Tracey Smith-Oliver

Thiazolidinedione derivatives are antidiabetic agents that increase the insulin sensitivity of target tissues in animal models of non-insulin-dependent diabetes mellitus. In vitro, thiazolidinediones promote adipocyte differentiation of preadipocyte and mesenchymal stem cell lines; however, the molecular basis for this adipogenic effect has remained unclear. Here, we report that thiazolidinediones are potent and selective activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor superfamily recently shown to function in adipogenesis. The most potent of these agents, BRL49653, binds to PPAR gamma with a Kd of approximately 40 nM. Treatment of pluripotent C3H10T1/2 stem cells with BRL49653 results in efficient differentiation to adipocytes. These data are the first demonstration of a high affinity PPAR ligand and provide strong evidence that PPAR gamma is a molecular target for the adipogenic effects of thiazolidinediones. Furthermore, these data raise the intriguing possibility that PPAR gamma is a target for the therapeutic actions of this class of compounds.

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7α-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRα and LXRβ, two orphan members of the nuclear receptor superfamily, are activated by 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7α-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis. Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7α-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRα and LXRβ, two orphan members of the nuclear receptor superfamily, are activated by 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7α-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis.

Often results from toxicological studies using rodent models cannot be directly extrapolated to probable effects in human beings. In order to examine the genotoxic potential of chemicals in human liver cells, a human hepatocyte DNA repair assay has been defined. Procedures were optimized to prepare primary cultures of human hepatocytes from discarded surgical material. On eight different occasions human hepatocyte cultures of sufficient viability to measure DNA repair were successfully prepared by collagenase perfusion techniques. The cells were allowed to attach to plastic or collagen substrata for periods of 1.5 to 24 h and subsequently incubated with [3H]thymidine and test chemicals for periods of 18 to 24 h. Chemically induced DNA repair, measured as unscheduled DNA synthesis, was quantitated autoradiographically. The following compounds were tested: 2-acetylaminofluorene, aflatoxin B1, 2-aminobenzyl alcohol, aniline, benzo(a)pyrene, carbon tetrachloride, chloroform, 2,4-diaminotoluene, 2,6-diaminotoluene, di(2-ethylhexyl)phthalate, dimethylnitrosamine, 1,6-dinitropyrene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, methyl chloride, 5-methylchrysene, mono(2-ethylhexyl)phthalate, 2-methyl-2-P-(1,2,3,4-tetrahydro-1-naphthyl)phenoxypropionic acid (nafenopin), beta-naphthylamine, nitrobenzene, 2-nitrobenzyl alcohol, 2-nitrotoluene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, unleaded gasoline, and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy-14,643). In only one of eight cases did some of the chemicals generally regarded as genotoxic fail to give a positive response. For purposes of comparison, all test chemicals were evaluated in the in vitro rat hepatocyte DNA repair assay. Individual-to-individual variation in the DNA repair response was far greater for the human cultures than for cultures derived from rats. For only three chemicals was there a qualitative difference in the response between the rodent and the human cells; beta-naphthylamine was positive in the rat but in none of the human cultures examined, whereas the opposite was seen for 2,6-diaminotoluene and 5-methylchrysene. Clofibric acid, mono(2-ethylhexyl)phthalate, and Wy-14,643 induced enzymes indicative of peroxisomal proliferation in primary rat hepatocyte cultures, but not in two human hepatocyte cultures. These results indicate that, in general, the in vitro rat hepatocyte DNA repair assay is a valid model for predicting potential genotoxic effects in human beings. However, rodent hepatocytes may not be appropriate for assessing the potential of chemicals to elicit nongenotoxic effects in human beings such as the induction of hepatocyte peroxisomal proliferation.

Di(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizer which has been reported to induce a statistically significant increase in the incidence of hepatocellular carcinomas in female Fischer-344 rats (8/50) when administered in the diet at 12 000 p.p.m. for two years. Numerous studies with cells in culture have failed to show any genotoxic activity associated with DEHP. Because DEHP induces multiple changes in the liver, such as peroxisomal proliferation, it was possible that these alterations could result in genotoxic effects in the treated whole animal that would not be seen in cells in culture. Accordingly, the ability of DEHP to induce DNA damage or repair was examined in rat hepatocytes in vivo and in vitro and in human hepatocytes in vitro. Unscheduled DNA synthesis was measured by incorporation of [3H]thymidine into primary hepatocyte cultures immediately isolated from treated animals or hepatocyte cultures incubated directly with DEHP. DNA damage was measured by alkaline elution of cellular DNA from the same cultures. In vivo-in vitro treatment regimens were: (i) female rats, 12 000 p.p.m. DEHP in the diet for 30 days; (ii) female rats, 12 000 p.p.m. in the diet for 30 days, followed by 500 mg/kg DEHP by gavage 2 h before sacrifice; (iii) male rats, 500 mg/kg DEHP by gavage 2, 12, 24, or 48 h before sacrifice; and (iv) male rats, 150 mg/kg/day by gavage for 14 days. In vitro conditions were 0.1, 1.0 and 10.0 mM DEHP in the cultures for 18 h. Primary cultures of human hepatocytes were prepared from freshly discarded surgical material and exposed to the same concentration of DEHP. Concentrations up to 0.5 mM mono(2-ethylhexyl)phthalate, a principal metabolite of DEHP, were also examined in the human hepatocyte assay. No chemically induced DNA damage or repair was observed in vivo or in vitro in rat or human hepatocytes under any of the conditions employed. However, an increase in the percentage of cells in S-phase in the animals given DEHP was observed. These data indicate that DEHP does not exhibit direct genotoxic activity in the animals even with a treatment regimen which eventually produced tumors in a long term bioassay, and that both rat and human hepatocytes are similar in their lack of a genotoxic response to DEHP exposure in culture.