Species-specific kinetics and zonation of hepatic DNA synthesis induced by ligands of PPARalpha.

Peroxisome proliferator-activated receptor alpha (PPARalpha) ligands evoke a profound mitogenic response in rodent liver, and the aim of this study was to characterize the kinetics of induction of DNA synthesis. The CAR ligand, 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene, caused induction of hepatocyte DNA synthesis within 48 h in 129S4/SvJae mice, but the potent PPARalpha ligand, ciprofibrate, induced hepatocyte DNA synthesis only after 3 or 4 days dosing; higher or lower doses did not hasten the DNA synthesis response. This contrasted with the rapid induction (24 h) reported by Styles et al., 1988, Carcinogenesis 9, 1647-1655. C57BL/6 and DBA/2J mice showed significant induction of DNA synthesis after 4, but not 2, days ciprofibrate treatment. Alderley Park and 129S4/SvJae mice dosed with methylclofenapate induced hepatocyte DNA synthesis at 4, but not 2, days after dosing and proved that inconsistency with prior work was not due to a difference in mouse strain or PPARalpha ligand. Ciprofibrate-induced liver DNA synthesis and growth was absent in PPARalpha-null mice and are PPARalpha dependent. In the Fisher344 rat, hepatocyte DNA synthesis was induced at 24 h after dosing, with a second peak at 48 h. Lobular localization of hepatocyte DNA synthesis showed preferential periportal induction of DNA synthesis in rat but panlobular zonation of hepatocyte DNA synthesis in mouse. These results characterize a markedly later hepatic induction of panlobular DNA synthesis by PPARalpha ligands in mouse, compared to rapid induction of periportal DNA synthesis in rat.

Both PPARgamma and PPARdelta influence sulindac sulfide-mediated p21WAF1/CIP1 upregulation in a human prostate epithelial cell line.

Nonsteroidal anti-inflammatory drugs (NSAIDs) including sulindac sulfide are known to exert cancer chemopreventative activity in a range of cell lines. This activity has been shown to involve the upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1. It is also known that NSAIDs can act as peroxisome proliferator-activated receptor (PPAR) agonists and antagonists. In this study, we show that sulindac sulfide acts both as a PPARgamma agonist and a PPARdelta antagonist in an immortalized prostatic epithelial cell line (PNT1A). We utilized siRNA technology to show that PPARgamma is required for both growth inhibition and p21WAF1/CIP1 upregulation in response to sulindac sulfide treatment in PNT1A cells. In addition, the overexpression of PPARdelta partially rescued these cells from growth inhibition and also dramatically inhibited sulindac sulfide-mediated p21WAF1/CIP1 upregulation. Together these data identify a novel link between PPARgamma/PPARdelta/p21WAF1/CIP1 and the cancer chemo-preventative properties of NSAIDs.

Peroxisome proliferator activated receptor alpha regulates a male-specific cytochrome P450 in mouse liver.

We set out to find if the strain-specific, male-specific hepatic expression of Cyp4a protein in mouse was due to expression of Cyp4a12 and to understand the genetic basis for reported differences in expression. 12-Lauric acid hydroxylase (LAH) activity was found to show higher levels in male ddY, but not C57Bl/6, mouse liver microsomes. The expression of Cyp4a12 mRNA was studied using RNAase protection assays in male and female liver and kidney of nine mouse strains. Cyp4a12 was found to be highly expressed in male liver and kidney, but at much lower levels in female liver and kidney, in all strains studied. Western blotting with an antibody specific for Cyp4a12 confirmed that Cyp4a12 was expressed in a male specific fashion in C57Bl/6 mouse liver. RNAase protection analysis for Cyp4a10 and 14 in ddY mice revealed that neither of these genes showed male-specific expression. To further investigate genetic factors that control male-specific Cyp4a12 expression, PPARalpha+/+ and -/- mice were studied, showing that total P450 and 12-LAH activity was male-specific in +/+, but not -/- mice. RNAase protection assays were used to confirm that Cyp4a12 was lower in -/- mice. However, the male-specific Slp and MUP-1 genes retained hepatic male-specific levels of expression in +/+ and -/- mice, showing that the decrease in Cyp4a12 was not a general effect on male-specific expression. Thus, PPARalpha has a specific effect on constitutive expression of Cyp4a12.

The effects of haloalkene cysteine conjugates on cytosolic free calcium levels in LLC-PK(1) cells--studies utilising digital imaging fluorescence microscopy.

The aim of this study was to examine the effect of haloalkene S-cysteine conjugates on cytosolic free Ca(2+) levels in renal epithelial cells using digital imaging fluorescence microscopy (DIFM). S-(1,2,3,4,4-pentachloro-1,3,-butadienyl)-L-cysteine (PCBC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) were both cytotoxic to LLC-PK(1) cells in culture. Prior treatment of the cells with aminooxyacetic acid (AOAA), an inhibitor of the enzyme cysteine conjugate beta-lyase, afforded complete protection against the toxicity at concentrations of PCBC up to 100 microM and DCVC up to 500 microM. The cytotoxicity produced by PCBC (100 microM) was time dependent with no loss of lactate dehydrogenase (LDH) into the medium being observed until 4 h after exposure, while removal of calcium from the medium prevented the toxicity. Addition of PCBC (100 microM) to LLC-PK(1) cells produced a small progressive increase in intracellular calcium ([Ca(2+)](i)) from 72+/-6 to 126+/-11 nM following 10 min of exposure. At this time there was a marked cellular heterogeneity in the calcium response with some cells showing marked increases in [Ca(2+)](i), while others cycled between low and high values and some just maintained basal levels. Exposure to PCBC (100 microM) for 1 h produced a more marked increase in [Ca(2+)](I), 469+/-46 nM, with all cells responding. The elevation in [Ca(2+)](i) was concentration-related with increases seen at concentrations of 5 microM PCBC and above. The increase in [Ca(2+)](i) produced by PCBC (100 microM) was prevented by treatment with AOAA, and markedly reduced by a nominally calcium free medium or the addition of the calcium chelator EGTA. DCVC (500 microM) also markedly elevated [Ca(2+)](i) following exposure for 1 h, this was also prevented by AOAA and a nominal calcium free medium. These findings indicate that elevation in [Ca(2+)](i) produced by PCBC in renal epithelial cells, is an early event in the cascade of signalling changes leading to renal cell death. The major source of calcium appears to be from increased influx although a small component is released from intracellular stores which my trigger a stress protein response.