<p>Potassium bromate (KBrO3) is a drinking water disinfection by-product and may represent a health hazard if found to be carcinogenic for humans, as was determined in rats (DeAngelo et al., 1998; Hayashi et al., 1986; Kurokawa, 1985; Kurokawa et al., 1985; Kurokawa et al., 1986a; Kurokawa et al., 1982; Kurokawa et al., 1983a; Kurokawa et al., 1987a; Kurokawa et al., 1983b; Kurokawa et al., 1986b; Ohno et al., 1982; Onodera et al., 1985). The purpose of this research was to determine the mechanism of toxicity. The peritoneal mesothelium is a (rat) target organ of potassium bromate carcinogenicity, but has not been studied due to the inherent difficulties of working with this one cell layer-thick tissue. Data from a two-year bioassay were used to more precisely map the location of origin, revealing that these tumors in the male F344 rat originate on the mesorchium of the tunica vaginalis testis, the mesosplenium, or at a point in between. An in vitro cell culture system was developed to study the mechanism of toxicity. It was demonstrated that KBrO3 caused cell cycle arrest and markedly increased the number of apoptotic cells. KBrO3 is a powerful oxidant and glutathione (GSH) is the major cellular antioxidant. Therefore, GSH-related responses were studied revealing mesothelial cells contained substantially less GSH than a human hepatocellular carcinoma cell line (Hep-G2). Studies employing GSH ester or N-acetyl cysteine (a GSH precursor) pre-treatment demonstrated abatement of toxicity in mesothelial, but not Hep-G2, cells. Experiments carried out to determine the chemical or enzymatic nature of the reaction between GSH and KBrO3 revealed differences between the reaction kinetics of the unbuffered and buffered chemical and cell-free/cell lysate reactions, probably due to reaction pH. Both chemical and cellular reactions exhibited a similar first step reaction between GSH and KBrO3; thus, enzyme participation is probably not required. Experiments using diethylmaleate, which depletes GSH by a reaction involving KBrO3, showed that GSH depletion greatly enhanced KBrO3 toxicity, indicating GSH glutathione S-transferase, buthionine sulfoximine (which prevents synthesis of GSH) and was protective. This does not support the hypothesis that the reaction of KBrO3 and GSH itself produces a radical/reactive species that oxidatively damages lipids, proteins and DNA. Rather, depletion of GSH likely precedes oxidative damage. Gene expression studies demonstrated that peritoneal mesothelial cells displayed expression changes in a discrete set of genes, including oxidative stress-responsive genes, after treatment with KBrO3 for four or 12 hours. Mesothelial cells severely damaged by five days KBrO3 treatment recovered from complete cell cycle arrest after four weeks and exhibited explosive growth, focus formation and altered morphology. The redox imbalance created by GSH depletion appears to mediate increased expression of known oxidative stress responsive genes (e.g., HO-1,GADD45, GADD153, QR), activation of transcription factors (AP-1 and NFkB) and down-regulation of cell cycle initiating cyclins (and up-regulation of the CDK inhibitor p21waf1/cip1) in KBrO3-mediated toxicity. These alterations may permit cell survival, as observed after severe toxicity, and may be accompanied by transforming mutations or clastogenic changes. Taken together, these data suggest that mesothelial cells represent a population susceptible to KBrO3-mediated toxicity in vitro, and suggest that tissue susceptibility in vivo plays a role in the nascence of mesotheliomas in the male F344 rat.<P>
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-20000509-143301 |
| Date | 10 May 2000 |
| Creators | Crosby, Lynn Marie |
| Contributors | Dr. Ross Leidy, Dr. Anthony DeAngelo, Dr. Robert C. Smart, Dr. Richard Miller, Dr. Sharon A. Meyer, Dr. Charles W. Qualls |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-20000509-143301 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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