Document Type

Dissertation

Date of Degree

Fall 2014

Degree Name

PhD (Doctor of Philosophy)

Degree In

Free Radical and Radiation Biology

First Advisor

Prabhat C. Goswami

Abstract

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are known to elicit adverse health effects including skin toxicity and cancer to animals and humans. 4-Monochlorobiphenyl (PCB3), a low-chlorinated airborne PCB conger is present in human blood and the environment. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of PCB3, has been shown to induce oxidative stress and toxicity in human mammary and prostate epithelial cells. These studies were designed to investigate and characterize the cellular responses to 4-ClBQ in HaCaT human skin keratinocytes. We found that 4-ClBQ treatment increased cellular reactive oxygen species (ROS) production, inhibited cell proliferation, and induced toxicity in HaCaT cells. Results from a Human Antioxidant Mechanism PCR array and quantitative RT-PCR assay showed that the mRNA levels of antioxidant gene selenoprotein P (sepp1) and catalase were significantly downregulated by the treatment, which correlated with evident decreases in their protein levels and catalase enzymatic activity. Pharmacological (sodium selenite supplementation) and molecular (sepp1overexpression) manipulation of SEPP1 expression significantly suppressed 4-ClBQ induced oxidative stress and toxicity. Additional results demonstrated that decreased catalase expression was associated with an inhibition in transcriptional coactivator peroxisome proliferator activated receptor Γ coactivator 1α (PGC-1α) expression. Overexpression of pgc-1α restored catalase expression and activity and consequently protected HaCaT cells from 4-ClBQ induced oxidative stress and toxicity. Furthermore, results from metabolic flux analysis using Seahorse XF96 Analyzer showed that 4-ClBQ treatment increased extracellular acidification rate, proton production rate, and oxygen consumption rate, which were associated with increases in glucose uptake and in the expression of glucose metabolism regulatory gene hexokinase 2, pyruvate kinase M2, and glucose-6-phosphate dehydrogenase (G6PD). G6PD is the rate-limiting enzyme of the pentose phosphate pathway. The enhanced expression of G6PD correlated with an increase in cellular glutathione content; and inhibition of G6PD activity sensitized HaCaT cells to 4-ClBQ induced toxicity, suggesting that the protective function of the pentose phosphate pathway is active in 4-ClBQ treated cells. Interestingly, we also found that 4-ClBQ selectively and significantly decreased mitochondrial complex II subunits C (sdhc) and D (sdhd) mRNA expression and subsequently reduced complex II activity leading to metabolic oxidative stress and toxicity, which were significantly suppressed by overexpressing sdhc and sdhd in HaCaT cells.

Taken together, findings from this project demonstrate that 4-ClBQ treatment increases ROS production through perturbing cellular metabolism and mitochondrial function and decreases antioxidant capacity by inhibiting SEPP1 and catalase expression in HaCaT cells. This imbalance due to increased mitochondrial prooxidant production and decreased antioxidant capacity leads to oxidative stress and toxicity. Importantly, antioxidant supplementation could abrogate 4-ClBQ induced toxicity, suggesting that antioxidants, especially nutrient-based manipulation of selenoproteins could be promising countermeasures for PCB induced adverse health effects in humans.

Public Abstract

Polychlorinated biphenyls (PCBs) are a large group of man-made chemicals, which had been widely used for a variety of industrial purposes owing to their extremely stable physical and chemical properties. Although intentional commercial production and applications of PCBs were banned over thirty years ago, PCB residues have been ubiquitously detected in the environment and biological organisms. Humans exposed to PCBs mainly through air, food, water, soil and dust. A long-term and cumulative exposure of PCBs in the environment leads to accumulation of PCBs in lipid tissues causing a wide spectrum of adverse health effects including skin toxicity and cancer. In these studies, we investigated the biological effects of PCB3 (present in human blood, commercial pigments, and Chicago air) and its quinone metabolite on human skin keratinocytes. Using cellular and molecular biological techniques, we identified antioxidant selenoprotein P (sepp1) as a previously unrecognized regulator of PCB3-quinone induced oxidative stress and toxicity. Additional results showed that PCB3-quinone perturbs cellular glucose metabolism and mitochondrial function causing oxidative stress and toxicity. Our results also suggest that mitochondria and cell metabolism could be the targets of PCBs. Since SEPP1 is a selenium-dependent antioxidant protein, results of this study support the rationale that antioxidants, especially nutrient-based manipulation of selenoproteins, could be promising countermeasures for PCB-induced adverse health effects in humans.

Keywords

publicabstract, AhR, Metabolic oxidative stress, Mitochondria, PCBs, PGC-1a, Selenoprotein P

Pages

xvii, 171 pages

Bibliography

Includes bibliographical references (pages 155-171).

Copyright

Copyright 2014 Wusheng Xiao

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