DOI

10.17077/etd.5qb7-ambg

Document Type

Dissertation

Date of Degree

Spring 2019

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Duffel, Michael W.

First Committee Member

Duffel, Michael W.

Second Committee Member

Kerns, Robert

Third Committee Member

Klingelhutz, Aloysius

Fourth Committee Member

Robertson, Larry

Fifth Committee Member

Wright, Michael

Abstract

Polychlorinated biphenyls (PCBs) are persistent, man-made toxicants that are linked to adverse health effects and diseases such as endocrine disruption, diabetes, obesity, cardiovascular effects, and cancer. Since their manufacturing began in 1929 for industrial use, and was banned in 1979, they have bioaccumulated in water, sediment, food, animals, humans and more. PCBs are also found in indoor air of older buildings and as inadvertent byproducts in the manufacture of paints and pigments. The lower chlorinated PCBs, those with fewer than 5 chlorine atoms, are readily metabolized to form hydroxylated PCBs (OH-PCBs) that are further converted to PCB-sulfates in reactions catalyzed by cytosolic sulfotransferases (SULTs).

Steroid sulfotransferases SULT1E1 and SULT2A1 participate in regulating the homeostasis of estrogens and androgens, respectively, through the deactivation of active hormones. The estrogen sulfotransferase (SULT1E1) is also a potential key player in adipogenesis. Recent literature has shown that downregulating expression of SULT1E1 in cells derived from humans and mice caused opposite effects, where adipogenesis was inhibited or stimulated, respectively. Adipogenesis is the maturation of preadipocytes into mature adipocytes, which is regulated by peroxisome proliferating-activator γ (PPARγ). Adipocytes are a main component of adipose tissue, which is important for energy homeostasis, organ protection, and thermoregulation. Adipose tissue also secretes various cytokines such as adiponectin. Adipose tissue dysfunction can result from adipocyte dysfunction, which can be caused by alterations in cell signaling.

The objective of this dissertation research was to determine if OH-PCBs and PCB-sulfates are inhibitors of SULT1E1 and SULT2A1 and if inhibition of SULT1E1 by OH-PCBs could potentially affect adipogenesis. We hypothesized that PCB metabolites would inhibit SULT1E1 and SULT2A1 and potentially affect adipogenesis in both human and murine cell models.

Using purified recombinant human SULT1E1 and SULT2A1, I found that 4’-OH-PCB 3, 4-OH-PCB 8, 4-OH-PCB 11, 4’-OH-PCB 25, and 4-OH-PCB 52 were potent inhibitors of the sulfation of representative substrates (7.0 nM estradiol for SULT1E1 and 1.0 µM dehydroepiandrosterone for SULT2A1, Figures 3-3 and 3-4, respectively). Moreover, 4-OH-PCB 11 and 4-OH-PCB 52 were the most potent inhibitors of SULT1E1 and SULT2A1 with IC50 values of 7.2 nM and 1.5 μM, tables 3-1 and 3-2, respectively. The least potent inhibitor of SULT1E1 was 4’-OH-PCB 3, with an IC50 of 1300 nM. The PCB-sulfates were not potent inhibitors for either enzyme. 4-OH-PCB 11 inhibited the sulfation of estradiol in the cytosol of both pre-adipocytes and fully differentiated adipocytes (Figure 4-9).

Immortalized human adipocytes were treated with 10 µM of triclosan (a known inhibitor of SULT1E1), 4’-OH-PCB 3 and 4-OH-PCB 11. Experiments included exposure to these toxicants for 1) 72 hours to preadipocytes, 2) 72 hours to preadipocytes followed by 11-day differentiation, 3) to differentiating adipocytes and for 48 hours post-differentiation. The lipid accumulation levels remained unaffected, as determined by microscopic imaging and quantification using AdipoRed. The mRNA expression levels of prominent adipogenic markers SULT1E1, PPARγ, and AdipoQ were measured using RT-Q-PCR. Changes in SULT1E1 and PPARγ expression were unaffected upon treatment before, during and after adipogenesis when compared to controls. However, the increase in AdipoQ expression was reduced upon treatment with 4-OH-PCB 11 in differentiated adipocytes and in preadipocytes exposed for 72 hours followed by 11-day differentiation (Figure 4-14). This could be an indicator of adipocyte dysfunction that was not manifested by a change in lipid accumulation.

Murine 3T3-L1 cells were also treated with 10 µM of triclosan, 4’-OH-PCB 3 and 4-OH-PCB 11 for 48 hours to preadipocytes, during 8-day differentiation and for 48 hours after differentiation. The mRNA expression levels of prominent markers of cardiovascular and adipogenesis functions, ACE2, PPARγ, FABP4, and AdipoQ were measured using RT-PCR. Compared to controls, the increase in AdipoQ expression was reduced following treatment of preadipocytes with triclosan and 4-OH-PCB 11 and subsequent differentiation (Figure 5-11). The increase in PPARγ expression remained either unchanged from controls or slightly stimulated in differentiating and differentiated adipocytes (Figures 5-11 and 5-13). Angiotensin-converting enzyme 2 (ACE2) expression was decreased compared to control values, upon treatment with 4’-OH-PCB 3 (Figure 5-12), while fatty acid binding protein 4 (FABP4) expression was stimulated to the same extent across all treatment groups in differentiating adipocytes (Figure 5-12).

The results, overall, show that these OH-PCBs did not affect lipid accumulation in human adipocytes, but they may affect other signaling pathways in adipogenesis. 4-OH-PCB 11 decreased adiponectin expression compared to the increase that was seen in unexposed differentiating human and mouse adipocytes. Adiponectin is secreted from adipose tissue, and this decrease could indicate a form of dysfunction. This finding is consistent with the results of the purified SULT1E1 study, where 4-OH-PCB 11 potently inhibited SULT1E1, but 4’-OH-PCB 3 did not (Figure 3-3 and Table 3-1). Thus, there is a potential for OH-PCBs to disrupt the expression of adiponectin and perhaps other vital adipokines and this could negatively affect adipose tissue function. Future studies will be needed to determine if these effects are indeed mediated by intracellular estradiol and SULT1E1. Moreover, the potential for in vivo disruption of circulating adiponectin by OH-PCBs and other toxicants that inhibit SULTs remains to be studied.

Keywords

Adipogenesis, Estrogen Sulfotransferase, Hydroxysteroid Sulfotransferase, Polychlorinated biphenyls

Pages

xxxiii, 199 pages

Bibliography

Includes bibliographical references (pages 168-194).

Copyright

Copyright © 2019 Victoria Shayla Parker

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