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Sphingosine-1-phosphate (S1P) binds to a family of sphingosine-1-phosphate G-protein-coupled receptors (S1P1-5). The interaction of S1P with these S1P receptors has a fundamental role in many physiological processes in the vascular and immune systems. Agonist-induced functional antagonism of S1P1 has been shown to result in lymphopenia. As a result, agonists of this type hold promise as therapeutics for autoimmune disorders. The previously disclosed differentiated S1P1 modulator BMS-986104 (1) exhibited improved preclinical cardiovascular and pulmonary safety profiles as compared to earlier full agonists of S1P1; however, it demonstrated a long pharmacokinetic half-life (T1/2 18 days) in the clinic and limited formation of the desired active phosphate metabolite. Optimization of this series through incorporation of olefins, ethers, thioethers, and glycols into the alkyl side chain afforded an opportunity to reduce the projected human T1/2 and improve the formation of the active phosphate metabolite while maintaining efficacy as well as the improved safety profile. These efforts led to the discovery of 12 and 24, each of which are highly potent, biased agonists of S1P1. These compounds not only exhibited shorter in vivo T1/2 in multiple species but are also projected to have significantly shorter T1/2 values in humans when compared to our first clinical candidate. In models of arthritis, treatment with 12 and 24 demonstrated robust efficacy.

[Image: see text] Efforts aimed at increasing the in vivo potency and reducing the elimination half-life of 1 and 2 led to the identification of aryl ether and thioether-derived bicyclic S1P(1) differentiated modulators 3–6. The effects of analogs 3–6 on lymphocyte reduction in the rat (desired pharmacology) along with pulmonary- and cardiovascular-related effects (undesired pharmacology) are described. Optimization of the overall properties in the aryl ether series yielded 3d, and the predicted margin of safety against the cardiovascular effects of 3d would be large enough for human studies. Importantly, compared to 1 and 2, compound 3d had a better profile in both potency (ED(50) < 0.05 mg/kg) and predicted human half-life (t(1/2) ∼ 5 days).

The kidney plays a critical role in the elimination of many xenobiotics, and drug-induced kidney injury is a risk factor in drug discovery and development. In addition, accumulation of nephrotoxic compounds, a process often controlled by xenobiotic transporters, is often a prerequisite to kidney injury. Such adverse events are dependent on many transporters, particularly those in the solute carrier and adenosine triphosphate-binding cassette superfamilies. This review details the current understanding of how kidney transporters contribute to toxic outcomes and highlights critical knowledge gaps regarding species differences that account for some lack of predictivity between preclinical animal models and human beings. The basic classification, physiological roles, and species differences of solute carrier and adenosine triphosphate-binding cassette transporters is reviewed, along with mechanistic details for drug-induced kidney injury involving transporters. The use of preclinical data (in vitro and in vivo), clinical data, and conventional as well as emerging tools for studying kidney transporter function are summarized. Finally, we highlight some challenges and opportunities to improve experimental approaches to support preclinical and clinical studies of kidney transporters and their role in nephrotoxicity.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life (T1/2) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P1. Herein, we describe our efforts to discover highly potent, partial agonists of S1P1 with a shorter T1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments ar...

Sphingosine-1-phosphate (S1P) binds to a family of sphingosine-1-phosphate G-protein-coupled receptors (S1P

Objective Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD). Methods A principal component analysis for all 91 metabolites measured indicated that metabolome perturbation is greater and less varied for humans than for rodents. Results Metabolome changes in human and rat NAFLD were greatest for the amino acid and bile acid metabolite families (e.g., asparagine, citrulline, gamma-aminobutyric acid, lysine); although, in many cases, the trends were reversed when compared between species (cholic acid, betaine). Conclusions Overall, these results indicate that metabolites of specific pathways may be useful biomarkers for NASH progression, although these markers may not correspond to rodent NASH models. The MCD model may be useful when studying certain end points of NASH; however, the metabolomics results indicate important differences between humans and rodents in the biochemical pathogenesis of the disease.

Recently, our research group reported the identification of prodrug amino-alcohol 2 as a potent and efficacious S1P1 receptor modulator. This molecule is differentiated preclinically over the marketed drug fingolimod (Gilenya 1), whose active phosphate metabolite is an S1P1 full agonist, in terms of pulmonary and cardiovascular safety. S1P1 partial agonist 2, however, has a long half-life in rodents and was projected to have a long half-life in humans. The purpose of this communication is to disclose highly potent partial agonists of S1P1 with shorter half-lives relative to the clinical compound 2. PK/PD relationships as well as their preclinical pulmonary and cardiovascular safety assessment are discussed.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P

We describe a highly efficient route for the synthesis of 4a (BMS-986104). A key step in the synthesis is the asymmetric hydroboration of trisubstituted alkene 6. Particularly given the known difficulties involved in this type of transformation (6 → 7), the current methodology provides an efficient approach to prepare this class of compounds. In addition, we disclose the efficacy of 4a in a mouse EAE model, which is comparable to 4c (FTY720). Mechanistically, 4a exhibited excellent remyelinating effects on lysophosphatidylcholine (LPC) induced demyelination in a three-dimensional brain cell culture assay.

Clinical validation of S1P receptor modulation therapy was achieved with the approval of fingolimod (Gilenya, 1) as the first oral therapy for relapsing remitting multiple sclerosis. However, 1 causes a dose-dependent reduction in the heart rate (bradycardia), which occurs within hours after first dose. We disclose the identification of clinical compound BMS-986104 (3d), a novel S1P1 receptor modulator, which demonstrates ligand-biased signaling and differentiates from 1 in terms of cardiovascular and pulmonary safety based on preclinical pharmacology while showing equivalent efficacy in a T-cell transfer colitis model.

This chapter is focused on the fundamental principles of toxic mechanisms of injury. Toxicity is influenced by the amount of toxicant that reaches the target organ, the reactivity of the compound itself or metabolites that are formed by major biotransformation systems, and the potential for altered cellular function or gene expression. There are many mechanisms and pathways by which toxicity can develop. Fundamental mechanisms of toxicity are presented in this chapter, with examples provided to illustrate the changes as appropriate.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing lung disease with limited effective treatment options. The LPA 1 pathway has been implicated in the etiology and pathogenesis of IPF and is a promising therapeutic target for fibrotic diseases. LPA 1 antagonists, including BMS‑986020 and BMS-986234, are being investigated for IPF. Differences in structure and pharmacology of LPA 1 antagonists could impact their efficacy and safety profile. In a Phase 2 trial, BMS-986020 compared with placebo significantly slowed lung function decline but, in some patients, showed hepatobiliary effects; the mechanisms underlying these effects were investigated in vitro and in vivo. In vitro, BMS-986020 inhibits bile acid and phospholipid transporters, BSEP (IC50=4.8 µM), MRP4 (6.2 µM), and MDR3 (7.5 µM), which may reduce bile acid and phospholipid efflux, and alter bile composition and flow. BMS-986020 altered bile homeostasis in vivo, yielding elevated systemic bile acids in rats and humans. In contrast, a structurally distinct LPA 1 antagonist BMS-986234, at projected clinically relevant concentrations, did not inhibit BSEP (IC50=19.6 µM), MRP4 (>50 µM), or MDR3 (>50 µM) in vitro, or inhibit bile acid efflux in human hepatocytes (≤50 µM). Additionally, BMS-986234 did not increase bile acids in rats or monkeys. In conclusion, the hepatobiliary effects observed with BMS‑986020 are likely off-target effects specific to this molecule and not mediated via antagonism of LPA 1 . These results suggest that structural variations in LPA 1 antagonists may result in different safety profiles in patients with IPF and other fibrotic diseases.

Organic anion transporting polypeptides (human: OATPs; rodent: Oatps) were thought to have important functions in bile acid (BA) transport. Oatp1a1, 1a4, and 1b2 are the three major Oatp1 family members in rodent liver. Our previous studies have characterized the BA homeostasis in Oatp1a1-null and Oatp1b2-null mice. The present study investigated the physiological role of Oatp1a4 in BA homeostasis by using Oatp1a4-null mice. Oatp1a4 expression is female-predominant in livers of mice, and thereby it was expected that female Oatp1a4-null mice will have more prominent changes than males. Interestingly, the present study demonstrated that female Oatp1a4-null mice had no significant alterations in BA concentrations in serum or liver, though they had increased mRNA of hepatic BA efflux transporters (Mrp4 and Ostα/β) and ileal BA transporters (Asbt and Ostα/β). In contrast, male Oatp1a4-null mice showed significantly altered BA homeostasis, including increased concentrations of deoxycholic acid (DCA) in serum, liver and intestinal contents. After feeding a DCA-supplemented diet, male but not female Oatp1a4-null mice had higher concentrations of DCA in serum and livers than their WT controls. This suggested that Oatp1a4 is important for intestinal absorption of secondary BAs in male mice. Furthermore, loss of Oatp1a4 function did not decrease BA accumulation in serum or livers of bile-duct-ligated mice, suggesting that Oatp1a4 is not likely a BA uptake transporter. In summary, the present study for the first time demonstrates that Oatp1a4 does not appear to mediate the hepatic uptake of BAs, but plays an important male-predominant role in secondary BA metabolism in mice.

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 were observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH.

The objective of this work was to investigate the mechanisms of hepatobiliary toxicity caused by thienopyrimidone MCHR1 antagonists using BMS-773174 as a tool molecule. Co-administration of the pan CYP inhibitor 1-aminobenzotriazole with BMS-773174 prevented hepatobiliary damage, and direct delivery of the diol metabolite BMS-769750 caused hepatobiliary toxicity, identifying the diol and possibly its downstream hydroxyacid (BMS-800754) metabolite as the toxic species. Rat liver gene expression revealed treatment-related changes in hepatic transporters and induction of oval cell-specific genes including deleted malignant tumor 1 (Dmbt1). The metabolites did not alter hepatic transporter activities, suggesting that transporter-mediated cholestasis was not involved. Because injury to biliary epithelium can result in adaptive hyperplasia, rat biliary epithelial cells (BECs) were isolated and exposed to the oxidative metabolites. BMS-769750 was cytotoxic to BECs, but not rat hepatocytes, suggesting a role of the diol in biliary epithelial injury. BMS-800754 was cytotoxic to rat hepatocytes therefore its contribution to hepatocyte injury in rats is a possibility. Induction of Dmbt1 in rat BECs was investigated because of its role in hepatic progenitor cell differentiation/proliferation during injury. Dmbt1 mRNA was induced by BMS-769750, but not BMS-800754 in BECs; this induction and cellular injury was confirmed with diol metabolites formed by other compounds with the same hepatobiliary liability. In conclusion, hepatobiliary injury by thienopyrimidinone MCHR1 antagonists was driven through a CYP-mediated bioactivation pathway. Induction of Dmbt1 mRNA coupled with cellular injury suggests that injury of biliary epithelium may be the first step toward an adaptive proliferative response causing BDH by these compounds.

We describe a highly efficient route for the synthesis of 4a (BMS-986104). A key step in the synthesis is the asymmetric hydroboration of trisubstituted alkene 6. Particularly given the known difficulties involved in this type of transformation (6 → 7), the current methodology provides an efficient approach to prepare this class of compounds. In addition, we disclose the efficacy of 4a in a mouse EAE model, which is comparable to 4c (FTY720). Mechanistically, 4a exhibited excellent remyelinating effects on lysophosphatidylcholine (LPC) induced demyelination in a three-dimensional brain cell culture assay.

LC-MS/MS has been widely applied to the quantitative analysis of tissue samples. However, one key remaining issue is that the extraction recovery of analyte from spiked tissue calibration standard and quality control samples (QCs) may not accurately represent the “true” recovery of analyte from incurred tissue samples. This may affect the accuracy of LC-MS/MS tissue bioanalysis. Here, we investigated whether the recovery determined using tissue QCs by LC-MS/MS can accurately represent the “true” recovery from incurred tissue samples using two model compounds: BMS-986104, a S1P1 receptor modulator drug candidate, and its phosphate metabolite, BMS-986104-P. We first developed a novel acid and surfactant assisted protein precipitation method for the extraction of BMS-986104 and BMS-986104-P from rat tissues, and determined their recoveries using tissue QCs by LC-MS/MS. We then used radioactive incurred samples from rats dosed with 3H-labeled BMS-986104 to determine the absolute total radioactivity recovery in six different tissues. The recoveries determined using tissue QCs and incurred samples matched with each other very well. The results demonstrated that, in this assay, tissue QCs accurately represented the incurred tissue samples to determine the “true” recovery, and LC-MS/MS assay was accurate for tissue bioanalysis. Another aspect we investigated is how the tissue QCs should be prepared to better represent the incurred tissue samples. We compared two different QC preparation methods (analyte spiked in tissue homogenates or in intact tissues) and demonstrated that the two methods had no significant difference when a good sample preparation was in place. The developed assay showed excellent accuracy and precision, and was successfully applied to the quantitative determination of BMS-986104 and BMS-986104-P in tissues in a rat toxicology study.

Objective Characteristic pathologic changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH), and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in the human condition, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD). Methods A principal components analysis for all 91 metabolites measured indicates that metabolome perturbation is greater and less varied for humans than for rodents. Results Metabolome changes in human and rat NAFLD were greatest for the amino acid and bile acid metabolite families (e.g. asparagine, citrulline, GABA, lysine); although, in many cases, the trends were reversed when compared between species (cholic acid, betaine). Conclusions Overall, these results indicate that metabolites of specific pathways may be useful biomarkers for NASH progression, although these markers may not correspond to rodent NASH models. The MCD model may be useful when studying certain endpoints of NASH; however, the metabolomics results indicate important differences between humans and rodents in the biochemical pathogenesis of the disease.

The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values22 µM were categorized as teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.

Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in livers of mice and is thought to transport bile acids (BAs) from blood into liver. Because Oatp1a1 expression is markedly decreased in mice after bile duct ligation (BDL). We hypothesized that Oatp1a1-null mice would be protected against liver injury during BDL-induced cholestasis due largely to reduced hepatic uptake of BAs. To evaluate this hypothesis, BDL surgeries were performed in both male wild-type (WT) and Oatp1a1-null mice. At 24 h after BDL, Oatp1a1-null mice showed higher serum alanine aminotransferase levels and more severe liver injury than WT mice, and all Oatp1a1-null mice died within 4 days after BDL, whereas all WT mice survived. At 24 h after BDL, surprisingly Oatp1a1-null mice had higher total BA concentrations in livers than WT mice, suggesting that loss of Oatp1a1 did not prevent BA accumulation in the liver. In addition, secondary BAs dramatically increased in serum of Oatp1a1-null BDL mice but not in WT BDL mice. Oatp1a1-null BDL mice had similar basolateral BA uptake (Na(+)-taurocholate cotransporting polypeptide and Oatp1b2) and BA-efflux (multidrug resistance-associated protein [Mrp]-3, Mrp4, and organic solute transporter α/β) transporters, as well as BA-synthetic enzyme (Cyp7a1) in livers as WT BDL mice. Hepatic expression of small heterodimer partner Cyp3a11, Cyp4a14, and Nqo1, which are target genes of farnesoid X receptor, pregnane X receptor, peroxisome proliferator-activated receptor alpha, and NF-E2-related factor 2, respectively, were increased in WT BDL mice but not in Oatp1a1-null BDL mice. These results demonstrate that loss of Oatp1a1 function exacerbates cholestatic liver injury in mice and suggest that Oatp1a1 plays a unique role in liver adaptive responses to obstructive cholestasis.

BDE47, BDE99 and BDE153 are the predominant polybrominated diphenyl ether (PBDE) congeners detected in humans and can induce drug metabolizing enzymes in the liver. We have previously demonstrated that several human liver organic anion transporting polypeptides (humans: OATPs; rodents: Oatps) can transport PBDE congeners. Mice are commonly used to study the toxicity of chemicals like the PBDE congeners. However, the mechanism of the hepatic PBDE uptake in mice is not known. Therefore, the purpose of the current study was to test the hypothesis that BDE47, BDE99, and BDE153 are substrates of mouse hepatic Oatps (Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1). We used Human Embryonic Kidney 293 (HEK293) cells transiently expressing individual Oatps and quantified the uptake of BDE47, BDE99, and BDE153. Oatp1a4, Oatp1b2, and Oatp2b1 transported all three PBDE congeners, whereas Oatp1a1 did transport none. Kinetic studies demonstrated that Oatp1a4 and Oatp1b2 transported BDE47 with the greatest affinity, followed by BDE99 and BDE153. In contrast, Oatp2b1 transported all three PBDE congeners with similar affinities. The importance of hepatic Oatps for the liver accumulation of BDE47 was confirmed using Oatp1a4-, and Oatp1b2-null mice.

Nuclear magnetic resonance (NMR)-based metabolomic profiling identified urinary 1- and 3-methylhistidine (1- and 3-MH) as potential biomarkers of skeletal muscle toxicity in Sprague-Dawley rats following 7 and 14 daily doses of 0.5 or 1mg/kg cerivastatin. These metabolites were highly correlated to sex-, dose- and time-dependent development of cerivastatin-induced myotoxicity. Subsequently, the distribution and concentration of 1- and 3-MH were quantified in 18 tissues by gas chromatography-mass spectrometry. The methylhistidine isomers were most abundant in skeletal muscle with no fiber or sex differences observed; however, 3-MH was also present in cardiac and smooth muscle. In a second study, rats receiving 14 daily doses of 1mg/kg cerivastatin (a myotoxic dose) had 6- and 2-fold elevations in 1- and 3-MH in urine and had 11- and 3-fold increases in 1- and 3-MH in serum, respectively. Selectivity of these potential biomarkers was tested by dosing rats with the cardiotoxicant isoproterenol (0.5mg/kg), and a 2-fold decrease in urinary 1- and 3-MH was observed and attributed to the anabolic effect on skeletal muscle. These findings indicate that 1- and 3-MH may be useful urine and serum biomarkers of drug-induced skeletal muscle toxicity and hypertrophy in the rat, and further investigation into their use and limitations is warranted.

In the present study, NMR-based urinary metabonomic profiles resulting from dosing with widely recognized microsomal enzyme inducers were evaluated in male rats. Wistar or Sprague–Dawley rats were dosed daily by oral gavage with phenobarbital (PB; 100 mg/kg), diallyl sulfide (DAS; 500 mg/kg), the investigational compound DMP-904 (150 mg/kg), or β-naphthoflavone (BNF; 100 mg/kg) for 4 days, and urine was collected daily for analysis. Compounds known to increase cytochrome P450 2B enzymes, including PB, DAS and DMP-904, increased the urinary excretion of gulonic and ascorbic acid in a time-dependent manner, reaching a maximum following 3–4 days of dosing. In contrast, BNF, an agent that induces primarily Cyp1A enzymes, did not increase gulonic or ascorbic acid excretion, despite inducing Cyp1A1 more than 200-fold. Given the metabonomic results, hepatic transcriptional changes in the regulation of ascorbic acid biosynthesis were determined by RT-PCR. All Cyp2B inducers increased hepatic mRNA levels of aldo-keto reductase 1A1, an enzyme that catalyzes the formation of gulonic acid from glucuronate with concurrent decreased expression of both regucalcin (Rgn), the enzyme responsible for conversion of gulonic acid to gulono-1, 4-lactone and gulonolactone oxidase (Gulo), the rate-limiting enzyme in ascorbate biosynthesis. These effects would be expected to increase levels of gulonic acid. In addition, Cyp2B inducers also increased hepatic expression of enzymes regulating ascorbic acid reutilization including glutaredoxin reductase (Glrx2) and thioredoxin reductase (Txnrd1). In contrast, BNF did not effect hepatic expression of any enzyme regulating gulonic or ascorbic acid biosynthesis. Thus, some microsomal enzyme inducers alter transcriptional regulation of ascorbic acid biosynthesis, and these changes are detected by noninvasive metabonomic profiling. However, not all microsomal enzyme inducers appear to alter ascorbic acid metabolism. Finally, the work illustrates how metabonomic results can direct additional studies to determine the biochemical mechanisms underlying changes in urinary metabolite excretion. Copyright © 2009 John Wiley & Sons, Ltd.

Humans and ecological species have been found to have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotypic effects similar to peroxisome proliferator chemicals (PPC) that work through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha). Recent studies indicate that along with PPAR alpha other nuclear receptors are required for transcriptional changes in the mouse liver after PFOA exposure including the constitutive activated receptor (CAR) and pregnane X receptor (PXR) that regulate xenobiotic metabolizing enzymes (XME). To determine the potential role of CAR/PXR in mediating effects of PFAAs in rat liver, we performed a meta-analysis of transcript profiles from published studies in which rats were exposed to PFOA or PFOS. We compared the profiles to those produced by exposure to prototypical activators of CAR, (phenobarbital (PB)), PXR (pregnenolone 16 alpha-carbonitrile (PCN)), or PPAR alpha (WY-14,643 (WY)). As expected, PFOA and PFOS elicited transcript profile signatures that included many known PPAR alpha target genes. Numerous XME genes were also altered by PFOA and PFOS but not WY. These genes exhibited expression changes shared with PB or PCN. Reexamination of the transcript profiles from the livers of chicken or fish exposed to PFAAs indicated that PPAR alpha, CAR, and PXR orthologs were not activated. Our results indicate that PFAAs under these experimental conditions activate PPAR alpha, CAR, and PXR in rats but not chicken and fish. Lastly, we discuss evidence that human populations with greater CAR expression have lower body burdens of PFAAs.

The hepatobiliary disposition of thyroxine (T4) was evaluated in Groningen Yellow transport deficient (TR(-)) rats lacking functional multidrug resistance-associated protein 2 (Mrp2; Abcc2). Male Wistar and TR(-) rats were dosed orally (4 days) with phenobarbital (PB; 100 mg/kg) or DMP 904 (200 mg/kg), after which T4 homeostasis and hepatic cytochromes P450, UDP-glucuronosyltransferase, xenobiotic transporters, and T4 glucuronidation were determined. Serum concentrations of T4 were approximately 50% higher in control TR(-) rats than Wistars. PB and DMP 904 increased hepatic levels of P450s and T4-glucuronidation (T4-G), and these changes were associated with decreased serum T4 levels in both strains. In Wistar but not TR(-) rats, DMP 904 increased thyroid stimulating hormone levels twofold. Hepatobiliary clearance of T4 was determined after intravenous infusion of [(125)I]T4 to rats dosed with PB and DMP 904 (4 days). PB and DMP 904 increased plasma clearance and hepatic uptake of [(125)I]T4 equivalents in Wistar but not TR(-) rats. Total biliary clearance (Cl(bile)) was approximately 0.85 and 0.2 ml/h in Wistar and TR(-) rats, respectively, with virtually no T4-G excreted in bile in TR(-) rats. Biliary clearance of unconjugated T4 was also lower in control TR(-) rats than in Wistars, although DMP 904 increased its biliary clearance in both strains. These results suggest that Mrp2 is likely to be responsible for biliary excretion of T4-G and contributes in part to excretion of T4. Decreased biliary clearance of T4 and metabolites in TR(-) rats mitigated but did not prevent drug-induced changes in serum T4, suggesting that other factors contribute to changes in T4 homeostasis in these rats.

Robust pharmaceutical treatment of obesity has been limited by the undesirable side-effect profile of currently marketed therapies. This paper describes the synthesis and optimization of a new class of pyrazinoisoindolone-containing, selective 5-HT2C agonists as antiobesity agents. Key to optimization of the pyrazinoisoindolone core was the identification of the appropriate substitution pattern and functional groups which led to the discovery of (R)-9-ethyl-1,3,4,10b-tetrahydro-7-trifluoromethylpyrazino[2,1-a]isoindol-6(2H)-one (58), a 5-HT2C agonist with >300-fold functional selectivity over 5-HT2B and >70-fold functional selectivity over 5-HT2A. Oral dosing of 58 reduced food intake in an acute rat feeding model, which could be completely reversed by a selective 5-HT2C antagonist and caused a reduction in body weight gain in a 4-day rat model.

Murine double minute 2 (Mdm2) negatively regulates p53 by mediating its ubiquitination and proteosomal degradation, and Mdm2 is recognized as a proto-oncogene. In the present study, hepatic gene expression patterns induced by phenobarbital (PB; 100 mg/kg) and pregnenolone 16alpha-carbonitrile (PCN, 100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.

Rats cotreated with lipopolysaccharide (LPS) and ranitidine (RAN) but not LPS and famotidine (FAM) develop hepatocellular injury in an animal model of idiosyncratic drug reactions. Evaluation of liver gene expression in rats given LPS and/or RAN led to confirmation that the hemostatic system, hypoxia, and neutrophils (PMNs) are critical mediators in LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression changes distinguish LPS/RAN-treated rats from rats given LPS or RAN alone and from those cotreated with LPS/FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg, iv) or its vehicle. Two hours thereafter they were given RAN (30 mg/kg, iv), FAM (either 6 mg/kg, a pharmacologically equi-efficacious dose, or 28.8 mg/kg, an equimolar dose, iv), or vehicle. They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity (2 and 6 h) and liver gene expression (2 h only). At a time before the onset of hepatocellular injury, hierarchical clustering distinguished rats treated with LPS/RAN from those given LPS alone. 205 probesets were expressed differentially to a greater or lesser degree only in LPS/RAN-treated rats compared to LPS/FAM or LPS alone, which did not develop liver injury. These included VEGF, EGLN3, MAPKAPK-2, BNIP3, MIP-2, COX-2, EGR-1, PAI-1, IFN-gamma, and IL-6. Expression of these genes was confirmed by real-time PCR. Serum concentrations of MIP-2, PAI-1, IFN-gamma, and IL-6 correlated with their respective gene expression patterns. Overall, the expression of several gene products capable of controlling requisite mediators of injury (i.e., hemostasis, hypoxia, PMNs) in this model were enhanced in livers of LPS/RAN-treated rats. Furthermore, enhanced expression of MAPKAPK-2 in RAN-treated rats and its target genes in LPS/RAN-treated rats suggests that p38/MAPKAPK-2 signaling is a regulation point for enhancement of LPS-induced gene expression by RAN.

In an animal model of drug idiosyncrasy, rats cotreated with nonhepatotoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) develop hepatocellular injury, whereas rats treated with LPS and famotidine (FAM) do not. The coagulation system and neutrophils (PMNs) are requisite mediators of LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression in LPS/RAN-treated rats requires coagulation system activation and that these changes are absent in rats given LPS and FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg i.v.) or its vehicle, and then 1 h later, they were treated with heparin (3000 U/kg) or its vehicle. One hour thereafter, they were given RAN (30 mg/kg), FAM (6 mg/kg, a pharmacologically equiefficacious dose, or 28.8 mg/kg, an equimolar dose), or vehicle (i.v.). They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity, coagulation system activation, and liver gene expression (2 h only). Statistical filtering of gene array results and real-time polymerase chain reaction identified groups of genes expressed in LPS/RAN-treated rats but not LPS/FAM-treated rats that were either changed or unchanged by heparin administration. For example, LPS/RAN-induced mRNA expression of the inflammatory mediators interleukin-6, cyclooxygenase-2, and macrophage inflammatory protein-2 (MIP-2) was reduced by anticoagulation. Enhancement of serum MIP-2 and plasminogen activator inhibitor-1 concentrations in LPS/RAN-treated rats was prevented by anticoagulation. The results suggest cross-talk between hemostasis-induced gene expression and inflammation (e.g., PMN function) in the genesis of hepatocellular injury in LPS/RAN-treated rats. In contrast, neither the expression of such genes nor hepatocellular necrosis occurred in rats treated with LPS/FAM.

Hepatotoxicity of coumarin is attributed to metabolic activation to an epoxide intermediate, coumarin 3,4-epoxide (CE). However, whereas rats are most susceptible to coumarin-induced hepatotoxicity, formation of CE is greatest in mouse liver microsomes, a species showing little evidence of hepatotoxicity. Therefore, the present work was designed to test the hypothesis that detoxification of CE is a major determinant of coumarin hepatotoxicity. CE can either rearrange spontaneously to o-hydroxyphenylacetaldehyde (o-HPA) or be conjugated with gluatathione (GSH). o-HPA is hepatotoxic and is further detoxified by oxidation to o-hydroxyphenylacetic acid (o-HPAA). In vitro experiments were conducted using mouse liver microsomes to generate a constant amount of CE, and cytosols from F344 rats, B6C3F1 mice, and human liver were used to characterize CE detoxification. All metabolites were quantified by HPLC methods with UV detection. In rats and mice, GSH conjugation occurred non-enzymatically and through glutathione-S-transferases (GSTs), and the kinetics of GSH conjugation were similar in rats and mice. In rat liver cytosol, oxidation of o-HPA to o-HPAA was characterized with a high affinity K(m) of approximately 12 microM, and a V(max) of approximately 1.5 nmol/min/mg protein. In contrast, the K(m) and V(max) for o-HPA oxidation in mouse liver cytosol were approximately 1.7 microM and 5 nmol/min/mg protein, respectively, yielding a total intrinsic clearance through oxidation to o-HPAA that was 20 times higher in mouse than in rats. Human cytosols (two separate pools) detoxified CE through o-HPA oxidation with an apparent K(m) of 0.84 microM and a V(max) of 5.7 nmol/min/mg protein, for a net intrinsic clearance that was more than 50 times higher than the rat. All species also reduced o-HPA to o-hydroxyphenylethanol (o-HPE), but this was only a major reaction in rats. In the presence of a metabolic reaction replete with all necessary cofactors, GSH conjugation accounted for nearly half of all CE metabolites in rat and mouse, whereas the GSH conjugate represented only 10% of the metabolites in human cytosol. In mouse, o-HPAA represented the major ring-opened metabolite, accounting for the remaining 50% of metabolites, and in human cytosol, o-HPAA was the major metabolite, representing nearly 90% of all CE metabolites. In contrast, no o-HPAA was detected in rats, whereas o-HPE represented a major metabolite. Collectively, these in vitro data implicate o-HPA detoxification through oxidation to o-HPAA as the major determinant of species differences in coumarin-induced hepatotoxicity.

Immunohistochemical methods have been widely used to determine the histogenesis of spontaneous and chemically-induced mouse lung tumors. Typically, antigens for either alveolar Type II cells or bronchiolar epithelial Clara cells are studied. In the present work, the morphological and immunohistochemical phenotype of a transgenic mouse designed to develop lung tumors arising from Clara cells was evaluated. In this model, Clara cell-specific transformation is accomplished by directed expression of the SV40 large T antigen (TAg) under the mouse Clara cell secretory protein (CC10) promoter. In heterozygous mice, early lesions at 1 month of age consisted of hyperplastic bronchiolar epithelial cells. These progressed to adenoma by 2 months as proliferating epithelium extended into adjacent alveolar spaces. By 4 months, a large portion of the lung parenchyma was composed of tumor masses. Expression of constitutive CC10 was diminished in transgenic animals at all time points. Only the occasional cell or segment of the bronchiolar epithelium stained positively for CC10 by immunohistochemistry, and all tumors were found to be uniformly negative for staining. These results were corroborated by Western blotting, where CC10 was readily detectable in whole lung homogenate from nontransgenic animals, but not detected in lung from transgenic animals at any time point. Tumors were also examined for expression of surfactant apoprotein C (SPC), an alveolar Type II cell-specific marker, and found to be uniformly negative for staining. These results indicate that, in this transgenic model, expression of CC10, which is widely used to determine whether lung tumors arise from Clara cells, was reduced and subsequently lost during Clara cell tumor progression.

The human relevance framework (HRF) outlines a four-part process, beginning with data on the mode of action (MOA) in laboratory animals, for evaluating the human relevance of animal tumors. Drawing on U.S. EPA and IPCS proposals for animal MOA analysis, the HRF expands those analyses to include a systematic evaluation of comparability, or lack of comparability, between the postulated animal MOA and related information from human data sources. The HRF evolved through a series of case studies representing several different MOAs. HRF analyses produced divergent outcomes, some leading to complete risk assessment and others discontinuing the process, according to the data available from animal and human sources. Two case examples call for complete risk assessments. One is the default: When data are insufficient to confidently postulate a MOA for test animals, the animal tumor data are presumed to be relevant for risk assessment and a complete risk assessment is necessary. The other is the product of a data-based finding that the animal MOA is relevant to humans. For the specific MOA and endpoint combinations studied for this article, full risk assessments are necessary for potentially relevant MOAs involving cytotoxicity and cell proliferation in animals and humans (Case Study 6, chloroform) and formation of urinary-tract calculi (Case Study 7, melamine). In other circumstances, when data-based findings for the chemical and endpoint combination studied indicate that the tumor-related animal MOA is unlikely to have a human counterpart, there is little reason to continue the risk assessment for that combination. Similarly, when qualitative considerations identify MOAs specific to the test species or quantitative considerations indicate that the animal MOA is unlikely to occur in humans, such hazard findings are generally conclusive and further risk assessment is not necessary for the endpoint-MOA combination under study. Case examples include a tumor-related protein specific to test animals (Case Study 3, d-limonene), the tumor consequences of hormone suppression typical of laboratory animals but not humans (Case Study 4, atrazine), and chemical-related enhanced hormone clearance rates in animals relative to humans (Case Study 5, phenobarbital). The human relevance analysis is highly specific for the chemical-MOA-tissue-endpoint combination under analysis in any particular case: different tissues, different endpoints, or alternative MOAs for a given chemical may result in different human relevance findings. By providing a systematic approach to using MOA data, the HRF offers a new tool for the scientific community's overall effort to enhance the predictive power, reliability and transparency of cancer risk assessment.

Incorporating mechanistic data into risk assessment is intended to improve the overall scientific validity, and thereby reduce the uncertainty, in human risk analysis. Relevant mechanistic data provides a link between the molecular and cellular event(s) and adverse outcome, and insight into mechanism of action is critical for accurate evaluation of dose-response relationships and inter-species extrapolation. This paper provides an example of applying mechanistic data to human risk assessment using the frameworks outlined by the International Programme on Chemical Safety (IPCS) and the U.S. Environmental Protection Agency (USEPA). Key components of the data are used to address the strength and consistency of the mechanism and to apply these data to human risk characterization.

Coumarin, a natural product and fragrance ingredient, is a well recognized rat liver toxicant, and dietary administration at toxic dosages increased the incidence of rat cholangiocarcinomas and parenchymal liver-cell tumors in a chronic bioassay. Hepatotoxicity in rats is site- and species-specific, and is thought to result from the formation of coumarin 3,4-epoxide and its rearrangement product, o-hydroxyphenylacetaldehyde (o-HPA). The goals of the current study were to describe the in vitro kinetics of the metabolic activation of coumarin, and determine whether species differences in susceptibility to liver injury correlate with coumarin bioactivation determined in vitro. Coumarin 3,4-epoxidation was quantified via the formation of o-HPA in pooled hepatic microsomes from female B6C3F1 mice, male F344 rats, and individual humans (n = 12 subjects), and the apparent kinetic constants for o-HPA production were calculated using nonlinear regression and fitting to either a one-enzyme or two-enzyme model. Eadie-Hofstee analyses indicated that o-HPA formation was biphasic in both rat and mouse liver. Although the apparent high affinity K:(m) in rat and mouse liver microsomes was 38.9 and 47.2 microM, respectively, the overall rate of o-HPA formation was far greater in mouse than in rat liver microsomes. Furthermore, the total clearance (CL(int)) of coumarin via o-HPA formation in mouse liver microsomes was 4-fold greater than in rat liver microsomes. Since mice are relatively resistant to hepatotoxicity, the data indicated that rates of o-HPA formation in rat and mouse liver microsomes were not directly predictive of liver toxicity in vivo, and further suggested that o-HPA detoxification played a role in modulating coumarin-mediated toxicity. The current studies also indicated that coumarin 3,4-epoxidation in human hepatic microsomes was minimal. In human liver microsomes (n = 12), the kinetics of o-HPA formation were best described by a single enzyme model, with the K(m) for o-HPA formation ranging from 1320-7420 microM. In the most active human sample, the intrinsic clearance of coumarin via the 3,4-epoxidation pathway was 1/9 and 1/38 that of the rat and mouse, respectively. The in vitro kinetics of o-HPA formation, and in particular, the large quantities of coumarin required for o-HPA production in human liver microsomes, strongly suggest that humans are unlikely to produce toxicologically relevant concentrations of this metabolite following low level coumarin exposures.

Diethanolamine (DEA), an alkanolamine used widely in industry, is hepatocarcinogenic in mice. The goal of this work was to determine whether DEA altered choline homeostasis in cultured cells, so as to ascertain whether the liver tumor response may be related to choline deficiency. CHO cells were cultured in Ham's F-12 medium containing DEA (0-1000 microgram/ml) and [(33)P]-phosphorus was used to label phospholipid pools. After 48 hours incubation, lipids were extracted and [(33)P]-labeled phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) accounted for 51 +/- 0.7% of the total lipid (33)P incorporation. DEA had no effect on cell number or total phospholipid biosynthesis, but it significantly decreased the incorporation of (33)P into PC at concentrations >/=50 microgram/ml. DEA (>/=20 microgram/ml) also inhibited the uptake of [(3)H]-choline into CHO cells, with 95% inhibition observed at 250 microgram/ml. To determine whether supplemental choline prevented PC synthesis inhibition by DEA, CHO cells were cultured with or without excess choline (30 mM) and DEA (500 microgram/ml). DEA reduced PC synthesis to 27 +/- 3% of total phospholipids, but had no effect on PC synthesis in choline-supplemented cells. When [(14)C]-DEA was incubated with CHO cells, it was also incorporated into the phospholipid fraction. Collectively, these results indicate that DEA reversibly inhibits PC synthesis by blocking choline uptake and competing for utilization in the CDP-choline pathway in CHO cells.

Coumarin is a known hepatotoxicant in laboratory animals, particularly rats. However, the mouse lung was identified as a major target organ in a chronic bioassay, with an oral gavage dosage of 200 mg/kg coumarin increasing the incidence of alveolar/bronchiolar adenomas and carcinomas. The purpose of the present work was to determine whether coumarin was acutely toxic in the mouse and rat lung. Male and female B6C3F1 mice were dosed orally by gavage with coumarin at 0, 10, 20, 50, 100, 150, and 200 mg/kg and lung toxicity was determined 24 h later by histological evaluation. The results indicated that coumarin dosages >/= 150 mg/kg caused selective injury to Clara cells in the distal bronchiolar epithelium. The time course of this injury was studied from 6 h to 7 days after a single dosage of coumarin (200 mg/kg). At 12 h after dosing, Clara cell swelling was apparent along with the onset of necrosis and bronchiolar epithelial disorganization. At 24-48 h, necrotic Clara cells were observed sloughed into the lumens of the terminal bronchioles, with concomitant thinning of the epithelium and flattening of the remaining ciliated cells. By 72-96 h, there was epithelial hypertrophy and hyperplasia, and by 7 days after dosing, the Clara cells had regenerated and the bronchiolar epithelial architecture appeared nearly normal. Unlike the mouse, oral administration of coumarin (200 mg/kg) caused severe hepatotoxicity in male F344 rats, seen histologically as centrilobular necrosis and associated with increases, up to 140-fold, in serum ALT, AST, and SDH levels. Clara cell toxicity was not observed in the distal bronchioles of treated rats. However, in the upper airways, coumarin treatment produced generalized epithelial necrosis involving both ciliated and nonciliated cells. 3,4-Dihydrocoumarin (DHC), which is not a mouse lung carcinogen, did not cause Clara cell injury when dosed to mice at 800 mg/kg. This finding suggests, because DHC lacks a 3,4-double bond, that bioactivation of coumarin to a 3,4-epoxide intermediate may contribute to mouse lung Clara cell toxicity. Collectively, the results indicate that coumarin is a Clara cell toxicant and establish the mouse lung as a target organ for coumarin toxicity. These new findings lay the foundation for studies to determine the mechanisms of coumarin-induced toxicity and carcincogenicity and to define the relevance of these effects to humans.

Mouse urinary protein (MUP) and alpha 2u-globulin are structurally homologous proteins that belong to a superfamily of ligand-binding proteins and represent the major urinary proteins excreted by adult male mice and rats, respectively. Although a variety of xenobiotics bind to alpha 2u-globulin and produce a male rat-specific hyaline droplet nephropathy, no endogenous ligand for this protein has been identified. Despite extensive sequence homology. MUP does not bind to hyaline droplet-inducing agents. While performing experiments with purified MUP, we observed that it presented with a strong, distinctive odor reminiscent of mouse urine. To determine whether this odor was the result of contamination or degradation or was attributed to an endogenous ligand bound to the protein, the protein was subjected to thermal desorption and any released volatile compounds were detected with a gas chromatograph equipped with an external sniff port and mass spectrometer. With this approach, two odorous compounds were detected at the sniff port by a human observer, but only one was present in sufficient mass to allow identification. This compound, which presented with the characteristic odor, was subsequently identified as 2-sec butyl-4,5-dihydrothiazole (DHT) by GC/MS/matrix isolation IR and NMR analyses. The identification of DHT was confirmed by comparing the chromatographic and spectral properties to those of the synthesized authentic compound. In direct contrast, purified urinary alpha 2u-globulin did not present with an obvious odor, and no volatile ligands were detected on this protein. Although DHT is a major endogenous ligand for MUP, it was also found to competitively inhibit the binding of [14C]d-limonene-1,2-epoxide to alpha 2u-globulin with relatively high affinity (Ki = 2.3 microM). When dosed orally to F344 rats, DHT (1 mmol/kg for 3 days) caused the characteristic exacerbation of hyaline droplets in male rat kidneys and increased renal levels of immunoreactive alpha 2u-globulin about threefold over control levels. These results indicate that despite structural homology, MUP and alpha 2u-globulin are distinguished by the presence of a volatile endogenous ligand only on the former, a distinction that may reflect differences in the physiological functions of the two proteins. Furthermore, although DHT can bind to both MUP and alpha 2u-globulin, renal toxicity was only observed in rats, thereby emphasizing the unique toxicological properties of alpha 2u-globulin in the development of hyaline droplet nephropathy.

Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone), are chemicals used as perfume ingredients in household products, cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene), another nitromusk, is not genotoxic but has been reported to produce mouse liver tumors in a chronic bioassay. In addition, MX has been shown to both induce and inhibit mouse liver cytochrome P450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2B enzyme activity is attributable to inactivation of the enzyme by a specific amine metabolite. MK is structurally similar to MX, but lacks the nitro substitution that is reduced to the inactivating amine metabolite. Therefore, we hypothesized that MK would induce, but not inhibit, CYP2B isozymes. To test this hypothesis, and to evaluate the effects of MK on mouse liver cytochrome P450 enzymes, two sets of experiments were performed. To evaluate the ability of MK to induce cytochromes P450, mice were dosed daily by oral gavage at dosages ranging from 5 to 500 mg/ kg MK for 7 days. This treatment resulted in a pleiotropic response in mouse liver, including increased liver weight, increased total microsomal protein, and centrilobular hepatocellular hypertrophy. At the highest dose tested, MK caused a 28-fold increase in CYP2B enzyme activity and a small (approximately 2-fold) increase in both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzyme activities over control levels. Protein and mRNA analyses confirmed the relative levels of induction for CYP2B, CYP1A, and CYP3A. In addition, the no-observable-effect level (NOEL) for CYP2B induction by MK was 20 mg/kg. To evaluate the ability of MK to inhibit phenobarbital-induced CYP2B activity, mice were given 500 ppm phenobarbital (PB) in the drinking water for 5 days to induce CYP2B isozymes, followed by a single equimolar (0.67 mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200 mg/kg), and microsomes were prepared 18 h later. While MX inhibited more than 90% of the PB-induced CYP2B activity in the microsomes, MK caused only a small (about 20%) reduction in PB-induced CYP2B enzyme activity. These results indicate that, like MX. MK is a PB-type inducer of mouse liver CYP2B isozymes, but unlike MX, MK does not effectively inhibit PB-induced CYP2B enzyme activity.

Musk xylene (MX) is a synthetic nitromusk perfume ingredient that, although uniformly negative in genotoxicity testing, causes liver tumors in B6C3F1 mice. MX is also capable of inducing cytochrome P450 enzymes in a manner similar to that of phenobarbital (PB), which suggests that epigenetic mechanisms may be involved in the carcinogenic response. At the same time, MX is metabolized in vivo by nitroreduction, a reaction catalyzed by intestinal flora that yields aromatic amine metabolites. These amine metabolites are also capable of inactivating CYP2B10, the major cytochrome P450 enzyme induced by MX treatment. In the study reported here, the monoamine metabolites of MX, o- and p-NH2-MX, were evaluated for their potential to induce CYP2B10 and CYP1A2 mRNAs. Northern blot analyses indicated that both amines markedly induced CYP2B10 mRNA, whereas CYP1A2 mRNA, the enzyme implicated in the bioactivation of aromatic amines and frequently induced by aromatic amines, was induced only slightly, a response that was not different from that seen with PB. Induction of CYP2B10 mRNA suggested that the amine metabolites may contribute to the enzyme induction profile seen with MX treatment. To test this hypothesis, mice were treated with broad-spectrum antibiotics (neomycin, tetracycline, and bacitracin) to eliminate the intestinal flora and prevent formation of o- and p-NH2-MX. In antibiotic-treated mice treated with MX (200 mg/kg) for 4 d, no evidence of microsomal enzyme induction was observed, including no increases in liver weight, total cytochrome P450 content, or CYP2B protein levels. These results indicate that the amine metabolites of MX are responsible for the enzyme induction seen after MX administration. Thus, the biochemical and molecular effects of amine metabolites of MX are markedly different from those of other aromatic amines but very similar to those of PB. Therefore, it appears that MX is a non-genotoxic chemical that may cause mouse liver tumors in a manner analogous to that of PB.

A reproducible, sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify hydroxytestosterone metabolites. The method features a simple one-step linear gradient of methanol in water (10%–60% methanol) for separation of the testosterone metabolites on a Supelcosil LC-18 column; metabolites are detected at 247 nm. This method provides a distinct advantage over previously developed assays in that the solvent gradient does not contribute to baseline changes throughout the chromatographic run. In this way, the flat baseline markedly improves robustness of the assay and simplifies peak integration and quantification. At the same time, resolution of 15 different steroid metabolites catalyzed by the cytochrome P-450 enzymes are readily separated in rat or mouse liver microsomes. An internal standard, cortexolone, was selected for use based on its structural and spectral similarity to the hydroxytestosterone metabolites, and quantification is based on the molar response for the testosterone/cortexolone peak area ratio. The limit of detection (LOD) is 1 pmol on-column with a limit of quantitation (LOQ) of 4 pmol on-column. The intraday repeatability is approximately 3%. This simplified procedure is straightforward and should greatly facilitate the routine use of the testosterone hydroxylation assay to measure cytochrome P-450 isozyme activity.

Musk xylene (MX) (1,3,5-trinitro-2-t-butylxylene) is a nitromusk perfume ingredient that although uniformly negative in a battery of genotoxicity tests, produces a high incidence of liver tumors in mice. The purpose of this work was to characterize the profile and dose–response relationship of microsomal enzyme induction following exposure to MX. MX was dosed by gavage to male B6C3F1 mice for 7 days at 0, 1, 5, 10, 20, 50, 100, and 200 mg/kg after which microsomes were prepared. At 200 mg/kg, MX increased liver weight by about 65% and increased microsomal cytochrome P-450 content 2-fold over control. MX increased microsomal activity forO-dealkylation of 7-ethoxy and 7-methoxyresorufin 4- and 2-fold, respectively, and increased theN-demethylation of erythromycin approximately 2-fold. These results were generally consistent with increased CYP1A1, 1A2, and 3A protein levels determined by Western blotting. In contrast, whereas no increase inO-dealkylation of 7-pentoxyresorufin (PROD) was observed, MX treatment increased CYP2B protein levels about 25-fold over control at 200 mg/kg. Furthermore, a single dosage of MX (200 mg/kg) increased Cyp2b-10 mRNA to a maximal level and with a time course similar to phenobarbital (PB). To study inhibition of CYP2B enzymesin vivo,mice were treated with PB (0.05% in drinking water for 5 days), then given a single dosage of corn oil or MX (200 mg/kg) at 2 or 18 hr before necropsy. PB treatment increased PROD activity 25-fold, and at 2 hr after MX treatment (associated with peak plasma levels of MX), there was no change in the PB-induced PROD activity. However, at 18 hr, MX treatment decreased PROD activity by 90%. Despite thein vivoinhibition,in vitrostudies indicated that MX did not cause mechanism-based inactivation of CYP2B enzymes. The potential for nitroreduction of MX (catalyzed by anaerobic intestinal bacteria) to contribute to the inhibition of CYP2B enzyme activity was evaluated in a separate group of PB-induced mice that were dosed orally with a regimen of broad spectrum antibiotics (neomycin, tetracyline, and bacitracin) to reduce gut flora prior to administration of MX. In these animals, MX (200 mg/kg) did not inhibit PB-induced PROD activity. In summary, MX treatment produced general hepatic changes consistent with induction of CYP2B enzymes in mice and caused a large increase in CYP2B protein and mRNA levels. These data indicate that MX is a PB-like inducer of cytochrome P-450 enzymes and may cause liver tumors in a manner analogous to PB. However, no increase in CYP2B enzyme activity was observed, suggesting that MX or metabolites of MX also inhibit this enzyme. When the intestinal flora was eliminated by antibiotic treatment, MX no longer inhibited the CYP2B enzyme, indicating that anaerobic bacteria are capable of metabolizing MX, and suggesting that amine metabolites formed by nitroreduction are involved in the inhibition of mouse CYP2B enzymes.

Since the first approval of interferon for the treatment of chronic hepatitis B virus (HBV) infection in 1992, six additional antivirals have been developed: pegylated interferon-alfa2a, and the oral antivirals lamivudine, adefovir, telbivudine, entecavir and tenofovir. The availability of animal models for HBV infection and hepatocyte cell culture led to the discovery and development of oral antivirals targeted at HBV polymerase and reverse transcriptase, which inhibit viral replication. The discovery and development of entecavir, the first oral anti-HBV drug with both potent antiviral activity and a high genetic barrier to resistance, took more than 10 years before it was first approved in the USA. Since then, multiple real-life studies have provided data consistent with the findings of the registration trials and the long-term rollover study in terms of efficacy, resistance, and safety. Data from the long-term follow-up of patients enrolled in the registration studies showed that treatment with entecavir can lead to significant improvements in liver histopathology, and recent cohort studies have demonstrated that treatment with entecavir may reduce disease progression and the development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B. In addition, real-life studies suggest that entecavir may reduce HCC recurrence and increase survival rates in patients with HBV-related HCC post-surgical resection.