Phthalic acid esters, which are widely dispersed in the environment, induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. Evidence that oxidative stress in involved has been presented; however, peroxisomal H2O2 generation does not increase in vivo following exposure to some of these chemicals, seriously questioning this interpretation. Therefore, important gaps in our knowledge exist and it follows that new ideas need to be evaluated before the mechanisms by which this important class of chemicals causes cancer become clear. Key findings from our laboratory showed that 2- ethylhexanol, a metabolite of the plasticizer di(ethylhexyl) phthalate (DEHP), was selectively toxic in metabolically active periportal regions of the liver lobule by uncoupling mitochondrial oxidative phosphorylation and that chronic treatment in vivo with the potent nongenotoxic carcinogen WY-14,643 caused uncoupling in perfused liver under conditions which lead ultimately to tumors. Based on these new findings, we postulate that highly lipophilic plasticizers and lipid-lowering drugs accumulate in mitochondrial membranes, uncouple oxidative phosphorylation and alter cellular energetics leading to increases in intracellular calcium. We further propose that calcium elevates eicosanoid and/or mitogenic cytokine production by Kupffer cells and increases cell turnover by mechanisms possible involving protein kinase c. We plan to focus on Kupffer cells because they are activated by calcium and because we have shown very recently that WY-14,643 cause elevate intracellular calcium much more efficiently that 2-ethylhexanol in culture. These hypotheses will be tested first by assessing whether the model compounds DEHP and WY-14,643 cause oxidative stress following chronic exposure in vivo by measuring H2O2 production in the perfused liver. Second, the accumulation of DEHP and WY-14,643 in hepatic subcellular fractions following treatment of rats for various times up to four months will be measured and uncoupling of oxidative phosphorylation in vitro and in vivo will be assessed. Third, intracellular free calcium will be determined from changes in FURA-2 fluorescence in Kupffer and parenchymal cells. In addition, eicosanoid and cytokine release will be measured. Kupffer cells will then be destroyed selectively by treatment with gadolinium chloride to test the hypothesis that they are critical to this sequence of events and are responsible for the elevation in cell turnover caused by WY-14,643. By testing these hypotheses rigorously, not only will much needed information on the mechanism of action of phthalates and lipid- lowering drugs be provided, but critical information which will be used to assess the health risk posed to humans by this important class of chemicals will be generated. GRANT=R01HD07503 The overall goals of the proposed research are to examine regulatory mechanisms that influence post-mitotic development of testicular germ cells. The major calcium binding protein in mammalian sperm is calspermin. We have cloned the cDNA encoding this protein. Analysis of the sequence and of the gene revealed that calspermin is derived from a transcription unit that also encodes a novel calcium-calmodulin dependent protein kinase. This cDNA has also been cloned and sequenced. Whereas the kinase mRNA is first detected in early primary spermatocytes and declines to a ow level following the last meiotic division, calspermin mRNA is first expressed in pachytene primary spermatocytes and continues to increase as cell complete meiosis and undergo terminal differentiation. We have isolated genomic DNA that encodes the entire transcription unit and propose to characterize the structural organization of the gene. We will then utilize transfected mammalian cells, a testicular transcription system and transgenic mice to determine the molecular mechanisms that control cell specific mRNA expression. The hypothesis is that alternative transcriptional initiation followed by a requisite unique RNA processing event are involved in calspermin production. Expression vectors have been used to produce calspermin and the kinase and to identify key amino acids required for function and/or regulation of activity. A constitutive kinase has been used to develop mammalian cell lines that express the enzyme in a regulated fashion and to identify a transcriptional DNA element that is trans-activated by the active enzyme. We propose to characterize the mechanism by which the overexpression of the kinase results in a G2 cell cycle block and the transcription factor responsive to the kinase. Transgenic mice will be utilized to evaluate the consequences of altering the concentration of the kinase on spermatogenesis and sperm motility. Different promoters will be employed to change the temporal expression and/or amount of the proteins during germ cell development. We predict that the kinase will play a role in the onset, progression or completion of meiosis whereas calspermin will be involved in terminal differentiation of the spermatid or sperm motility.
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