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Clopidogrel and prasugrel are antiplatelet therapies commonly used to treat patients with cardiovascular disease. They are both pro-drugs requiring biotransformation into active metabolites. It has been proposed that a genetic variant Q192R (rs662 A>G) in PON1 significantly alters the biotransformation of clopidogrel and affects clinical outcomes; however, this assertion has limited support. The relationship between this variant and clinical outcomes with prasugrel has not been studied. We genotyped PON1 Q192R in 275 healthy subjects treated with clopidogrel or prasugrel and 2922 patients with an ACS undergoing PCI randomized to treatment with clopidogrel or prasugrel in the TRITON-TIMI 38 trial. A meta-analysis was performed including 13 studies and 16,760 clopidogrel-treated patients. Among clopidogrel-treated subjects, there were no associations between Q192R and active drug metabolite levels (P = 0.62) or change in platelet aggregation (P = 0.51). Consistent with these results, in clopidogrel-treated patients in TRITON-TIMI 38, there was no association between Q192R and the rates of CV death, myocardial infarction, or stroke (RR 11.2 %, QR 8.6 %, and QQ 9.3 %; P = 0.66) or stent thrombosis (RR 2.4 %, QR 0.7 %, and QQ 1.6 %, P = 0.30), with patients with the putative at-risk Q variant having numerically lower event rates. Likewise, among prasugrel-treated subjects, there were no associations between Q192R and active drug metabolite levels (P = 0.88), change in platelet aggregation (P = 0.97), or clinical outcomes (P = 0.72). In a meta-analysis, the Q variant was not significantly associated with MACE (QQ vs. RR 1.22, 95 % CI 0.84-1.76) or stent thrombosis (QQ vs. RR OR 1.36, 95 % CI 0.77-2.38). Furthermore, when restricted to the validation studies, the OR (95 % CI) for MACE and stent thrombosis were 0.99 (0.77-1.27) and 1.23 (0.74-2.03), respectively. In the present study, the Q192R genetic variant in PON1 was not associated with the pharmacologic or clinical response to clopidogrel, nor was it associated with the response to prasugrel. The meta-analysis reinforced a lack of a significant association between Q192R and cardiovascular outcomes in clopidogrel-treated patients.

Background: Certain alleles of the CYP2C19 gene are associated with higher platelet reactivity and increased ischemic events among patients treated with clopidogrel. However, the relationship of CYP2C19 genotype and outcomes in medically managed patients with acute coronary syndromes (ACS) is not known., Objectives: This study sought to assess the effect of CYP2C19 genotype on ischemic outcomes in patients with ACS initially managed medically without revascularization who were randomized to either clopidogrel or prasugrel., Methods: We classified patients as extensive metabolizers (EM) or reduced metabolizers (RM) based on CYP2C19 genotype and evaluated ischemic outcomes and platelet reactivity. Among 9,326 patients enrolled from 2008 to 2011, 5,736 participated in the genetics cohort; of these, 2,236 had platelet function testing data., Results: There was no association between CYP2C19 metabolizer status (EM vs. RM) and the primary composite endpoint of cardiovascular death, myocardial infarction (MI), or stroke (hazard ratio [HR]: 0.86). EM and RM patients had similar rates of the primary endpoint whether treated with prasugrel (HR: 0.82) or clopidogrel (HR: 0.91; p for interaction = 0.495). After adjusting for clinical and treatment variables, EM patients had a lower risk of MI versus RM patients (HR: 0.80), but risks of other outcomes were similar. RM patients had significantly higher mean P2Y12 reaction units versus EM patients when treated with clopidogrel (39.93), but not with prasugrel (3.87)., Conclusions: CYP2C19 metabolizer status is not associated with the composite outcome of cardiovascular death, MI, or stroke in medically managed ACS patients treated with clopidogrel or prasugrel. Our findings do not support routine CYP2C19 genetic testing in this population. (A Comparison of Prasugrel and Clopidogrel in Acute Coronary Syndrome Subjects [TRILOGY ACS]; NCT00699998)., (Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: A straightforward, reproducible blood-based test that predicts age dependent risk of Alzheimer's disease (AD) could be used as an enrichment tool for clinical development of therapies. This study evaluated the prognostic performance of a genetics-based biomarker risk algorithm (GBRA) established on a combination of Apolipoprotein E (APOE)/Translocase of outer mitochondrial membrane 40 homolog (TOMM40) genotypes and age, then compare it to cerebrospinal fluid (CSF) biomarkers, neuroimaging and neurocognitive tests using data from two independent AD cohorts., Methods: The GBRA was developed using data from the prospective Bryan-ADRC study (n=407; 86 conversion events (mild cognitive impairment (MCI) or late onset Alzheimer's disease (LOAD)). The performance of the algorithm was tested using data from the ADNI study (n=660; 457 individuals categorized as MCI or LOAD)., Results: The positive predictive values (PPV) and negative predictive values (NPV) of the GBRA are in the range of 70-80%. The relatively high odds ratio (approximately 3-5) and significant net reclassification index (NRI) scores comparing the GBRA to a version based on APOE and age alone support the value of the GBRA in risk prediction for MCI due to LOAD. Performance of the GBRA compares favorably with CSF and imaging (fMRI) biomarkers. In addition, the GBRA "high" and "low" AD-risk categorizations correlated well with pathological CSF biomarker levels, PET amyloid burden and neurocognitive scores., Conclusions: Unlike dynamic markers (i.e., imaging, protein or lipid markers) that may be influenced by factors unrelated to disease, genomic DNA is easily collected, stable, and the technical methods for measurement are robust, inexpensive, and widely available. The performance characteristics of the GBRA support its use as a pharmacogenetic enrichment tool for LOAD delay of onset clinical trials, and merits further evaluation for its clinical utility in evaluating therapeutic efficacy.

Background: Several studies have demonstrated a lower apolipoprotein E4 (APOE ε4) allele frequency in African-Americans, but yet an increased age-related prevalence of AD. An algorithm for prevention clinical trials incorporating TOMM40'523 (Translocase of Outer Mitochondria Membrane) and APOE depends on accurate TOMM40'523-APOE haplotypes., Methods: We have compared the APOE and TOMM40'523 phased haplotype frequencies of a 9.5 kb TOMM40/APOE genomic region in West African, Caucasian, and African-American cohorts., Results: African-American haplotype frequency scans of poly-T lengths connected in phase with either APOE ε4 or APOE ε3 differ from both West Africans and Caucasians and represent admixture of several distinct West African and Caucasian haplotypes. A new West African TOMM40'523 haplotype, with APOE ε4 connected to a short TOMM40'523 allele, is observed in African-Americans but not Caucasians., Conclusion: These data have therapeutic implications for the age of onset risk algorithm estimates and the design of a prevention trial for African-Americans or other mixed ethnic populations., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

High on-treatment platelet reactivity (HPR) has been identified as an independent risk factor for ischaemic events. The randomised, double-blind, TRIPLET trial included a pre-defined comparison of HPR in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) following a placebo/600-mg clopidogrel loading dose (LD) immediately before a subsequent prasugrel 60-mg or 30-mg LD. Platelet reactivity was assessed using the VerifyNow® P2Y12 assay (P2Y12 Reaction Units, PRU) within 24 hours (h) following the placebo/clopidogrel LD (immediately prior to prasugrel LD), and at 2, 6, 24, 72 h following prasugrel LDs. The impact of CYP2C19 predicted metaboliser phenotype (extensive metabolisers [EM] and reduced metabolisers [RM]) on HPR status was also assessed. HPR (PRU ≥240) following the clopidogrel LD (prior to the prasugrel LD) was 58.5% in the combined clopidogrel LD groups. No significant difference was noted when stratified by time between the clopidogrel and prasugrel LDs (≤6 hs vs>6 h). At 6 h following the 2nd loading dose in the combined prasugrel LD groups, HPR was 7.1%, with 0% HPR by 72 h. There was no significant effect of CYP2C19 genotype on pharmacodynamic (PD) response following either prasugrel LD treatments at any time point, regardless of whether it was preceded by a clopidogrel 600-mg LD. In conclusion, in this study, patients with ACS intended for PCI showed a high prevalence of HPR after clopidogrel 600-mg LD regardless of metaboliser status. When prasugrel LD was added, HPR decreased substantially by 6 h, and was not seen by 72 h.

We compared results obtained with the Nanosphere Verigene® System, a novel point-of-care (POC) genetic test capable of analysing 11 CYP2C19 variants within 3 hours, to an established, validated genotyping method (Affymetrix™ DMET+; reference assay) for identifying extensive and reduced metabolisers of clopidogrel. Based on genotyping, patients (N=82) with stable coronary artery disease on clopidogrel 75 mg daily were defined as extensive metabolisers (*1/*1, *1/*17, *17/*17), reduced metabolisers (*1/*2, *1/*8, *2/*2, *2/*3), or of indeterminate metaboliser status (*2/*17). Pharmacokinetic exposure to clopidogrel's active metabolite and pharmacodynamic measures with P2Y12 reaction units (PRU) (VerifyNow®P2Y12 assay) and VASP PRI (PRI) were also assessed. There was a 99.9% overall concordance of marker-level data between the Nanosphere Verigene and DMET+ systems in identifying the CYP2C19 variants and 100% agreement in classifying the patients as extensive (n=59) or reduced metabolisers (n=15). Extensive metabolisers had significantly higher active metabolite exposure than reduced metabolisers (LS means 12.6 ng*h/ml vs 7.7 ng*h/ml; p<0.001). Extensive metabolisers also had lower PRU (LS means 158 vs 212; p=0.003) and VASP PRI (LS means 48% vs 63%, p=0.01) compared to reduced metabolisers. Rates of high on-treatment platelet reactivity were higher in reduced metabolisers compared to extensive metabolisers (VASP PRI ≥ 50%: 79% vs 47%; PRU >235: 33% vs 16%). The Nanosphere Verigene CBS system identified 11 CYP2C19 alleles in less than 3 hours with a high degree of accuracy when compared to a conventional method, and was further validated against pharmacokinetic and pharmacodynamic phenotypes.

Background: Endogenous opioid-mediated reward pathways may play a role in the development and maintenance of alcohol dependence. This study tested whether LY2196044, an opioid receptor antagonist, in combination with medical management would reduce drinking in alcohol-dependent patients., Methods: This was a multicenter, outpatient, randomized, double-blind, parallel, and placebo-controlled trial with a 16-week treatment period. Patients (N = 375) were alcohol-dependent, treatment-seeking adults. Patients were randomly assigned to once-daily LY2196044 (final doses of 125 or 250 mg/d) or placebo. DNA samples were collected at baseline. At each visit, patients underwent safety assessments, laboratory testing, efficacy measures, and medical management. Blood samples were also obtained for pharmacokinetic testing. The primary measure was the change from baseline in the percent heavy drinking days (HDD). Secondary efficacy measures were percent days abstinent per month and number of drinks per day., Results: The treatment difference in change from baseline in % HDD between LY2196044 and placebo was not statistically significant (-43.02 vs. -38.72%, respectively; p = 0.12). There was a trend toward greater change from baseline in the percent days abstinent per month for the LY2196044 group compared with the placebo group (33.49 vs. 28.12%, respectively; p = 0.051). The decrease from baseline for mean number of drinks per day was statistically significantly greater in the LY2196044 group compared with the placebo group (-5.37 vs. -4.66 drinks per day, respectively; p = 0.013). LY2196044-treated patients who were dopamine receptor type 4-variable number tandem repeat L carriers had greater reductions in % HDD (p = 0.0565), increased percent days abstinent (p = 0.0496), and reduced drinks per day (p = 0.0069) than placebo-treated L carriers. The safety profile for LY2196044 appeared similar to that of other opioid antagonists., Conclusions: The results from this proof-of-concept clinical trial warrant further evaluation of LY2196044 for the treatment of alcohol dependence., (Copyright © 2013 by the Research Society on Alcoholism.)

The ɛ4 allele of the apolipoprotein E (APOE) gene is currently the strongest and most highly replicated genetic factor for risk and age of onset of late-onset Alzheimer's disease (LOAD). Using phylogenetic analysis, we have identified a polymorphic poly-T variant, rs10524523, in the translocase of outer mitochondrial membrane 40 homolog (TOMM40) gene that provides greatly increased precision in the estimation of age of LOAD onset for APOE ɛ3 carriers. In two independent clinical cohorts, longer lengths of rs10524523 are associated with a higher risk for LOAD. For APOE ɛ3/4 patients who developed LOAD after 60 years of age, individuals with long poly-T repeats linked to APOE ɛ3 develop LOAD on an average of 7 years earlier than individuals with shorter poly-T repeats linked to APOE ɛ3 (70.5 ± 1.2 years versus 77.6 ± 2.1 years, P=0.02, n=34). Independent mutation events at rs10524523 that occurred during Caucasian evolution have given rise to multiple categories of poly-T length variants at this locus. On replication, these results will have clinical utility for predictive risk estimates for LOAD and for enabling clinical disease prevention studies. In addition, these results show the effective use of a phylogenetic approach for analysis of haplotypes of polymorphisms, including structural polymorphisms, which contribute to complex diseases.

Aims/hypothesis: Several susceptibility genes for type 2 diabetes have been discovered recently. Individually, these genes increase the disease risk only minimally. The goals of the present study were to determine, at the population level, the risk of diabetes in individuals who carry risk alleles within several susceptibility genes for the disease and the added value of this genetic information over the clinical predictors., Methods: We constructed an additive genetic score using the most replicated single-nucleotide polymorphisms (SNPs) within 15 type 2 diabetes-susceptibility genes, weighting each SNP with its reported effect. We tested this score in the extensively phenotyped population-based cross-sectional CoLaus Study in Lausanne, Switzerland (n = 5,360), involving 356 diabetic individuals., Results: The clinical predictors of prevalent diabetes were age, BMI, family history of diabetes, WHR, and triacylglycerol/HDL-cholesterol ratio. After adjustment for these variables, the risk of diabetes was 2.7 (95% CI 1.8-4.0, p = 0.000006) for individuals with a genetic score within the top quintile, compared with the bottom quintile. Adding the genetic score to the clinical covariates improved the area under the receiver operating characteristic curve slightly (from 0.86 to 0.87), yet significantly (p = 0.002). BMI was similar in these two extreme quintiles., Conclusions/interpretation: In this population, a simple weighted 15 SNP-based genetic score provides additional information over clinical predictors of prevalent diabetes. At this stage, however, the clinical benefit of this genetic information is limited.

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its response gene, Acyl CoA synthetase 5 (ACSL5), which has an important role in fatty acid metabolism, may affect weight loss in response to caloric restriction. Therefore, we aimed to determine whether these genes were involved in the interindividual response to dietary treatment. Genotypic/phenotypic comparisons were made between selected obese women from the quintiles losing the most (diet responsive, n = 74) and the quintiles losing the least (diet-resistant, n = 67) weight in the first 6 weeks of a 900-kcal formula diet. Two common PPARgamma single nucleotide polymorphisms, Pro(12)Ala and C1431T, and eight polymorphisms across the ACSL5 gene were selected for single locus and haplotypic association analyses. The PPARgamma Pro(12)Ala single nucleotide polymorphism was associated with diet resistance (odds ratio = 3.48, 95% confidence interval = 1.41 to 8.56, p = 0.03), and the rs2419621, located in the 5'untranslated region of the ACSL5 gene, displayed the strongest association with diet response (odds ratio = 3.45, 95% confidence interval = 1.61 to 7.69, p = 0.001). Skeletal muscle ACSL5 mRNA expression was significantly lower in carriers of the wildtype compared with the variant rs2419621 allele (p = 0.03). Our results suggest a link between PPARgamma2 and ACSL5 genotype and diet responsiveness.

We conducted a genome-wide scan using variance components linkage analysis to localize quantitative-trait loci (QTLs) influencing triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol, and total cholesterol (TC) levels in 3,071 subjects from 459 families with atherogenic dyslipidemia. The most significant evidence for linkage to TG levels was found in a subset of Turkish families at 11q22 [logarithm of the odds ratio (LOD)=3.34] and at 17q12 (LOD=3.44). We performed sequential oligogenic linkage analysis to examine whether multiple QTLs jointly influence TG levels in the Turkish families. These analyses revealed loci at 20q13 that showed strong epistatic effects with 11q22 (conditional LOD=3.15) and at 7q36 that showed strong epistatic effects with 17q12 (conditional LOD=3.21). We also found linkage on the 8p21 region for TG in the entire group of families (LOD=3.08). For HDL-C levels, evidence of linkage was identified on chromosome 15 in the Turkish families (LOD=3.05) and on chromosome 5 in the entire group of families (LOD=2.83). Linkage to QTLs for TC was found at 8p23 in the entire group of families (LOD=4.05) and at 5q13 in a subset of Turkish and Mediterranean families (LOD=3.72). These QTLs provide important clues for the further investigation of genes responsible for these complex lipid phenotypes. These data also indicate that a large proportion of the variance of TG levels in the Turkish population is explained by the interaction of multiple genetic loci.

264W94 was designed to inhibit the ileal bile acid transporter (IBAT). Evaluated in vitro, 264W94 dose-dependently inhibited sodium-dependent uptake of 10 micro M [(3)H]taurocholic acid (TC) by rat and monkey brush border membrane vesicles with IC(50)s of 0.24 micro M and 0.41 micro M, and had a competitive profile with K(i) of 0.2 micro M against TC in Chinese hamster ovary cells expressing human IBAT. In distal ileum in situ, 1-10 micro M of 264W94 rapidly decreased uptake of 3mM TC by 24-39%, with corresponding decreases in biliary recovery. In rats and mice in vivo, oral 264W94 decreased absorption of TC analog, 23,25-(75)Se-homocholic acid taurine ((75)SeHCAT; quantitated in feces), with ED(30) of 0.02 mg/kg bid. (75)SeHCAT traced through the GI-tract revealed that peak (97%) inhibition of (75)SeHCAT absorption by the distal quarter of small intestine occurred at 4 h after single dose of 264W94 (0.1 mg/kg). Inhibition of IBAT by 264W94 in rats was associated with compensatory, same-day, 4-fold induction of hepatic cholesterol 7alpha-hydroxylase (CYP7A1) activity, exhibiting normal diurnal fluctuation for 3 days of dosing. In diet induced hypercholesterolemic rats, 264W94 (0.03-1.0 mg/kg bid) dose-dependently reduced serum LDL+VLDL cholesterol up to 61%. In conclusion, 264W94 is a potent new cholesterol lowering agent that acts through inhibition of IBAT and exhibits activity in a human model.

Expression pharmacogenomics includes differential gene expression (DGE) profiling of drug responses in model systems to generate a set of differentially modulated drug-responsive genes which can serve as a surrogate measure for drug action. In this manner, expression pharmacogenomics bridges the fields of genomics and medicinal chemistry. Additionally, modulated genes can be organized into metabolic and signaling pathways that highlight the mechanism of drug activity in a selected tissue. Here, we describe the application of expression pharmacogenomics to characterize a drug response in the clinically relevant in vivo model, the Sprague-Dawley rat. Following oral dosing of rats with GW9578, a novel synthetic peroxisome proliferator activated receptor alpha (PPAR alpha) ligand indicated for lipid disorders, we applied GeneCalling, a differential mRNA transcript profiling technique, to rat liver cDNA. Following GW9578 treatment, 2.4% of the rat liver genes were differentially expressed. We confirmed the sequence identity of 50 distinctly modulated genes. DGE was observed among genes representative of at least six discrete metabolic pathways. Furthermore, we observed up-regulation of 20 genes involved in mitochondrial, peroxisomal and microsomal fatty acid oxidation, consistent with molecular biological and clinical data indicating PPAR alpha ligand principal efficacy to be through increasing fatty acid metabolism. Those pathways regulated in our study that are potentially contributory to target effect, non-target adverse effects, or of unknown consequence include xenobiotic detoxification and steroid modification. Finally, comprehensive drug response profiling can lead to the serendipitous discovery of novel disease indications. In this case, these results suggest a potential novel indication for GW9578 in the treatment of X-linked adrenoleukodystrophy. We have shown, therefore, that the organization of DGE results into metabolic and signaling pathways can elucidate mechanisms of pharmacologically desired (i.e., efficacious) and, where appropriate, undesired (i.e., potentially deleterious) effects.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists, including the glitazone class of drugs, are insulin sensitizers that reduce glucose and lipid levels in patients with type 2 diabetes mellitus. To more fully understand the molecular mechanisms underlying their therapeutic actions, we have characterized the effects of the potent, tyrosine-based PPAR gamma ligand GW1929 on serum glucose and lipid parameters and gene expression in Zucker diabetic fatty rats. In time-course studies, GW1929 treatment decreased circulating FFA levels before reducing glucose and triglyceride levels. We used a comprehensive and unbiased messenger RNA profiling technique to identify genes regulated either directly or indirectly by PPAR gamma in epididymal white adipose tissue, interscapular brown adipose tissue, liver, and soleus skeletal muscle. PPAR gamma activation stimulated the expression of a large number of genes involved in lipogenesis and fatty acid metabolism in both white adipose tissue and brown adipose tissue. In muscle, PPAR gamma agonist treatment decreased the expression of pyruvate dehydrogenase kinase 4, which represses oxidative glucose metabolism, and also decreased the expression of genes involved in fatty acid transport and oxidation. These changes suggest a molecular basis for PPAR gamma-mediated increases in glucose utilization in muscle. In liver, PPAR gamma activation coordinately decreased the expression of genes involved in gluconeogenesis. We conclude from these studies that the antidiabetic actions of PPAR gamma agonists are probably the consequence of 1) their effects on FFA levels, and 2), their coordinate effects on gene expression in multiple insulin-sensitive tissues.

Aims/hypothesis: Although retinoid X receptor (RXR) and peroxisome proliferator activated receptor-gamma (PPARgamma) agonists have antidiabetic effects in hyperinsulinaemic animals, little information exists on their effects after pancreatic beta-cell failure. Thus, we examined if RXR and PPARgamma agonists alter distinct metabolic pathways in animals suffering from impaired insulin secretion., Methods: Adverse side effects and antidiabetic responses were measured in db/db mice treated from 14-16 weeks of age with the RXR agonist, LG100268, and/or the PPARgamma agonists, BRL49653 or GW1929., Results: In animals treated with LG100268 or BRL49653, serum glucose, glycohaemoglobin and the cardiovascular risk factor, fibrinogen, decreased to the same extent. Both of these agonists were equally effective at increasing insulin accumulation in beta cells, although neither agent had an effect on serum insulin concentrations. In contrast, the RXR agonist was less effective than the PPARgamma agonists at lowering serum triglycerides and non-esterified fatty acids and increasing interscapular brown fat and body weight. Further, LG100268 increased serum alkaline phosphatase and liver mass, hepatic fat accumulation, lauric acid hydroxylase activity, catalase-immunostaining and peroxisomal number more than the PPARgamma agonists. Moreover, co-treatment with the RXR and PPARgamma agonists reduced glucose, triglycerides, non-esterified fatty acids and cholesterol more than either agent alone., Conclusion/interpretation: These data suggest 1) RXR and PPARgamma agonists decrease islet degeneration, cardiovascular risk and cachexia during later stages of diabetes, 2) RXR agonists are less effective than PPARgamma agonists at decreasing serum lipids and causing weight gain and 3) RXR agonists have a more pronounced effect on liver metabolism (e.g. peroxisome accumulation and hepatomegaly) than PPARgamma agonists.

Peroxisome proliferator-activated receptors (PPARs) alpha and gamma are key regulators of lipid homeostasis and are activated by a structurally diverse group of compounds including fatty acids, eicosanoids, and hypolipidemic drugs such as fibrates and thiazolidinediones. While thiazolidinediones and 15-deoxy-Delta12, 14-prostaglandin J2 have been shown to bind to PPARgamma, it has remained unclear whether other activators mediate their effects through direct interactions with the PPARs or via indirect mechanisms. Here, we describe a novel fibrate, designated GW2331, that is a high-affinity ligand for both PPARalpha and PPARgamma. Using GW2331 as a radioligand in competition binding assays, we show that certain mono- and polyunsaturated fatty acids bind directly to PPARalpha and PPARgamma at physiological concentrations, and that the eicosanoids 8(S)-hydroxyeicosatetraenoic acid and 15-deoxy-Delta12,14-prostaglandin J2 can function as subtype-selective ligands for PPARalpha and PPARgamma, respectively. These data provide evidence that PPARs serve as physiological sensors of lipid levels and suggest a molecular mechanism whereby dietary fatty acids can modulate lipid homeostasis.

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. 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.

The hypothesis that mitochondrial sterol 27-hydroxylase plays a role in the sterol-mediated down-regulation of LDL receptor activity was evaluated in HepG2 cells. 27-Hydroxycholesterol was found to be more potent at suppressing LDL receptor activity than cholesterol (IC50 values of 8 mu M and 142 mu M for 27-hydroxycholesterol and cholesterol, respectively) when the sterols were delivered to cells from 2-hydroxypropyl-beta-cyclodextrin (beta-CD)-solubilized solutions. Cyclosporin, an immunosuppressant which has been shown to inhibit the 27-hydroxylation of sterols, was used to assess whether the formation of endogenous 27-hydroxycholesterol was required for the cholesterol-induced suppression of LDL receptor activity. Cyclosporin dose-dependently inhibited the 27-hydroxylation of cholesterol by HepG2 mitochondria (Ki = 0.25 mu M) and HepG2 cell cultures (IC50 = 1 mu M). At 1 mu M, cyclosporin had no effect on LDL receptor activity, and did not prevent the suppression of LDL receptor activity caused by: 1) the addition of beta-CD-solubilized cholesterol, 2) the receptor-mediated uptake of beta-VLDL, or 3) the inhibition of cholesterol esterification. In contrast, 10 mu M cyclosporin was found to inhibit the esterification of cholesterol and to increase the cellular level of free cholesterol resulting in suppression of LDL receptor activity. These results suggest that if mitochondrial sterol 27-hydroxylase plays a role in the regulation of LDL receptor activity, it is not through the formation of potent regulatory oxysterols, but through its effects on the availability and/or size of the free cholesterol pool regulating LDL receptor activity.

Glutathione S-transferases (GSTs) play an important role in the detoxification of diverse electrophilic chemicals, including anticancer drugs. Gene-specific oligonucleotide probes were developed to monitor the expression of individual GST mRNAs in livers of adult male rats treated with drugs and other chemical modulators of GST expression. Northern blot analysis of total liver RNA using probes specific for individual GSTs belonging to classes alpha (GSTs Ya1, Ya2, Yc), mu (GSTs Yb1, Yb2, Yb3), pi (GST Yp), and GSTms demonstrated the expression in liver of all but Yp mRNA. Kidney GST expression was at least as high as that in liver for GSTs Ya1, Yc, and Yp, while it was substantially lower but still detectable for GSTs Ya2, Yb2, and GSTms. Several of the liver GST class alpha mRNAs, in particular Ya2, were inducible by pretreatment of rats with phenobarbital or isosafrole. In contrast, dexamethasone preferentially induced Yb1, Yb2, and Ya2, while two other inducers of liver drug metabolism, isoniazid and clofibrate, were less effective with respect to GST induction. GSTms mRNA was induced to a small extent or not at all by the agents tested. Treatment of adult male rats with the anticancer drug cisplatin increased liver expression of GST Yc mRNA and suppressed Ya1 mRNA levels with little or no major effect on several other GST mRNAs. Western blot analysis of liver cytosols prepared from the cisplatin-treated rats revealed corresponding changes in GST Yc and Ya protein levels. Comparable changes in liver GST Ya1 and Yc expression were effected by the cisplatin analogue iproplatin but not by carboplatin or transplatin. This pattern of response to these platinum drugs is comparable to that seen with respect to platinum drug-induced gonadal toxicity and modulation of liver cytochrome P450 expression, suggesting a common mechanistic basis for these diverse effects of platinum anticancer drugs on hepatic enzymes of drug metabolism. Together, these studies demonstrate the utility of oligonucleotide probes for phenotyping liver tissue for the expression of GST enzymes that can contribute to anticancer drug metabolism and resistance. They also raise the possibility of drug-drug interactions involving cisplatin and alkylating agent anticancer drugs that can be metabolized in liver by alpha-class GSTs.

Growth hormone (GH) differentially regulates the expression of several male-specific and female-specific liver cytochrome P450 mRNAs as a function of its sex-dependent ultradian secretory pattern. Pulsatile GH release stimulates expression of the male-specific P450 2C11, while a continuous GH secretion pattern suppresses expression of 2C11 and stimulates the expression of the female-specific P450 2C12. To help define the level at which GH regulates the expression of 2C11 and 2C12 mRNA, liver nuclear RNA samples isolated from rats differing in GH status were analyzed for 2C11 and 2C12 hnRNAs by hybridization to 2C11 and 2C12 gene-specific exonic oligonucleotide probes, as well as exon/intron junction probes. The 2C11 and 2C12 hnRNAs were found to be responsive to circulating GH profiles in a manner indistinguishable from the corresponding mature, cytoplasmic mRNAs, with no 2C12 mRNA precursors found in untreated male or hypophysectomized female liver nuclei, and no 2C11 mRNA precursors in untreated female or hypophysectomized male liver nuclei. Thus, transport of 2C11 and 2C12 RNA to the cytoplasm and cytoplasmic mRNA stability are unlikely to be important GH-regulated control points for sex-specific P450 RNA expression. Run-on transcription analysis further established that GH regulates the sex-specific expression of the 2C11 and 2C12 genes at the level of transcript initiation. Transcription was also shown to be the major step for regulation of the male-specific P450 2A2 RNA, whose expression, unlike 2C11, is not obligatorily dependent on pulsatile GH release. In vitro footprinting analysis of 2C11 and 2C12 promoter fragments incubated with liver nuclear proteins isolated from rats differing in GH status revealed several sex- and GH-dependent differences in DNase cleavage patterns ("hypersensitivity sites"), demonstrating that GH can regulate specific protein-DNA interactions in the 5'-flanking sequences of these two genes. In vitro transcription assays driven by 2C11 and 2C12 5'-flanking DNA sequences fused to TATAA box-G-less cassette template constructs did not, however, faithfully mimic the sex-specific transcription of the 2C11 and 2C12 genes, indicating that additional cis-elements or trans-acting factors may be required to achieve the transcriptional regulation of these genes that occurs in vivo.

The induction of liver cytochrome P450 4A-catalyzed fatty acid omega-hydroxylase activity by clofibrate and other peroxisome proliferators has been proposed to be causally linked to the ensuing proliferation of peroxisomes in rat liver. Since female rats are less responsive than males to peroxisome proliferation induced by clofibrate, the influence of gender and hormonal status on the basal and clofibrate-inducible expression of the 4A P450s was examined. Northern blot analysis using gene-specific oligonucleotide probes revealed that in the liver, P450 4A1 and 4A3 mRNAs are induced to a much greater extent in male as compared to female rats following clofibrate treatment, whereas P450 4A2 mRNA is altogether absent from female rat liver. Male-specific expression of P450 4A2 mRNA was also observed in kidney. Western blot analysis indicated that a similar sex dependence characterizes both the basal expression and the clofibrate inducibility of the corresponding P450 4A proteins. This suggests that the lower responsiveness of female rats to clofibrate-induced peroxisome proliferation may reflect the lower inducibility of the P450 4A fatty acid hydroxylase enzymes in this sex. Investigation of the contribution of pituitary-dependent hormones to the male-specific expression of 4A2 revealed that this P450 mRNA is fully suppressed in liver following exposure to the continuous plasma growth hormone profile that characterizes adult female rats; in this and other regards liver P450 4A2 is regulated in a manner that is similar, but not identical to, P450 3A2, a male-specific testosterone 6 beta-hydroxylase. In contrast, kidney 4A2 expression, although also male-specific, was not suppressed by continuous growth hormone treatment, but was regulated by pathways that, in part, involve testosterone as a positive regulator. The male-specific expression of liver and kidney P450 4A2 is thus under the control of distinct pituitary-dependent hormones acting in a tissue-specific manner.

Treatment of male rats with the anticancer drug cisplatin leads to feminization of the profile of cytochrome P-450 and other microsomal enzymes involved in steroid hormone and drug metabolism (G.A. LeBlanc, and D.J. Waxman, J. Biol. Chem., 263: 15732-15739, 1988). The present study uses the rat model to evaluate the differential effects of cisplatin treatment on liver microsomal enzymes between genders, and also examines whether the modulation of enzyme activities by cisplatin and its analogues involves changes in P-450 gene expression. While cisplatin treatment of male rats caused a severalfold increase in female-predominant hepatic enzymes, including testosterone 5 alpha-reductase and testosterone 7 alpha-hydroxylase (P-450 form 2A1), it partially decreased the expression of these enzymes in females. The reduced expression of these estrogen-dependent enzymes in females may derive from the loss of circulating estradiol that was shown to occur in response to cisplatin treatment. Analysis of mRNA levels of individual P-450 forms revealed that the effects of cisplatin on P-450-catalyzed steroid hydroxylase activities in both male and female rats are primarily operative through the drug's effects on P-450 mRNA expression. P-450-dependent cyclophosphamide activation was significantly compromised in male rats after cisplatin administration; however, this activity was not altered in cisplatin-treated females. This sex-dependent effect of cisplatin was due to its suppression of P-450 form 2C11, a male-specific P-450 that is a major contributor to microsomal cyclophosphamide bioactivation in male rat liver. The clinically active cisplatin analogue iproplatin elicited effects very similar to those of cisplatin, while carboplatin and transplatin did not have significant effects on hepatic P-450 expression. Together, these findings demonstrate that the response of rat liver to cisplatin-induced changes in hepatic P-450 enzyme profiles and cyclophosphamide bioactivation capacity differs between the sexes, and in addition, these effects can be minimized by use of carboplatin in place of cisplatin.

Cholesterol 7 alpha-hydroxylase (P-450 Ch7 alpha) catalyzes the first and rate-limiting step in the hepatic conversion of cholesterol to bile acids. P-450 Ch7 alpha activity in rat liver is regulated at three independent levels: (a) feedback inhibition by bile acids (long term regulation); (b) midterm regulation through the diurnal cycle; (c) short term modulation by hormones and dietary factors. P-450 Ch7 alpha was purified to apparent homogeneity and in active form (turnover number = 10-15 min-1 P-450(-1)) from cholestyramine-fed female rats, and rabbit anti-P-450 Ch7 alpha polyclonal antibodies were then prepared. Liver microsomes were isolated from rats fed normal diet or diet containing the bile acid sequestrant cholestyramine and were then killed at either the apex (midnight) or nadir (noon) of the diurnal rhythm of P-450 Ch7 alpha activity. Direct comparison of microsomal P-450 Ch7 alpha enzyme activity levels with P-450 Ch7 alpha protein (Western blotting) and mRNA levels (Northern and slot blots) revealed that the 2.5-3-fold induction of P-450 Ch7 alpha activity with cholestyramine feeding can be fully accounted for by an increase in P-450 Ch7 alpha protein and mRNA. Turnover numbers of 7-9 nmol of 7 alpha-hydroxycholesterol/min/nmol of microsomal P-450 Ch7 alpha were observed for both induced and uninduced animals. Similarly, the postmidnight decrease in enzyme activity could be generally accounted for by a decrease in P-450 Ch7 alpha protein and mRNA, suggesting that these species have relatively short half-lives. The short term regulation of P-450 Ch7 alpha was examined following treatment with the cholesterol precursor mevalonic acid. A 2.5-fold increase in hepatic microsomal P-450 Ch7 alpha activity occurred within 150 min and was accompanied by a significant elevation of P-450 Ch7 alpha mRNA (up to 3-6-fold increase). These findings establish that hepatic cholesterol 7 alpha-hydroxylase activity is regulated in response to long term, midterm, and short term control factors primarily at a pretranslational level and that this regulation is of greater importance than proposed mechanisms based on allosteric effects of bile acids on P-450 Ch7 alpha protein, changes in cholesterol availability, or reversible phosphorylation of a putative P-450 Ch7 alpha phosphoprotein.

Oligonucleotide probes that distinguish between two closely related mRNAs encoding steroid 6 beta-hydroxylases of rat P-450 gene family CYP3A were used to individually assess their responsiveness to pituitary hormone regulation. Northern blot analysis revealed that the elevation of immunoreactive P-450 IIIA2 in livers of hypophysectomized rats reflects an elevation of the constitutive, male-specific P-450 IIIA2 (P-450 2a) and not an induction of the drug-inducible P-450 IIIA1 (P-450p). P-450 IIIA2 mRNA levels in intact adult male rats were found to be markedly reduced by GH administered as a continuous infusion at levels as low as 1 mU/h, indicating that GH acts at a pretranslational step to suppress expression of this P-450 enzyme. In hypophysectomized male rats, however, this same hormone treatment was only partially effective at suppressing P-450 IIIA2 mRNA and protein, suggesting that other pituitary-dependent factors contribute to the suppression observed in the intact rats. Further analysis revealed that T4, but not ACTH or human CG, can act in concert with GH to effect a more complete suppression of hepatic P-450 IIIA2 mRNA and protein in hypophysectomized rats. T4 also suppressed the expression of another GH-regulated, male-specific hepatic enzyme, designated P-450 IIA2 (P-450 RLM2), particularly in hypophysectomized female rats. In contrast, the GH-responsive P-450 IIA1 (P-450 3) was much less affected by T4 treatment. Thus, while T4 can modulate P-450 IIIA2 expression, it does not serve as a universal regulator for hepatic expression of GH-responsive P-450s.

Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were essentially free of other proteins as judged by SDS/PAGE. Partial reconstitution of 7-ethoxycoumarin de-ethylase activity was achieved using rabbit NADPH: cytochrome P450 reductase and purified preparations containing P450-B.