HLM-beads: Rapid Assessment of Nonspecific Binding to Human Liver Microsomes Using Magnetizable Beads.

In early drug development, drug-drug interaction risk is routinely assessed using human liver microsomes (HLMs). Nonspecific binding of drugs to HLMs can affect the determination of accurate enzyme parameters (Km, Ki, KI). Previously, we described a novel in vitro model consisting of HLMs bound to magnetizable beads [HLM-magnetizable-beads system (HLM-beads)]. The HLM-beads enable rapid separation of HLMs from incubation media by applying a magnetic field. Here, HLM-beads were further characterized and evaluated as a tool to assess HLM nonspecific binding of small molecules. The free fractions (fu,mic) of 13 compounds (chosen based on their pKa values) were determined using HLM-beads under three HLM concentrations (0.025, 0.50, and 1.0 mg/ml) and compared with those determined by equilibrium dialysis. Most fu,mic values obtained using HLM-beads were within 0.5- to 2-fold of the values determined using equilibrium dialysis. The highest fold difference were observed for high binders itraconazole and BIRT2584 (1.9- to 2.9-fold), as the pronounced adsorption of these compounds to the equilibrium dialysis apparatus interfered with their fu,mic determination. Correlation and linear regression analysis of the fu,mic values generated using HLM-beads and equilibrium dialysis was conducted. Overall, a good correlation of fu,mic values obtained by the two methods were observed, as the r and R2 values from correlational analysis and linear regression analysis were >0.9 and >0.89, respectively. These studies demonstrate that HLM-beads can produce comparable fu,mic values as determined by equilibrium dialysis while reducing the time required for this type of study from hours to only 10 minutes and compound apparatus adsorption. SIGNIFICANCE STATEMENT: This work introduces a new method of rapidly assessing nonspecific microsomal binding using human liver microsomes bound to magnetizable beads.

In vitro-in vivo extrapolation of metabolic clearance using human liver microsomes: factors showing variability and their normalization.

In vitro-in vivo extrapolation (IVIVE) using human liver microsomes has been widely used to predict metabolic clearance, but some of the factors used in the process of prediction show variability for the same compound: notably, microsomal intrinsic clearance values corrected by the unbound fraction (CLint, u), physiological parameters used for scale-up, and the source of in vivo clearance data.The purpose of this study was to assess the correlation between in vitro and in vivo CLint with a focus on factors showing variability using four cytochrome P450 (CYP)3A substrates.We surveyed in vivo clearance values in literature and also determined the microsomal CLint, u values. A scaling factor (SFdirect) was defined as in vivo CLint divided by the microsomal CLint, u, which ranged from 1190 to 2310 (mg protein per kg body weight). The application of a mean SFdirect of 1600 (mg protein per kg body weight) and further normalization by the microsomal CLint, u values of midazolam, the most commonly used substrate, resulted in improved prediction accuracy for CLint, u values from various microsomal batches.The results suggest the normalization of variability might be useful for predicting the in vivo CLint.

Utilizing Pediatric Physiologically Based Pharmacokinetic Models to Examine Factors That Contribute to Methadone Pharmacokinetic Variability in Neonatal Abstinence Syndrome Patients.

Chronic intrauterine exposure to psychoactive drugs often results in neonatal abstinence syndrome (NAS). NAS is the symptomatic drug withdrawal in newborns that generally occurs after in utero chronic opioid exposure. Methadone is an opioid analgesic commonly prescribed for pharmacologic management of NAS. It exhibits high pharmacokinetic (PK) variability. The current study used physiologically based PK modeling to predict the PK profile of methadone in 20 newborns treated for NAS. The physiologically based PK simulations adequately predicted the PK profile of the clinical data for 45% of the patients. Sensitivity analyses were conducted to explore contributing factors to methadone PK variability. The data suggest that P450 enzymatic activity impacts the clearance of methadone in virtual adults and neonates, while the contribution of cardiac output may be negligible. Understanding maturational and/or pharmacogenetic changes in cytochrome P450 enzymatic activity may further explain the large PK variability of methadone in newborns with NAS and will help individualized treatment.

Role of Cell Death in Toxicology: Does It Matter How Cells Die?

My research activity started with studies on drug metabolism in rat liver microsomes in the early 1960s. The CO-binding pigment (cytochrome P450) had been discovered a few years earlier and was subsequently found to be involved in steroid hydroxylation in adrenal cortex microsomes. Our early studies suggested that it also participated in the oxidative demethylation of drugs catalyzed by liver microsomes, and that prior treatment of the animals with phenobarbital caused increased levels of the hemoprotein in the liver, and similarly enhanced rates of drug metabolism. Subsequent studies of cytochrome P450-mediated metabolism of toxic drugs in freshly isolated rat hepatocytes characterized critical cellular defense systems and identified mechanisms by which accumulating toxic metabolites could damage and kill the cells. These studies revealed that multiple types of cell death could result from the toxic injury, and that it is important to know which type of cell death results from the toxic injury.

New Psychoactive Substances 3-Methoxyphencyclidine (3-MeO-PCP) and 3-Methoxyrolicyclidine (3-MeO-PCPy): Metabolic Fate Elucidated with Rat Urine and Human Liver Preparations and their Detectability in Urine by GC-MS, "LC-(High Resolution)-MSn" and "LC-(High Resolution)-MS/MS".

BACKGROUND: 3-Methoxyphencyclidine (3-MeO-PCP) and 3-methoxyrolicyclidine (3-MeOPCPy) are two new psychoactive substances (NPS). The aims of the present study were the elucidation of their metabolic fate in rat and pooled human liver microsomes (pHLM), the identification of the cytochrome P450 (CYP) isoenzymes involved, and the detectability using standard urine screening approaches (SUSA) after intake of common users' doses using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-multi-stage mass spectrometry (LC-MSn), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). METHODS: For metabolism studies, rat urine samples were treated by solid phase extraction or simple precipitation with or without previous enzymatic conjugate cleavage. After analyses via LC-HR-MSn, the phase I and II metabolites were identified. RESULTS: Both drugs showed multiple aliphatic hydroxylations at the cyclohexyl ring and the heterocyclic ring, single aromatic hydroxylation, carboxylation after ring opening, O-demethylation, and glucuronidation. The transferability from rat to human was investigated by pHLM incubations, where Odemethylation and hydroxylation were observed. The involvement of the individual CYP enzymes in the initial metabolic steps was investigated after single CYP incubations. For 3-MeO-PCP, CYP 2B6 was responsible for aliphatic hydroxylations and CYP 2C19 and CYP 2D6 for O-demethylation. For 3-MeO-PCPy, aliphatic hydroxylation was again catalyzed by CYP 2B6 and O-demethylation by CYP 2C9 and CYP 2D6 Conclusions: As only polymorphically expressed enzymes were involved, pharmacogenomic variations might occur, but clinical data are needed to confirm the relevance. The detectability studies showed that the authors' SUSAs were suitable for monitoring the intake of both drugs using the identified metabolites.

Harmonised high throughput microsomal stability assay.

INTRODUCTION: Prediction of human pharmacokinetics from in vitro assays and pre-clinical data is an integral part of drug discovery. In vitro stability metabolic studies can provide an estimate of in vivo hepatic intrinsic clearance through inclusion of biological scaling factors. Many labs have personalised stability protocols including marker compounds and have adopted QC criteria and assay limits to ensure data integrity. Contract research organisations (CRO's) provide integrated drug discovery support to academic and pharmaceutical/biotechnology institutions to progress their in-house projects. The majority of these clients have established in-house protocols with associated criteria to ensure data consistency between in-house and external labs. METHODS: In this study, numerous assay variables were condensed into one harmonised assay format and a range of compounds with diverse physicochemical properties were evaluated. The protocols were diverse with respect to the following attributes: buffer, microsomal concentration and species strain. RESULTS: Comparison of human lots in vitro CLint between the traditional and consolidated assay formats showed a good correlation with no significant difference. A clear relationship was demonstrated between strains. Interpretation of in vitro intrinsic clearance between the strains for each species was consistent. Using strict classes of in vitro hepatic intrinsic clearance values (<50, 50-100, >150µL/min/mg protein) comparisons across different conditions such as, assay variables, human lots, mouse and rat strains showed >80% agreement. DISCUSSION: A high throughput assay was developed that enables the simultaneous measurement of CLint using mouse, rat and human hepatic microsomes (consolidated assay). By condensing all possible variables into one assay format, consistent data were obtained during head to head tests.

Effect of N-methyl deuteration on metabolism and pharmacokinetics of enzalutamide.

BACKGROUND: The replacement of hydrogen with deuterium invokes a kinetic isotope effect. Thus, this method is an attractive way to slow down the metabolic rate and modulate pharmacokinetics. PURPOSE: Enzalutamide (ENT) acts as a competitive inhibitor of the androgen receptor and has been approved for the treatment of metastatic castration-resistant prostate cancer by the US Food and Drug Administration in 2012. To attenuate the N-demethylation pathway, hydrogen atoms of the N-CH3 moiety were replaced by the relatively stable isotope deuterium, which showed similar pharmacological activities but exhibited favorable pharmacokinetic properties. METHODS: We estimated in vitro and in vivo pharmacokinetic parameters for ENT and its deuterated analog (d3-ENT). For in vitro studies, intrinsic primary isotope effects (K H/K D) were determined by the ratio of intrinsic clearance (CLint) obtained for ENT and d3-ENT. The CLint values were obtained by the substrate depletion method. For in vivo studies, ENT and d3-ENT were orally given to male Sprague Dawley rats separately and simultaneously to assess the disposition and metabolism of them. We also investigated the main metabolic pathway of ENT by comparing the rate of oxidation and hydrolysis in vitro. RESULTS: The in vitro CLint (maximum velocity/Michaelis constant [V max/K m]) of d3-ENT in rat and human liver microsomes were 49.7% and 72.9% lower than those of the non-deuterated compound, corresponding to the K H/K D value of ~2. The maximum observed plasma concentration, C max, and area under the plasma concentration -time curve from time zero to the last measurable sampling time point (AUC0-t) were 35% and 102% higher than those of ENT when orally administered to rats (10 mg/kg). The exposure of the N-demethyl metabolite M2 was eightfold lower, whereas that of the amide hydrolysis metabolite M1 and other minor metabolites was unchanged. The observed hydrolysis rate of M2 was at least ten times higher than that of ENT and d3-ENT in rat plasma. CONCLUSION: ENT was mainly metabolized through the "parent→M2→M1" pathway based on in vitro and in vivo elimination behavior. The observed in vitro deuterium isotope effect translated into increased exposure of the deuterated analog in rats. Once the carbon-hydrogen was replaced with carbon-deuterium (C-D) bonds, the major metabolic pathway was retarded because of the relatively stable C-D bonds. The systemic exposure to d3-ENT can increase in humans, so the dose requirements can be reduced appropriately.

Single-Nucleotide Polymorphisms in Cytochrome P450 2E1 (CYP2E1) 3'-Untranslated Region Affect the Regulation of CYP2E1 by miR-570.

Human cytochrome P450 2E1 (CYP2E1) catalyzes the metabolism of numerous xenobiotics, including acetaminophen and ethanol. CYP2E1 expression is known to be extensively regulated by post-transcriptional and post-translational mechanisms. A previous study had reported that a single-nucleotide polymorphism (SNP) 1561A>G in the 3'-untranslated region (3'-UTR) of CYP2E1 leads to a decreased CYP2E1 mRNA level in human peripheral blood mononuclear cells. In this study, we examined the possibility that microRNA(s) (miR) may be involved in the SNP-mediated modulation of CYP2E1 expression. Genotyping and sequencing analyses revealed that another SNP, 1556T>A, in the 3'-UTR was in complete linkage disequilibrium with the SNP 1561A>G. We termed the alleles with 1556T and 1561A or 1556A and 1561G haplotype I or II, respectively. A luciferase assay revealed that miR-570 recognizes the CYP2E1 3'-UTR of haplotype I but not haplotype II. Human embryonic kidney 293 (HEK293) cell lines stably expressing human CYP2E1 that included the 3'-UTR of haplotype I or II (HEK/2E1(I) or HEK/2E1(II) cells, respectively) were established. Overexpression of miR-570 significantly decreased the CYP2E1 protein level in the HEK/2E1(I) cells but not in the HEK/2E1(II) cells. In seven human livers with diplotype I/I, the CYP2E1 protein levels were inversely correlated with the miR-570 levels, but no relationship was observed in 25 human livers with diplotypes I/II and II/II. Collectively, it was demonstrated that human CYP2E1 was regulated by miR-570 in a genotype-dependent manner. This report describes the first proof that SNP in 3'-UTR of human P450 affects binding of miRNA to modulate the expression in the liver.

Targeting human central nervous system protein kinases: An isoform selective p38αMAPK inhibitor that attenuates disease progression in Alzheimer's disease mouse models.

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150's exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.

Dibenzazepines and dibenzoxazepines as sodium channel blockers.

We have identified two related series of dibenzazepine and dibenzoxazepine sodium channel blockers, which showed good potency on Nav1.7 in FLIPR-based and electrophysiological functional assays.

Pharmacokinetics and drug interactions of eslicarbazepine acetate.

Eslicarbazepine acetate (ESL) is a novel once-daily antiepileptic drug (AED) approved in Europe since 2009 that was found to be efficacious and well tolerated in a phase III clinical program in adult patients with partial onset seizures previously not controlled with treatment with one to three AEDs, including carbamazepine (CBZ). ESL shares with CBZ and oxcarbazepine (OXC) the dibenzazepine nucleus bearing the 5-carboxamide substitute, but is structurally different at the 10,11 position. This molecular variation results in differences in metabolism, preventing the formation of toxic epoxide metabolites such as carbamazepine-10,11-epoxide. Unlike OXC, which is metabolized to both eslicarbazepine and (R)-licarbazepine, ESL is extensively converted to eslicarbazepine. The systemic exposure to eslicarbazepine after ESL oral administration is approximately 94% of the parent dose, with minimal exposure to (R)-licarbazepine and OXC. After ESL oral administration, the effective half-life (t(1/2,eff) ) of eslicarbazepine was 20-24 h, which is approximately two times longer than its terminal half-life (t(1/2)). At clinically relevant doses (400-1,600 mg/day) ESL has linear pharmacokinetics (PK) with no effects of gender or moderate liver impairment. However, because eslicarbazepine is eliminated primarily (66%) by renal excretion, dose adjustment is recommended for patients with renal impairment. Eslicarbazepine clearance is induced by phenobarbital, phenytoin, and CBZ and it dose-dependently decreases plasma exposure of oral contraceptive and simvastatin.

Discovery and SAR of small molecule PAR1 antagonists.

High-throughput screening resulted in the identification of a small molecule inhibitor of PAR1. Optimisation of the initial hit led to the discovery of compounds 34 and 49, which displayed antithrombotic activity in an arteriovenous shunt model in the rat after iv administration.

Impact of CYP2B6 polymorphism on hepatic efavirenz metabolism in vitro.

OBJECTIVES: To determine the influence of cytochrome P450 2B6 (CYP2B6) genotype on the rate of oxidative efavirenz metabolism in human liver microsomes. MATERIALS & METHODS: Formation rates of 8-hydroxyefavirenz, 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz were determined in vitro with efavirenz as a substrate (10 microM) in a large panel of human liver microsomes (n = 87) that were genotyped for variants of the CYP2B6 gene and phenotyped for CYP2B6 protein expression and bupropion hydroxylation. RESULTS: Efavirenz 8-hydroxylation, the major route of efavirenz clearance, was detected in all samples, exhibiting an overall interindividual variability of 44.7-fold; 8,14-dihydroxyefavirenz and 7-hydroxyefavirenz were also detected in most samples. The formation rate of 8-hydroxyefavirenz correlated significantly with CYP2B6 protein (Spearman's r(S) = 0.54; p < 0.0001) and bupropion hydroxylase activity (r(S) = 0.73; p < 0.0001). Compared with the *1/*1 genotype, efavirenz 8-hydroxylation was significantly lower in samples with *1/*6 and *6/*6 genotype, which also had significantly decreased CYP2B6 protein (Mann-Whitney test, p < 0.05). A decrease in CYP2B6 protein was also observed in samples with *1/*5 and *5/*6 genotypes, but this did not result in significant reduction of efavirenz metabolism, probably due to differences in specific activity of the protein variants. Lower CYP2B6 protein and activity, as well as efavirenz 8-hydroxylation was also found in several samples with rarer genotypes. We found no effect of gender and age on any of the phenotypes tested, but prior exposure to carbamazepine markedly increased CYP2B6 protein expression and activity as well as efavirenz 8-hydroxylation. CONCLUSIONS: We have shown that CYP2B6 genetic polymorphism markedly influences the metabolism of efavirenz in human liver microsomes. Importantly, the CYP2B6*6 allele harboring the SNPs c.516G>T [Q172H] and c.785A>G [K262R] was significantly associated with a pronounced decrease in CYP2B6 expression and activity, as well as a low rate of efavirenz 8-hydroxylation. These results represent a first step towards elucidating the mechanism by which this allele identifies patients exhibiting very high efavirenz plasma concentrations.

Evidence for a large conductance voltage gated cationic channel in rough endoplasmic reticulum of rat hepatocytes.

In this work, we report the single channel characterization of a voltage gated cationic channel from rough endoplasmic reticulum (RER) membranes of rat hepatocytes incorporated into a planar lipid bilayer. The channel was found to be cation selective with a main conductance of 598+/-20 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage dependent with a voltage for half activation (V(1/2)) of 38 mV and an effective gating charge z of -6.66. Adding either 4-AP (5 mM) or ATP (2.5 mM) to the side corresponding to the cell internal medium caused a strong inhibition of the channel activity. This channel is likely to be involved in maintaining proper cation homeostasis in the RER of hepatocytes.

In vitro and in vivo efficacy and in vitro metabolism of 1-phenyl-3-aryl-2-propen-1-ones against Plasmodium falciparum.

Investigation of a series of 1-phenyl-3-aryl-2-propen-1-ones resulted in the identification of nine inhibitors with submicromolar efficacy against at least one Plasmodium falciparum strain in vitro. These inhibitors were inactive when given orally in a Plasmodium berghei infected mouse model. Significant compound degradation occurred upon their exposure to a liver microsome preparation, suggesting metabolic instability may be responsible for the lack of activity in vivo.

A mechanical force contributes to the "osmotic swelling" of brush-border membrane vesicles.

Brush-border membrane vesicles and an osmotic swelling assay have been used extensively to monitor the pore-forming activity of Bacillus thuringiensis toxins. After a hypertonic shock, Manduca sexta midgut brush-border membrane vesicles shrink rapidly and reswell partially to a volume that depends on membrane permeability and toxin concentration rather than regaining their original volume as expected from theoretical models. Because efflux of buffer from the vesicles, as they shrink, could contribute to this phenomenon, vesicles were mixed with a hypertonic solution of the buffer with which they were loaded. Under these conditions, they are not expected to reswell, since the same solute is present on both sides of the membrane. Nevertheless, with several buffers, vesicles reswelled readily, an observation that demonstrates the involvement of an additional restoration force. Reswelling also occurred when, in the absence of toxin, the buffers were replaced by glucose, a solute that diffuses readily across the membrane, but did not occur with rat liver microsomes, despite their permeability to glucose. Unexpected swelling was also observed with rabbit jejunum brush-border membrane vesicles, suggesting that the cytoskeleton, present in brush-border membrane vesicles but absent from microsomes, could be responsible for the restoration force.

Antimalarial activity of N-alkyl amine, carboxamide, sulfonamide, and urea derivatives of a dispiro-1,2,4-trioxolane piperidine.

With an aim to identify a dispiro-1,2,4-trioxolane with high oral activity and good physicochemical properties, 27 derivatives of an achiral piperidine trioxolane were synthesized; most were potent antimalarial peroxides with IC(50)s ranging from 0.20 to 7.0 ng/mL. The oral efficacies of two of these were superior to artesunate and comparable to artemether. The attractive chemical simplicity of these compounds is balanced only by an apparent metabolic susceptibility.

Defensive nature of Sargassum polycystum (Brown alga) against acetaminophen-induced toxic hepatitis in rats: role of drug metabolizing microsomal enzyme system, tumor necrosis factor-alpha and fate of liver cell structural integrity.

AIM: To assess the defensive nature of Sargassum polycystum (S. polycystum) (Brown alga) against acetaminophen (AAP)-induced changes in drug metabolizing microsomal enzyme system, tumor necrosis factor (TNF-alpha) and fine structural features of the liver during toxic hepatitis in rats. METHODS: Male albino Wistar strain rats used for the study were randomly categorized into 4 groups. Group I consisted of normal control rats fed with standard diet. Group II rats were administered with acetaminophen (800 mg/kg body weight, intraperitoneally). Group III rats were pre-treated with S. polycystum extract alone. Group IV rats were orally pre-treated with S. polycystum extract (200 mg/kg body weight for 21 d) prior to acetaminophen induction (800 mg/kg body weight, intraperitoneally). Serum separated and liver was excised and microsomal fraction was isolated for assaying cytochrome P450, NADPH Cyt P450 reductase and b(5). Serum TNF-alpha was detected using ELISA. Fine structural features of liver were examined by transmission electron microscopy. RESULTS: Rats intoxicated with acetaminophen showed considerable impairment in the activities of drug metabolizing microsomal enzymes, such as cytochrome P450, NADPH Cyt P450 reductase and b(5) when compared with the control rats. The rats intoxicated with acetaminophen also significantly triggered serum TNF-alpha when compared with the control rats. These severe alterations in the drug metabolizing enzymes were appreciably prevented in the rats pretreated with S. polycystum. The rats pretreated with S. polycystum showed considerable inhibition in the elevation of TNF-alpha compared to the rats intoxicated with acetaminophen. The electron microscopic observation showed considerable loss of structural integrity of the endoplasmic reticulum, lipid infiltration and ballooning of mitochondria in the acetaminophen-intoxicated rats, whereas the rats treated with S. polycystum showed considerable protection against acetaminophen-induced alterations in structural integrity. CONCLUSION: These observations suggest that the animals treated with S. polycystum extract may have the ability to protect the drug metabolizing enzyme system and mitochondrial functional status from free radical attack, thereby showing its defense mechanism in protecting hepatic cells from acetaminophen toxic metabolite N-acetyl-para-benzoquinone-imine (NAPQI).

Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis.

Effectively assessing subtle hepatic metabolic functions by novel non-invasive tests might be of clinical utility in scoring NAFLD (non-alcoholic fatty liver disease) and in identifying altered metabolic pathways. The present study was conducted on 39 (20 lean and 19 obese) hypertransaminasemic patients with histologically proven NAFLD {ranging from simple steatosis to severe steatohepatitis [NASH (non-alcoholic steatohepatitis)] and fibrosis} and 28 (20 lean and eight overweight) healthy controls, who underwent stable isotope breath testing ([(13)C]methacetin and [(13)C]ketoisocaproate) for microsomal and mitochondrial liver function in relation to histology, serum hyaluronate, as a marker of liver fibrosis, and body size. Compared with healthy subjects and patients with simple steatosis, NASH patients had enhanced methacetin demethylation (P=0.001), but decreased (P=0.001) and delayed (P=0.006) ketoisocaproate decarboxylation, which was inversely related (P=0.001) to the degree of histological fibrosis (r=-0.701), serum hyaluronate (r=-0.644) and body size (r=-0.485). Ketoisocaproate decarboxylation was impaired further in obese patients with NASH, but not in patients with simple steatosis and in overweight controls. NASH and insulin resistance were independently associated with an abnormal ketoisocaproate breath test (P=0.001). The cut-off value of 9.6% cumulative expired (13)CO(2) for ketoisocaproate at 60 min was associated with the highest prediction (positive predictive value, 0.90; negative predictive value, 0.73) for NASH, yielding an overall sensitivity of 68% and specificity of 94%. In conclusion, both microsomal and mitochondrial functions are disturbed in NASH. Therefore stable isotope breath tests may usefully contribute to a better and non-invasive characterization of patients with NAFLD.

Behavioral and neurochemical effects induced by subchronic combined exposure to toluene at 40 ppm and noise at 80 dB-A in rats.

We investigated whether exposure to noise, in addition to its well-known potentiating effect on toluene-induced ototoxicity, may also exacerbate behavioral disturbances and brain neurochemical alterations produced by subchronic exposure to low toluene concentration. To test this hypothesis, we evaluated whether subchronic combined exposure (16 weeks, 104 h per week) to noise at 80 dB-A and toluene at 40 ppm potentiates the recently reported neurotoxic effects of subchronic exposure to 40 ppm toluene. Locomotor and rearing activities, sensitization to narcosis induced by acute toluene at high concentration, and tyrosine and tryptophan hydroxylase activities in the caudate-putamen and hippocampus were investigated in both male and female rats. Our results confirm that subchronic exposure to 40 ppm toluene significantly decreases rearing activity and leads to a sensitization to toluene-induced narcosis, as evaluated by loss of righting reflex, but fails to demonstrate any adverse effect of noise, alone or in combination with toluene. Given that toluene has addictive properties, the lack of potentiating behavioral and neurochemical effect of noise is discussed with regards to a recent study that has shown that methamphetamine neurotoxicity is potentiated by exposure to loud noise.