic enzymes than their male counterparts. Additionally, as the BPAF concentration increased from 0ppm (vehicle) to 250 ppm to 1000ppm, the gene expression proportionally increased in both males and females. Lower expression of metabolic genes indicates lesser metabolic activity and breakdown of BPAF in females compared to males. Thus, females may be retaining the BPAF in their body at higher amounts. To investigate if accumulation of BPAF had an impact on the intestinal epithelial repair, a scratch assay was performed. To demonstrate the effects of BPAF toxicity on the epithelial layer repair, T-84 epithelial cells were grown in the 24 well plates. Upon confluency, a scratch was made and exposed to BPAF concentrations of 1000 μg/ml, 250 μg/ml, 25 μg/ml, 2.5 μg/ml, 0.25 μg/ml, or control. Results of this study show that scratched wells introduced to 0.25 μg/ml BPAF and 2.5 μg/ml BPAF were able to completely heal the scratch in the span of 9 days or less. Healing time decreased as concentrations of BPAF in wells increased, with some wells introduced to 0.25 μg/ml completely almost healing within 5 days. Wells introduced to 25 μg/ml did not completely heal by the ninth day but had begun to close. Scratched wells exposed to 250 μg/ml did not heal and cell death was noted as early as the third day. Wells introduced to 1000 μg/ml did not heal at all, experiencing cell death as early as the first day after exposure. These preliminary results emphasize the need to assess sex-dependent impact of BPAF pertaining to the healing of intestinal injury.