Hepatic Transcript Profiles of Cytochrome P450 Genes Predict Sex Differences in Drug Metabolism.

Safety assessments of new drug candidates are an important part of the drug development and approval process. Often, possible sex-associated susceptibilities are not adequately addressed, and better assessment tools are needed. We hypothesized that hepatic transcript profiles of cytochrome P450 (P450) enzymes can be used to predict sex-associated differences in drug metabolism and possible adverse events. Comprehensive hepatic transcript profiles were generated for F344 rats of both sexes at nine ages, from 2 weeks (preweaning) to 104 weeks (elderly). Large differences in the transcript profiles of 29 drug metabolizing enzymes and transporters were found between adult males and females (8-52 weeks). Using the PharmaPendium data base, 41 drugs were found to be metabolized by one or two P450 enzymes encoded by sexually dimorphic mRNAs and thus were candidates for evaluation of possible sexually dimorphic metabolism and/or toxicities. Suspension cultures of primary hepatocytes from three male and three female adult rats (10-13 weeks old) were used to evaluate the metabolism of 11 drugs predicted to have sexually dimorphic metabolism. The pharmacokinetics of the drug or its metabolite was analyzed by liquid chromatography/tandem mass spectrometry using multiple reaction monitoring. Of those drugs with adequate metabolism, the predicted significant sex-different metabolism was found for six of seven drugs, with half-lives 37%-400% longer in female hepatocytes than in male hepatocytes. Thus, in this rat model, transcript profiles may allow identification of potential sex-related differences in drug metabolism. SIGNIFICANCE STATEMENT: The present study showed that sex-different expression of genes coding for drug metabolizing enzymes, specifically cytochrome P450s, could be used to predict sex-different drug metabolism and, thus, provide a new tool for protecting susceptible subpopulations from possible adverse drug events.

A randomized controlled laboratory study on the long-term effects of methylphenidate on cardiovascular function and structure in rhesus monkeys.

BACKGROUND: Whether long-term methylphenidate (MPH) results in any changes in cardiovascular function or structure can only be properly addressed through a randomized trial using an animal model which permits elevated dosing over an extended period of time. METHODS: We studied 28 male rhesus monkeys (Macaca mulatta) approximately 7 years of age that had been randomly assigned to one of three MPH dosages: vehicle control (0 mg/kg, b.i.d., n = 9), low dose (2.5 mg/kg, b.i.d., n = 9), or high dose (12.5 mg/kg, b.i.d., n = 10). Dosage groups were compared on serum cardiovascular and inflammatory biomarkers, electrocardiograms (ECGs), echocardiograms, myocardial biopsies, and clinical pathology parameters following 5 years of uninterrupted dosing. RESULTS: With the exception of serum myoglobin, there were no statistical differences or apparent dose-response trends in clinical pathology, cardiac inflammatory biomarkers, ECGs, echocardiograms, or myocardial biopsies. The high-dose MPH group had a lower serum myoglobin concentration (979 ng/mL) than either the low-dose group (1882 ng/mL) or the control group (2182 ng/mL). The dose response was inversely proportional to dosage (P = .0006). CONCLUSIONS: Although the findings cannot be directly generalized to humans, chronic MPH exposure is unlikely to be associated with increased cardiovascular risk in healthy children.

Cytotoxicity of 34 FDA approved small-molecule kinase inhibitors in primary rat and human hepatocytes.

Of the 34 FDA approved oral small-molecule kinase inhibitors (KI), 23 (68%) have warnings for hepatotoxicity in product labeling. To better understand the mechanisms of KI hepatotoxicity and whether such effects can be predicted, we examined 34 KIs for cytotoxicity in primary rat and human hepatocytes. The hepatocytes were treated with KIs at ten concentrations normalized to maximal therapeutic blood levels (Cmax). At 5 and 24 h post treatment, lactate dehydrogenase or alanine aminotransferase leakage, caspase 3/7 activities and cellular adenosine triphosphate levels were measured. At 1 to 100-fold Cmax, while 5 KIs were neither toxic to human nor rat hepatocytes, 3 KIs showed similar cytotoxicity in both species and 26 KIs showed species-biased cytotoxicity, with 16 KIs being more toxic to human hepatocytes and 10 KIs being more toxic to rat hepatocytes. At concentrations of 1-, 2.5-, 5-, 10-, 100-fold Cmax, the number of cytotoxic KIs in human hepatocytes was 4, 8, 11, 14 and 27, respectively, and the corresponding number in rat hepatocytes was 1, 4, 9, 12 and 27, respectively. When hepatocyte cytotoxicity at 100-fold Cmax was used to predict KI clinical hepatotoxicity reflected in product labeling, the accuracy was 0.65 with human hepatocytes and 0.59 with rat cells. When the criterion of daily dose ≥100 mg or Cmax ≥1.1 µM was used to predict KI hepatotoxicity, the accuracy was 0.56 or 0.47, respectively. These results suggest both indirect and direct drug-induced hepatocyte toxicity may contribute to the mechanisms of KI-induced hepatotoxicity seen clinically and use of primary hepatocytes is a useful in vitro model to help predict such toxicity.

Drug-Induced Liver Injury in Children: Clinical Observations, Animal Models, and Regulatory Status.

Drug-induced liver injury in children (cDILI) accounts for about 1% of all reported adverse drug reactions throughout all age groups, less than 10% of all clinical DILI cases, and around 20% of all acute liver failure cases in children. The overall DILI susceptibility in children has been assumed to be lower than in adults. Nevertheless, controversial evidence is emerging about children's sensitivity to DILI, with children's relative susceptibility to DILI appearing to be highly drug-specific. The culprit drugs in cDILI are similar but not identical to DILI in adults (aDILI). This is demonstrated by recent findings that a drug frequently associated with aDILI (amoxicillin/clavulanate) was rarely associated with cDILI and that the drug basiliximab caused only cDILI but not aDILI. The fatality in reported cDILI studies ranged from 4% to 31%. According to the US Food and Drug Administration-approved drugs labels, valproic acid, dactinomycin, and ampicillin appear more likely to cause cDILI. In contrast, deferasirox, isoniazid, dantrolene, and levofloxacin appear more likely to cause aDILI. Animal models have been explored to mimic children's increased susceptibility to valproic acid hepatotoxicity or decreased susceptibility to acetaminophen or halothane hepatotoxicity. However, for most drugs, animal models are not readily available, and the underlying mechanisms for the differential reactions to DILI between children and adults remain highly hypothetical. Diagnosis tools for cDILI are not yet available. A critical need exists to fill the knowledge gaps in cDILI. This review article provides an overview of cDILI and specific drugs associated with cDILI.

Effects of 31 FDA approved small-molecule kinase inhibitors on isolated rat liver mitochondria.

The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I-V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1-2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.

Potential of extracellular microRNAs as biomarkers of acetaminophen toxicity in children.

UNLABELLED: Developing biomarkers for detecting acetaminophen (APAP) toxicity has been widely investigated. Recent studies of adults with APAP-induced liver injury have reported human serum microRNA-122 (miR-122) as a novel biomarker of APAP-induced liver injury. The goal of this study was to examine extracellular microRNAs (miRNAs) as potential biomarkers for APAP liver injury in children. Global levels of serum and urine miRNAs were examined in three pediatric subgroups: 1) healthy children (n=10), 2) hospitalized children receiving therapeutic doses of APAP (n=10) and 3) children hospitalized for APAP overdose (n=8). Out of 147 miRNAs detected in the APAP overdose group, eight showed significantly increased median levels in serum (miR-122, -375, -423-5p, -30d-5p, -125b-5p, -4732-5p, -204-5p, and -574-3p), compared to the other groups. Analysis of urine samples from the same patients had significantly increased median levels of four miRNAs (miR-375, -940, -9-3p and -302a) compared to the other groups. Importantly, correlation of peak serum APAP protein adduct levels (an indicator of the oxidation of APAP to the reactive metabolite N-acetyl-para-quinone imine) with peak miRNA levels showed that the highest correlation was observed for serum miR-122 (R=0.94; p<0.01) followed by miR-375 (R=0.70; p=0.05). CONCLUSION: Our findings demonstrate that miRNAs are increased in children with APAP toxicity and correlate with APAP protein adducts, suggesting a potential role as biomarkers of APAP toxicity.

Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis.

The tyrosine kinase inhibitor regorafenib was approved by regulatory agencies for cancer treatment, albeit with strong warnings of severe hepatotoxicity included in the product label. The basis of this toxicity is unknown; one possible mechanism, that of mitochondrial damage, was tested. In isolated rat liver mitochondria, regorafenib directly uncoupled oxidative phosphorylation (OXPHOS) and promoted calcium overload-induced swelling, which were respectively prevented by the recoupler 6-ketocholestanol (KC) and the mitochondrial permeability transition (MPT) pore blocker cyclosporine A (CsA). In primary hepatocytes, regorafenib uncoupled OXPHOS, disrupted mitochondrial inner membrane potential (MMP), and decreased cellular ATP at 1h, and triggered MPT at 3h, which was followed by necrosis but not apoptosis at 7h and 24h, all of which were abrogated by KC. The combination of the glycolysis enhancer fructose plus the mitochondrial ATPase synthase inhibitor oligomycin A abolished regorafenib induced necrosis at 7h. This effect was not seen at 24h nor with the fructose or oligomycin A separately. CsA in combination with trifluoperazine, both MPT blockers, showed similar effects. Two compensatory mechanisms, activation of AMP-activated protein kinase (AMPK) to ameliorate ATP shortage and induction of autophagy to remove dysfunctional mitochondria, were found to be mobilized. Hepatocyte necrosis was enhanced either by the AMPK inhibitor Compound C or the autophagy inhibitor chloroquine, while autophagy inducer rapamycin was strongly cytoprotective. Remarkably, all toxic effects were observed at clinically-relevant concentrations of 2.5-15µM. These data suggest that uncoupling of OXPHOS and the resulting ATP shortage and MPT induction are the key mechanisms for regorafenib induced hepatocyte injury, and AMPK activation and autophagy induction serve as pro-survival pathways against such toxicity.

Identification of a metabolic biomarker panel in rats for prediction of acute and idiosyncratic hepatotoxicity.

It has been estimated that 10% of acute liver failure is due to "idiosyncratic hepatotoxicity". The inability to identify such compounds with classical preclinical markers of hepatotoxicity has driven the need to discover a mechanism-based biomarker panel for hepatotoxicity. Seven compounds were included in this study: two overt hepatotoxicants (acetaminophen and carbon tetrachloride), two idiosyncratic hepatotoxicants (felbamate and dantrolene), and three non-hepatotoxicants (meloxicam, penicillin and metformin). Male Sprague-Dawley rats were orally gavaged with a single dose of vehicle, low dose or high dose of the compounds. At 6 h and 24 h post-dosing, blood was collected for metabolomics and clinical chemistry analyses, while organs were collected for histopathology analysis. Forty-one metabolites from previous hepatotoxicity studies were semi-quantified and were used to build models to predict hepatotoxicity. The selected metabolites were involved in various pathways, which have been noted to be linked to the underlying mechanisms of hepatotoxicity. PLS models based on all 41 metabolite or smaller subsets of 6 (6 h), 7 (24 h) and 20 (6 h and 24 h) metabolites resulted in models with an accuracy of at least 97.4% for the hold-out test set and 100% for training sets. When applied to the external test sets, the PLS models predicted that 1 of 9 rats at both 6 h and 24 h treated with idiosyncratic liver toxicants was exposed to a hepatotoxic chemical. In conclusion, the biomarker panel might provide information that along with other endpoint data (e.g., transcriptomics and proteomics) may diagnose acute and idiosyncratic hepatotoxicity in a clinical setting.

Inhibition of cytochrome P450s enhances (+)-usnic acid cytotoxicity in primary cultured rat hepatocytes.

(+)-Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non-isoenzyme-selective inhibitor SKF-525A (20 µM), or the CYP1A inhibitor alpha-naphthoflavone (10 µM), or the CYP3A inhibitor ketoconazole (25 µM), the cytotoxicity of UA at 3~6 µM after 3~20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA-induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 µM, showed no effects in parallel experiments. These data demonstrate that UA is bio-transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Metabolomics evaluation of the effects of green tea extract on acetaminophen-induced hepatotoxicity in mice.

Green tea has been purported to have beneficial health effects including protective effects against oxidative stress. Acetaminophen (APAP) is a widely used analgesic drug that can cause acute liver injury in overdose situations. These studies explored the effects of green tea extract (GTE) on APAP-induced hepatotoxicity in liver tissue extracts using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Mice were orally administered GTE, APAP or GTE and APAP under three scenarios. APAP alone caused a high degree of hepatocyte necrosis associated with increases in serum transaminases and alterations in multiple metabolic pathways. The time of GTE oral administration relative to APAP either protected against or potentiated the APAP-induced hepatotoxicity. Dose dependent decreases in histopathology scores and serum transaminases were noted when GTE was administered prior to APAP; whereas, the opposite occurred when GTE was administered after APAP. Similarly, metabolites altered by APAP alone were less changed when GTE was given prior to APAP. Significantly altered pathways included fatty acid metabolism, glycerophospholipid metabolism, glutathione metabolism, and energy pathways. These studies demonstrate the complex interaction between GTE and APAP and the need to employ novel analytical strategies to understand the effects of dietary supplements on pharmaceutical compounds.

Evaluating effects of penicillin treatment on the metabolome of rats.

Penicillin (PEN) V, a well-known antibiotic widely used in the treatment of Gram-positive bacterial infections, was evaluated in this study. LC/MS- and NMR-based metabolic profiling were employed to examine the effects of PEN on the host's metabolic phenotype. Male Sprague Dawley rats were randomly divided into groups that were orally administered either 0.5% methylcellulose vehicle, 100 or 2400mg PEN/kg body weight once daily for up to 14 consecutive days. Urine, plasma and tissue were collected from groups sacrificed at 6h, 24h or 14d. The body fluids were subjected to clinical chemistry and metabolomics analysis; the tissue samples were processed for histopathology. The only notable clinical chemistry observation was that gamma glutamyltransferase (GGT) significantly decreased at 24h for both dose groups, and significantly decreased at 14d for the high-dose groups. Partial least squares discriminant analysis scores plots of the metabolomics data from urine and plasma samples showed dose- and time-dependent grouping patterns. Time- and dose-dependent decreases in urinary metabolites including indole-containing metabolites (such as 3-methyldioxyindole sulfate generated from bacterial metabolism of tryptophan), organic acids containing phenyl groups (such as hippuric acid, phenyllactic acid and 3-hydroxyanthranilic acid), and metabolites conjugated with sulfate or glucuronide (such as cresol sulfate and aminophenol sulfate) indicated that the gut microflora population was suppressed. Decreases in many host-gut microbiota urinary co-metabolites (indole- and phenyl-containing metabolites, amino acids, vitamins, nucleotides and bile acids) suggested gut microbiota play important roles in the regulation of host metabolism, including dietary nutrient absorption and reprocessing the absorbed nutrients. Decreases in urinary conjugated metabolites (sulfate, glucuronide and glycine conjugates) implied that gut microbiota might have an impact on chemical detoxification mechanisms. In all, these results clearly show that metabolic profiling is a useful tool to better understand the effects of the antibiotic penicillin has on the gut microbiota and the host.

Mouse liver protein sulfhydryl depletion after acetaminophen exposure.

Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.

Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes.

Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1-100µM for 30min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1µM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1µM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.

Green tea extract can potentiate acetaminophen-induced hepatotoxicity in mice.

Green tea extract (GTE) has been advocated as a hepatoprotective compound and a possible therapeutic agent for acetaminophen (APAP) overdose. This study was conducted to determine if GTE can provide protection against APAP-induced hepatotoxicity. Three different exposure scenarios were tested. The first involved administering APAP (150 mg/kg, orally) to mice followed 6h later by GTE (500 or 1000 mg/kg). The other two involved administering GTE prior to the APAP dose. GTE (500 or 1000 mg/kg, orally) was administered 3h prior to APAP (200 mg/kg, orally) or for three consecutive days (once-daily) followed by APAP (300 mg/kg) on the fourth day. Indices of hepatotoxicity were assessed 24h after the APAP dose. GTE potentiated APAP-induced hepatotoxicity when administered after the APAP dose. GTE caused significant glutathione depletion and this effect likely contributed to the observed potentiation. In contrast, GTE provided protection against APAP-induced hepatotoxicity when administered prior to the APAP dose. GTE dramatically decreased APAP covalent binding to protein indicating that less reactive metabolite was available to cause hepatocellular injury. These results highlight the potential for drug-dietary supplement interactions and the importance of testing multiple exposure scenarios to adequately model different types of potential interactions.

Identification of urinary microRNA profiles in rats that may diagnose hepatotoxicity.

Circulating microRNAs (miRNAs) have emerged as novel noninvasive biomarkers for several diseases and other types of tissue injury. This study tested the hypothesis that changes in the levels of urinary miRNAs correlate with liver injury induced by hepatotoxicants. Sprague-Dawley rats were administered acetaminophen (APAP) or carbon tetrachloride (CCl(4)) and one nonhepatotoxicant (penicillin/PCN). Urine samples were collected over a 24 h period after a single oral dose of APAP (1250 mg/kg), CCl(4) (2000 mg/kg), or PCN (2400 mg/kg). APAP and CCl(4) induced liver injury based upon increased serum alanine and aspartate aminotransferase levels and histopathological findings, including liver necrosis. APAP and CCl(4) both significantly increased the urinary levels of 44 and 28 miRNAs, respectively. In addition, 10 of the increased miRNAs were in common between APAP and CCl(4). In contrast, PCN caused a slight decrease of a different nonoverlapping set of urinary miRNAs. Cluster analysis revealed a distinct urinary miRNA pattern from the hepatotoxicant-treated groups when compared with vehicle controls and PCN. Analysis of hepatic miRNA levels suggested that the liver was the source of the increased urinary miRNAs after APAP exposure; however, the results from CCl(4) were equivocal. Computational analysis was used to predict target genes of the 10 shared hepatotoxicant-induced miRNAs. Liver gene expression profiling using whole genome microarrays identified eight putative miRNA target genes that were significantly altered in the liver of APAP- and CCl(4)-treated animals. In conclusion, the patterns of urinary miRNA may hold promise as biomarkers of hepatotoxicant-induced liver injury.

Changes in mouse liver protein glutathionylation after acetaminophen exposure.

The role of protein glutathionylation in acetaminophen (APAP)-induced liver injury was investigated in this study. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase and aspartate aminotransferase levels and liver necrosis in a dose-dependent manner. The ratio of GSH to GSSG was decreased in a dose-dependent manner, suggesting that APAP produced a more oxidizing environment within the liver. Despite the increased oxidation state, the level of global protein glutathionylation was decreased at 1 h and continued to decline through 24 h. Immunohistochemical localization of glutathionylated proteins showed a complex dynamic change in the lobule zonation of glutathionylated proteins. At 1 h after APAP exposure, the level of glutathionylation decreased in the single layer of hepatocytes around the central veins but increased mildly in the remaining centrilobular hepatocytes. This increase correlated with the immunohistochemical localization of APAP covalently bound to protein. Thereafter, the level of glutathionylation decreased dramatically over time in the centrilobular regions with major decreases observed at 6 and 24 h. Despite the overall decreased glutathionylation, a layer of cells lying between the undamaged periportal region and the damaged centrilobular hepatocytes exhibited high levels of glutathionylation at 3 and 6 h in all samples and in some 24-h samples that had milder injury. These temporal and zonal pattern changes in protein glutathionylation after APAP exposure indicate that protein glutathionylation may play a role in protein homeostasis during APAP-induced hepatocellular injury.

Hepatic cytochrome P450s attenuate the cytotoxicity induced by leflunomide and its active metabolite A77 1726 in primary cultured rat hepatocytes.

The Black Box Warning section of the U.S. drug label for leflunomide was recently updated to include stronger warnings about potential hepatotoxicity from this novel anti-arthritis drug. Because metabolic activation is a key mechanism for drug-induced hepatotoxicity, we examined whether leflunomide and its major metabolite, A77 1726, are cytotoxic to primary rat hepatocytes and whether their toxicity is modulated by hepatic cytochrome P450s (CYPs). As measured by lactate dehydrogenase leakage, time-dependent cytotoxicity was observed at 250-500 µM for leflunomide and 330-500 µM for A77 1726 within 20 h. Unexpectedly, three nonisoenzyme-specific CYP inhibitors, including SKF-525A, metyrapone, and 1-aminobenzotriazole, did not reduce but remarkably enhanced the cytotoxicity of leflunomide or A77 1726. SKF-525A pretreatment notably rendered hepatocytes susceptible to as low as 15 µM leflunomide or A77 1726. Three isoenzyme-specific CYP inhibitors including alpha-naphthoflavone, ticlopidine, and ketoconazole that mainly target CYP1A, CYP2B/2C, and CYP3A, respectively, also enhanced the cytotoxicity. A strong synergistic effect, similar to SKF-525A alone, was noted using a combination of all three of the isoenzyme-specific inhibitors. Hepatocytes pretreated with the CYP inducer dexamethasone for 24 h exhibited decreased cytotoxicity to leflunomide and A77 1726. At the concentrations tested, the CYP inhibitors and inducer showed no cytotoxicity. These data demonstrate that the parent forms of leflunomide and A77 1726 are more toxic to hepatocytes than their poorly characterized metabolites, indicating that the metabolic process of leflunomide is a detoxification step rather than an initiating event leading to toxicity.

Novel resuscitation strategy for pulmonary contusion after severe chest trauma.

BACKGROUND: Adenosine A2a receptor stimulation can increase coronary perfusion and also reduce leukocyte-mediated inflammatory responses in some conditions. Hextend is a novel colloid solution that may have antioxidant properties. All these actions might be beneficial after severe chest trauma, but have never been investigated. To fill these gaps, this study evaluated the therapeutic potential of a novel adenosine A2a agonist during fluid resuscitation from severe chest trauma with either standard-of-care crystalloid or Hextend. METHODS: Anesthetized, ventilated swine received unilateral, blunt trauma to the right chest via captive bolt gun, followed by a 10- to 12-mL/kg arterial hemorrhage. After 25 minutes of shock, ATL-146e was started (10 ng/kg/min intravenously for 180 minutes). After an additional 5 minutes, the minimum amount of either colloid (Hextend, 5% hetastarch in lactate-buffered, balanced electrolyte solution) or crystalloid (lactated Ringer's [LR] solution) was administered to maintain mean arterial pressure > 70 mm Hg and heart rate < 100 beats/min and to correct lactate for 180 minutes postinjury. Cardiopulmonary function was monitored and serial bronchoalveolar lavage samples were analyzed for protein, leukocyte infiltration, and expression of cyclooxygenase (COX)-1 and COX-2 isozymes as markers of the inflammatory cascade. RESULTS: Fluid requirements were reduced by half with Hextend compared with LR (p < 0.05). ATL-146e in either Hextend or LR transiently increased cardiac output, cardiac contractility, and systemic oxygen delivery (all p < 0.05). Pao(2)/Fio(2) ratio was 50 to 100 higher and bronchoalveolar lavage leukocytes were reduced by half with Hextend versus LR (both p < 0.05), but there was no added effect of ATL-146e. COX-1 expression was induced in macrophages (Mphis), whereas COX-2 was induced in neutrophils. Neither Hextend nor ATL-146e reduced COX expression. CONCLUSION: Hextend reduced the volume for initial resuscitation, which may offer logistical advantages in prehospital field conditions or whenever there is limited medical resources or prolonged transport times; ATL-146e improved early cardiac performance without causing hypotension or bradycardia; when administered 25 to 30 minutes after injury, neither Hextend nor ATL-146e altered inflammatory changes in pulmonary Mphis or infiltrating PMNs; and further studies are needed to determine whether these short-term benefits correlate with long-term outcome.