Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library.

Background: The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown.

Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway.

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7alpha-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRalpha and LXRbeta, 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 7alpha-hydroxylase gene.

An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma).

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.

Elevated levels of TNF in the joints of adjuvant arthritic rats.

The primary purpose of this study was to determine whether local levels of tumor necrosis factor (TNF) were elevated in chronically inflamed joints in rats with adjuvant-induced arthritis (AA). We also wished to develop methodology for the quantitative measurement of joint TNF, and to examine the effects of known anti-inflammatory agents on joint TNF levels. TNF levels were measured in joints from AA rats taken during the systemic phase (day 20) of arthritic disease.

The induction of DNA-strand breaks and unscheduled DNA synthesis in F-344 rat hepatocytes following in vivo administration of caprolactam or benzoin.

Benzoin (ZOIN) and caprolactam (CAP) were administered by gavage to Fischer 344 rats at a dose of 750 mg/kg and the hepatocytes isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled DNA synthesis (UDS). Neither ZOIN nor CAP induced SB or UDS in hepatocytes, however ZOIN did induce an increase in the fraction of cells in S-phase 24 h after treatment.

Use of primary cultures of human hepatocytes in toxicology studies.

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.

Measurement of unscheduled DNA synthesis in primary cultures of adult mouse epidermal keratinocytes.

Skin is a major target tissue for many environmental carcinogens. In order to provide a tool to study the role of DNA-repair processes in relation to DNA damage and carcinogenesis in this tissue, we have developed an assay that measures chemically induced DNA repair as unscheduled DNA synthesis (UDS) using cultures of adult mouse epidermal keratinocytes (MEKs) from SENCAR mice. Primary MEKs were prepared and incubated for 24 h in the presence of the test chemical and 10 microCi/ml [3H]thymidine.

Failure of the peroxisome proliferator WY-14,643 to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment.

Peroxisome proliferator hepatocarcinogens lack genotoxic activity in numerous in vitro assays using non-target cells which do not respond with peroxisome proliferation. Therefore, the effect of in vivo treatment with WY-14,643 on DNA repair was quantitated in rat hepatocytes, the target cell for carcinogenesis. Palmitoyl CoA oxidase and carnitine acetyltransferase activities in isolated hepatocytes were elevated by WY-14,643 (50 mg/kg/day by gavage for up to 5 consecutive days) and by WY-14,643 (0.

The potential role of chemically induced hyperplasia in the carcinogenic activity of the hypolipidemic carcinogens.

Di(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizing agent resulting in substantial human exposure and environmental contamination. In a chronic bioassay, high doses of DEHP induced hepatocellular carcinomas in female Fischer-344 rats and male and female B6C3F1 mice. Thus, there is considerable concern as to the species specificity, mechanism of action, and human risk assessment of DEHP.

Correlation of the carcinogenic potential of di(2-ethylhexyl)phthalate (DEHP) with induced hyperplasia rather than with genotoxic activity.

It has been reported that in a long-term feeding study 12,000 ppm of di(2-ethylhexyl)phthalate (DEHP) in the diet produced hepatocellular carcinomas in male and female F-344 rats while 6000 ppm DEHP produced the same tumor type in male and female B6C3F1 mice. In terms of the actual numbers of animals with tumors DEHP produced a greater response in mice than rats. DEHP and its principal hydrolysis product, mono(2-ethylhexyl)phtalate (MEHP) produce multiple effects in the animal such as liver peroxisomal proliferation and hyperplasia.

Assessment of the covalent binding potential of 2,2,4-trimethylpentane to rat alpha 2u-globulin.

Subchronic exposure of male rats to the nephrotoxin 2,2,4-trimethylpentane (TMP) causes an accumulation of protein droplets in the epithelial cells of the renal cortex. Experimental evidence suggests that these droplets contain alpha 2u-globulin, a low-molecular-weight protein found specifically in the urine of male rats. It has been proposed that aldehyde metabolites of TMP form Schiff base adducts with the lysine groups of alpha 2u-globulin and thereby inhibit renal lysosomal processing of the protein.

Assessment of unscheduled and replicative DNA synthesis in rat kidney cells exposed in vitro or in vivo to unleaded gasoline.

Unleaded gasoline (UG) induces renal toxicity and neoplasia in male but not female rats after chronic inhalation exposure. Before a meaningful determination of the potential human health risk of UG can be made, it is imperative that the mechanism responsible for its carcinogenic action be understood. The purpose of the present investigation was to determine whether the induction of kidney tumors by UG is related to genotoxic or to cell-proliferative effects.

Effect of peroxisome proliferator carcinogens on unscheduled DNA synthesis in rat hepatocytes determined by autoradiography.

The potent peroxisome proliferator hepatocarcinogens WY-14,643, BR-931, and nafenopin, as well as mono(ethylhexyl)phthalate (MEHP), the principle metabolite of the weaker hepatocarcinogen di(2-ethylhexyl)phthalate) (DEHP), were evaluated in the in vitro rat hepatocyte DNA repair assay by quantitative autoradiography. None of these carcinogens induced unscheduled DNA synthesis (UDS). These results failed to confirm the previously reported induction of UDS by WY-14,643 and BR-931 as determined by nuclear liquid scintillation counting.

Unscheduled DNA synthesis in rat tracheal epithelial cells, hepatocytes and spermatocytes following exposure to methyl chloride in vitro and in vivo.

Measurement of DNA repair as unscheduled DNA synthesis (UDS) in vitro following exposure in vivo in multiple tissues from the same treated animal can provide valuable information relating to the tissue- and organ-specificity of chemically induced DNA damage. UDS was evaluated in primary cultures of rat tracheal epithelial cells, hepatocytes and pachytene spermatocytes after exposure in vitro to methyl chloride (MeCl), and after isolation from the same treated animal following inhalation exposure in vivo. Concentrations of 1-10% MeCl in vitro induced UDS in hepatocytes and spermatocytes, but not in tracheal epithelial cells.

Assessment of unscheduled and replicative DNA synthesis in hepatocytes treated in vivo and in vitro with unleaded gasoline or 2,2,4-trimethylpentane.

In a recent chronic inhalation exposure study, unleaded gasoline (UG) produced kidney tumors in male rats and liver tumors in female mice, but did not increase the incidence of liver tumors in male mice or rats of either sex. To examine the possible basis for this pattern of hepatocarcinogenesis, unscheduled DNA synthesis (UDS) as an indicator of genotoxic activity and replicative DNA synthesis (RDS) as an indicator of cell proliferation were measured in rat and mouse hepatocytes following in vivo and in vitro exposures to UG and 2,2,4-trimethylpentane (TMP), a nephrotoxic component of UG. Primary hepatocyte cultures, prepared from cells isolated from Fischer-344 rats, B6C3F1 mice, or human surgical material, were incubated with [3H]thymidine and the test agent.

Induction of DNA repair in rat spermatocytes and hepatocytes by 1,2-dibromoethane: the role of glutathione conjugation.

1,2-Dibromoethane (EDB) is a widely used industrial chemical, and a well-known mutagen and carcinogen. EDB is biotransformed either by cytochrome P450-dependent oxidation, leading to the formation of bromoacetaldehyde, or by enzyme-catalyzed conjugation with glutathione, giving rise to reactive half-sulfur mustard compounds and their derivatives. In vitro mutagenicity and DNA binding studies suggest that the latter pathway is the primary source of genotoxic metabolites from EDB.

Lack of genotoxic activity of di(2-ethylhexyl)phthalate (DEHP) in rat and human hepatocytes.

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.