Tracey Smith-Oliver

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 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.

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. RESULTS A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

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. Using the L929 bioassay, joint extracts from AA rats had significantly greater TNF levels (1054 +/- 147 pg/g tissue) than joint extracts from normal rats (110 +/- 42 pg/g tissue). Administration of ibuprofen failed to significantly inhibit TNF levels in the joint at a time point when paw swelling was significantly reduced. The immunomodulating agents, methotrexate, cyclosporin A (CSA) and HWA486 profoundly inhibited both joint TNF levels and paw swelling. The specificity of this assay for TNF was supported by studies with a polyclonal rabbit anti-mouse TNF antibody which neutralized 78-87% of the TNF activity in the joint extracts. Our studies demonstrate a quantitative increase in local TNF expression in adjuvant arthritis and support a role for TNF in chronic inflammation.

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. DEHP belongs to a class of agents described as hypolipidemic hepatocarcinogens. These chemicals share the ability to induce hepatic peroxisomal proliferation and range from very weak to very potent hepatocarcinogens. Unlike most identified carcinogens, the hypolipidemic carcinogens lack DNA reactivity in sensitive cell culture systems such as the Ames test. It has been proposed that active oxygen radicals, produced as a result of peroxisomal proliferation, induce DNA damage. While this is an attractive hypothesis, no genotoxic activity has been observed in hepatocytes with peroxisomal proliferation in treated animals. Another biological activity shared by this class of compounds is their ability to stimulate liver growth or hyperplasia. This additive hyperplasia results from direct mitogenic stimulation rather than regenerative growth following liver toxicity. This hyperplasia can be dramatic, with liver to body weight ratios from treated animals reaching two to three times normal. The degree of induced hyperplasia correlates well with the carcinogenic potency of these agents, whereas genotoxicity does not correlate at all. Increased cellular growth may result in spontaneous mutational events or proniotional effects. While some feedback mechanism eventually inhibits liver growth, it is possible that key genes related to the regulation of cellular growth and cancer remain stimulated during continued administration of the chemical. Thus, determination of hyperplastic activity represents an attractive first-step approach to the short-term detection and study of the mode of action of nongenotoxic carcinogens.

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. UDS was measured by quantitative autoradiography as net nuclear grains (NG). By similar methods, UDS and RDS (S-phase cells) were measured in hepatocytes isolated from rats and mice treated by gavage with TMP (500 mg/kg) or UG (100 to 5,000 mg/kg). A dose-related increase in UDS activity was observed in rat hepatocytes treated in vitro with 0.05 to 0.10% (v/v) UG. These doses were, however, toxic in both mouse and human hepatocyte cultures. Weak UDS activity was observed in hepatocytes isolated from male and female mice treated 12 hr previously with UG. No UDS was induced in rat hepatocytes treated in vivo or in vitro with TMP. Twenty- and fourfold increases in the percentage of cells in S-phase were observed 24 hr after treatment with TMP in male and female mice, respectively, as compared to a fivefold increase in male rats. UG increased the percentage of S-phase cells in male mice by ninefold but failed to induce RDS in females. Thus, there appears to be genotoxic compounds in UG that can be detected in cultured hepatocytes and in the livers of exposed mice. The lack of UDS activity in rat liver was consistent with the reported lack of liver tumors in chronically exposed rats. However, neither the UDS nor the RDS responses in mice exposed by gavage correlated to the sex-specific pattern of liver tumors observed in the 2-year bioassay.

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. Unscheduled DNA synthesis (UDS), as an indicator of genotoxicity, was measured autoradiographically as the incorporation of [3H]thymidine in isolated rat kidney cells following in vivo or in vitro exposure to UG. As an indicator of proliferative activity, cells in S-phase were quantitated in isolated cell preparations obtained from exposed rats. UG was administered to rats by inhalation (2000 ppm) or by gavage (up to 5000 mg/kg). The ability of the in vivo/in vitro kidney cell UDS assay to detect genotoxicants was verified using a variety of compounds. No UDS activity was elicited by UG under any of the conditions employed, including inhalation exposure to a concentration that produced kidney tumors in the 2-year bioassay. A five- to eightfold increase in the percentage of cells in S-phase was observed in male rats exposed to UG for 18 days either by inhalation or by gavage. Cell turnover was not markedly enhanced in identically treated female rats. These data indicate that UG does not evoke UDS in the rat kidney even after exposures that, in all probability, resulted in greater tissue concentrations of UG components than was realized in the long-term inhalation bioassay. The sex-specific induction of replicative DNA synthesis in the kidney paralleled the carcinogenic activity of UG, suggesting that induced cell turnover may be an important factor in the carcinogenic action of this motor fuel.

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. Hydroxyurea (HU, 10 mM) is commonly employed to inhibit replicative DNA synthesis (RDS) when using scintillation counting techniques for UDS. Autoradiographs revealed incomplete inhibition of RDS by HU.

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. Inhalation exposure to MeCl in vivo (3000-3500 ppm 6 h/day for 5 successive days) failed to induce DNA repair in any cell type. In vivo exposure to 15 000 ppm MeCl for 3 h also failed to induce UDS in tracheal epithelial cells and spermatocytes, but did cause a marginal increase in UDS in hepatocytes. Thus, MeCl appears to be a weak, direct-acting genotoxicant. While activity could be measured in hepatocytes and spermatocytes directly in vitro, only extremely high concentrations of MeCl elicited a response in the whole animal, and then only in hepatocytes.

Assessment of the covalent binding potential of 2,2,4-trimethylpentane to rat α2u-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. Accordingly, the ability of TMP and its metabolites to covalently bind to alpha 2u-globulin was examined. As a model, a [14C] formaldehyde-alpha 2u-globulin Schiff base was formed. This protein adduct was stabilized by reduction with cyanoborohydride and could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein analysis by SDS-PAGE demonstrated that hepatocytes isolated from male Fischer-344 rats produced significant quantities of alpha 2u-globulin in culture, whereas hepatocytes from female rats did not. A 15-hr exposure of metabolically competent, primary cultures of male rat hepatocytes to [14C] TMP (0.1 and 0.5%, v/v), followed by reduction with cyanoborohydride, dialysis, and analysis with SDS-PAGE, revealed no evidence of radiolabeled alpha 2u-globulin. When [14C]TMP was administered to an adult male Fischer-344 rat (300 mg/kg, ig) 22, 16, and 10 hr before sacrifice, 16% of the administered radioactivity was eliminated in the urine as TMP metabolites. Analysis as above showed no TMP-derived radioactivity in fractions containing alpha 2u-globulin from liver, blood, kidney cortex, or urine. The absence of a detectable covalent interaction between TMP and alpha 2u-globulin following in vitro or in vivo exposure suggests that a TMP-alpha 2u-globulin adduct is not responsible for the excessive formation of protein droplets in the renal cortex of exposed male rats.