Structure-activity relationships for halobenzene induced cytotoxicity in rat and human hepatocytes.

Halobenzenes are ubiquitous environmental contaminants, which are hepatotoxic in both rodents and humans. The molecular mechanism of halobenzene hepatotoxicity was investigated using Quantitative structure-activity relationships (QSAR) and accelerated cytotoxicity mechanism screening (ACMS) techniques in rat and human hepatocytes. The usefulness of isolated hepatocytes for prediciting in vivo xenobiotic toxicity was reassessed by correlating the LC(50) of 12 halobenzene congeners in phenobarbital (PB) induced rat hepatocytes in vitro determined by ACMS to the hepatotoxicities reported in vivo in PB-induced male Sprague-Dawely (SD) rats. A high correlation (r(2)=0.90) confirmed the application of hepatocytes as a "gold standard" for toxicity testing in vitro. QSARs were derived to determine the physico-chemcial variables that govern halobenzene toxicity in PB-induced rat, normal rat and human hepatocytes. We found that toxicity in normal rat and normal human hepatocytes both strongly correlate with hydrophobicity (logP), ease of oxidation (E(HOMO), energy of the highest molecular orbital) and on the asymmetric charge distribution according to arrangement of halogen substituents (dipole moment, mu). This suggests that halobenzene interaction with cytochrome P450 for oxidation is the metabolic activating path for toxicity and is similar in both species. In PB-induced rat hepatocytes the QSAR derivation is changed, where halobenzene toxicity strongly correlates to logP and dipole moment, but not E(HOMO). The changed QSAR suggests that oxidation is no longer the rate-limiting step in the cytotoxic mechanism when CYP2B/3A levels are increased, confirming CYP450 oxidation as the metabolic activating step under normal conditions.

An estimate of the number of hepatocyte donors required to provide reasonable estimates of human hepatic clearance from in vitro experiments.

Cryopreserved human hepatocytes in suspension were used to estimate in vivo hepatic clearances for six different drugs. In vitro intrinsic clearances were measured on the basis of substrate depletion. The number of different hepatocyte donors required for a reasonable estimate of in vivo hepatic clearance--within twice or (1/2) of the actual value--was determined. Depending upon the desired level of confidence, anywhere from 9-20 donors are required by this method.

New cytochrome P450 2D6*56 allele identified by genotype/phenotype analysis of cryopreserved human hepatocytes.

Genotype/phenotype analysis with human hepatocytes has identified a new inactive CYP2D6 allele, CYP2D6*56. Cryopreserved human hepatocytes from 51 livers were evaluated for CYP2D6 activity with dextromethorphan as the probe substrate. Hepatocyte lots that lacked CYP2D6 activity were further evaluated for CYP2D6 expression and known genetic variations, including CYP2D6*2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *14, *15, *17, *18, *19, *20, *25, *26, *29, *30, *35, *40, *41, *43, and various multiple copy CYP2D6 alleles (*1xn, *2xn, and *4xn) by the AmpliChip CYP450 prototype microarray (Roche Molecular Systems, Inc., Branchburg, NJ). Two discrepancies were uncovered between the CYP2D6 genotype and activity by this approach. In one sample, a previously unreported 3201C 224 T transition in exon 7 resulted in Arg344(CGA) being replaced by a stop codon (TGA), resulting in a CYP2D6 enzyme lacking the terminal 153 amino acids. This allele was given the designation of CYP2D6*56 and the GenBank accession number DQ282162. The lack of CYP2D6 activity in cryopreserved hepatocytes and microsomes found in the second sample, despite a normal level of CYP2D6 expression and a genotype (*10/*1) predictive of normal CYP2D6 activity, was attributed to enzyme inactivation by an unknown metabolite. The identification and characterization of the CYP2D6*56 allele indicates that commercial cryopreserved human hepatocytes may provide a valuable means to rapidly identify genetic variations with functional relevance. This integrated approach of identifying alleles and examining allele relationships to gene expression and function could be of tremendous value to understanding the mechanism responsible for functional differences in gene variation. The commercial availability of human cryopreserved hepatocytes also makes this potential readily available to any who are interested in it, not just those with access to private liver banks.

Prediction of hepatic clearance using cryopreserved human hepatocytes: a comparison of serum and serum-free incubations.

Cryopreserved human hepatocytes have been used to predict hepatic in-vivo clearance. Physiologically-based direct scaling methods generally underestimate human in-vivo hepatic clearance. Cryopreserved human hepatocytes were incubated in 100% serum and in serum-free medium to predict the in-vivo hepatic clearance of six compounds (phenazone (antipyrine), bosentan, mibefradil, midazolam, naloxone and oxazepam). Monte Carlo simulations were performed in an attempt to incorporate the variability and uncertainty in the measured parameters to the prediction of hepatic clearance. The intrinsic clearance (CL(int)) and the associated variability of the six compounds decreased in the presence of serum and the values were reproducible across donors. The predicted CL(hep, in-vivo) obtained with hepatocytes from donors incubated in serum was more accurate than the prediction obtained in the absence of serum. For example, the CL(hep, in-vivo) of mibefradil in donor GNG was 4.27 mL min(-1) kg(-1) in the presence of serum and 0.46 mL min(-1) kg(-1) in the absence of serum (4.88 mL min(-1) kg(-1) observed in-vivo). Using the results obtained in this study together with an extended data set (26 compounds), the clearance of 77% of the compounds was predicted within a 2-fold error in the absence of serum. In the presence of serum, 85% of the compounds were successfully predicted within a 2-fold error. In conclusion, cryopreserved human hepatocyte suspensions represented a convenient and predictive model to assess human drug clearance.

Human and animal hepatocytes in vitro with extrapolation in vivo.

Human and animal hepatocytes are now being used as an in vitro technique to aid drug discovery by predicting the in vivo metabolic pathways of drugs or new chemical entities (NCEs), identifying drug-metabolizing enzymes and predicting their in vivo induction. Because of the difficulty of establishing whether the cytotoxic susceptibility of human hepatocytes to xenobiotics/drugs in vitro could be used to predict in vivo human hepatotoxicity, a comparison of the susceptibility of the hepatocytes of human and animal models to six chemical classes of drugs/xenobiotics in vitro have been related to their in vivo hepatotoxicity and the corresponding activity of their metabolizing enzymes. This study showed that the cytotoxic effectiveness of 16 halobenzenes towards rat hepatocytes in vitro using higher doses and short incubation times correlated well with rat hepatotoxic effectiveness in vivo with lower doses/longer times. The hepatic/hepatocyte xenobiotic metabolizing enzyme activities of various animal species and human have been reviewed for use by veterinarians and research scientists. Where possible, recommendations have been made regarding which animal hepatocyte model is most applicable for modeling the susceptibility to xenobiotic induced hepatotoxicity of those humans with slow versus rapid metabolizing enzyme polymorphisms. These recommendations are based on the best human fit for animal drug/xenobiotic metabolizing enzymes in terms of activity, kinetics and substrate/inhibitor specificity. The use of human hepatocytes from slow versus rapid metabolizing individuals for drug metabolism/cytotoxicity studies; and the research use of freshly isolated rat hepatocytes and "Accelerated Cytotoxicity Mechanism Screening" (ACMS) techniques for identifying drug/xenobiotic reactive metabolites are also described. Using these techniques the molecular hepatocytotoxic mechanisms found in vitro for seven classes of xenobiotics/drugs were found to be similar to the rat hepatotoxic mechanisms reported in vivo.

Catechol-O-methyltransferase low activity genotype (COMTLL) is associated with low levels of COMT protein in human hepatocytes.

A single nucleotide polymorphism in the COMT (catechol-O-methyltransferase) gene that alters the amino acid sequence at codon 108 of S-COMT from val to met (val108met polymorphism) has been associated with a number of diseases and neuropsychiatric disorders. Several studies have shown that the met108 allele (COMTL) is associated with three to four-fold lower levels of COMT activity, compared to the val108 allele (COMTH), in extracts of human erythrocytes, liver and kidney tissue. We hypothesized that the differences in COMT activity observed in these studies were due to differing levels of COMT protein in cells and tissues with varying COMT genotypes. In order to address this, we obtained hepatocytes from 31 Caucasian female donors and determined their COMT genotype, COMT activity and COMT protein levels. We found that both cytosolic COMT activity and cytosolic COMT protein levels are lower in hepatocytes from COMTLL individuals, and that COMT activity levels correlate with COMT protein levels. Therefore, lower COMT activity seen in tissues and cells with the COMTLL genotype is likely due to lower COMT protein levels compared with tissues and cells from COMTHH individuals.

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes.

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.

Maintenance of liver functions in rat hepatocytes cultured as spheroids in a rotating wall vessel.

Rat hepatocytes were cultured initially as spheroids on culture plates and then transferred into a rotating wall vessel (high-aspect ratio vessel [HARV]) for further culturing. Morphological evaluation based on electron microscopy showed that hepatocyte spheroids cultured for 30 d in the HARV had a compact structure with tight cell-cell junctions, numerous smooth and rough endoplasmic reticulum, intact mitochondria, and bile canaliculi lined with microvilli. The viability and differentiated properties of the hepatocytes cultured in the HARV were further substantiated by the presence of both phase I oxidation and phase II conjugation drug-metabolizing enzyme activities, as well as albumin synthesis. Homogenates prepared from freshly isolated hepatocytes and hepatocytes cultured in the HARV showed similar cytochrome P450 2B activities measured as pentoxyresorufin-O-dealkylase and testosterone 16beta-hydroxylase. Further, intact hepatocytes cultured in the HARV were found to metabolize chlorzoxazone to 6-hydroxychlorzoxazone; dextromethorphan to dextrorphan, 3-methoxymorphinan, and 3-hydroxymorphinan; midazolam to 1-hydroxymidazolam and 4-hydroxymidazolam; and 7-hydroxycoumarin to its glucuronide and sulfate conjugates. In conclusion, we found that hepatocyte spheroids could be cultured in a HARV to retain cellular and physiological properties of the intact liver, including drug-metabolizing enzyme activities, plasma protein production, and long-term (1 mo) maintenance of viability and cellular function.

Differential in vitro hepatotoxicity of troglitazone and rosiglitazone among cryopreserved human hepatocytes from 37 donors.

We report here our studies on troglitazone and rosiglitazone cytotoxicity in human hepatocytes isolated from multiple donors to investigate factors responsible for individual differences in sensitivity to the known hepatotoxicity of these antidiabetic drugs. Using cellular adenosine triphosphate (ATP) content as an endpoint, cytotoxicity of both drugs was evaluated in cryopreserved human hepatocytes from 37 donors. We confirmed reports of others that troglitazone was cytotoxic to human hepatocytes using cellular ATP content as an endpoint. In addition, we found that rosiglitazone, although less toxic in the study population, was cytotoxic to hepatocytes in some donors (EC(50)<100 microM). ATP content, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolism, depletion of intracellular glutathione, Alamar Blue metabolism, and neutral red uptake were used as endpoints in a single donor study using freshly isolated human hepatocytes. Troglitazone appeared to be more toxic than rosiglitazone by all endpoints. From the demographic data provided to us for each donor, we were able to establish no direct correlation between cytotoxicity (expressed as EC(50) values) and age, sex, smoking status, or alcohol consumption. We conclude that troglitazone and rosiglitazone are differentially toxic to human hepatocytes, and that toxicity may be independent of age, sex, tobacco use, and alcohol use.

Identification of glutathione conjugates of troglitazone in human hepatocytes.

Troglitazone (TGZ) is an orally active antihyperglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus. Several cases of liver failure following TGZ administration led to its withdrawal from the market. The mechanism of toxicity is still not understood. The formation of toxic metabolites is believed to play an important role. Herein, we report the biotransformation of TGZ in human hepatocytes. TGZ at 50 microM concentration was incubated with cryopreserved human hepatocytes. Four metabolites were found-glucuronide, sulfate, and two glutathione (GSH) conjugates of TGZ. The two GSH metabolites could be conjugation at the 6-hydroxychromane nucleus and the thiazolidinedione ring. Alternatively, the conjugation could be one of the two rings, with the two GSH metabolites are diastereomers. The sulfate conjugate was the major metabolite found. The cytochrome P450 (CYP) inhibitors furafylline (CYP1A1/2), omeprazole (CYP2C19), ketoconazole (CYP3A4), and sulfaphenazole (CYP2C9) had no inhibitory effect on the TGZ metabolism suggesting that several P450s may play a role in the TGZ metabolic pathway. Previous studies in our laboratory have shown a large interindividual variation between different donors in cytotoxicity after dosing with TGZ. Based on EC(50) values, donors were classified as sensitive or resistant. The sensitive human donors were found to form significantly less troglitazone GSH conjugates and glucuronides than the resistant donors.