Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) alpha is equally abundant in the liver and the testis, whereas PPAR gamma and retinoic acid receptor (RAR) alpha are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RAR alpha and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPAR alpha and PPAR gamma in Sertoli cells, but they decreased the nuclear localization of RAR alpha, as previously shown. Both PPAR alpha and PPAR gamma were in the nucleus and cytoplasm of liver cells, but RAR alpha was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RAR alpha, PPAR alpha, and PPAR gamma in these organs.

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We characterize a novel microsome system that forms highmolecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16␣-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg 2ϩ , or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.Cytochromes P450 (P450) constitute a superfamily of cysteine thiolate enzymes that catalyze a diverse array of biochemical reactions (Nelson et al., 1996). CYP3A is a subfamily of integral membrane proteins that are expressed at high levels in the endoplasmic reticulum (ER) of liver and intestine. In humans, CYP3A is important in the metabolism of approximately 50% of all prescribed drugs as well as lipophilic hormones such as testosterone (Guengerich, 1999). As a result of this broad substrate specificity and high level of expression, drug-drug interactions resulting from altered CYP3A activity are well documented (Thummel and Wilkinson, 1998).After heme destruction and denaturation by reactive metabolites, CYP3A and CYP2E1 have been reported to be degraded by the ubiquitin-proteasome system (Correia et al., 1992;Tierney et al., 1992). Even in the absence of denaturation by reactive metabolites, these two P450s have relatively short half-lives (ϳ7-10 h) compared with most other P450s or resident ER proteins (typical t 1/2 of 18 -36 h) (Watkins et al., 1987;Koop and Tierney, 1990). Polyubiquitination commonly marks a protein for degradation by the 26S proteasome (Brodsky and McCracken, 1999;Schwartz and Ciechanover, 1999). Covalent linkage of ubiquitin typically requires the sequential action of a three-enzyme system. The first ...

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A polyclonal antibody was made to a liver cytochrome P450 purified from di-(2-ethyl-hexyl)phthalate (DEHP)-treated Sprague-Dawley rats and was used to identify the CYP4A forms in liver and kidney cortex microsomes of control rats and rats treated with this peroxisome proliferator. Three clearly separated major protein bands were recognized on western blots in liver microsomes of control male rats or male rats treated with a single dose of DEHP, which, based on the description of relative mobility, tissue specificity, and sex dependent expression of CYP4A forms (Sundseth and Waxman (1992). J. Biol. Chem., 267, 801-810), correspond to the migration pattern of forms 4A1, 4A2, and 4A3 in clofibrate-treated rats. The administration of DEHP for 2 or 3 days caused a loss of resolution of two of the protein bands. The protein band corresponding to 4A2 was absent in liver or kidney cortex microsomes of DEHP-treated or control female rats and was not always visible in the livers of control male rats. The purified P450DEHP supported the hydroxylation of arachidonic acid at both the 19- and 20-carbon atoms with turnover rates of 1.4 +/- 0.2 and 22.7 +/- 2.5 nmoles per minute per nmol P450, respectively. No measurable amounts of hydroxylated products were obtained when prostaglandin E1, leukotriene B4, or testosterone were used as substrates. Another member of the CYP4 family, 4B1 from rabbit lung microsomes, was also recognized by this antibody on western blot analysis; however, rabbit lung form 4A4 showed only minimal cross-reactivity with this antibody.