M. Teresa Donato

Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism: focus on in vitro studies.

Drug metabolism is the major determinant of drug clearance and, because of polymorphic or inducible expression of drug-metabolising cytochrome P450s (CYPs), is the factor most frequently responsible for interindividual differences in pharmacokinetics. A number of well characterised CYP substrates and inhibitors have been identified that allow precise measurements of individual CYP isoforms. Their use, alone or in combination, facilitates the phenotype characterisation of hepatocytes in vitro and in vivo.Two procedures are used for in vitro investigation of the metabolic profile of a drug: incubation with microsomes and incubation with metabolically competent cells. The major limitation of microsomes is that they express phase I activities, but only part of phase II activities, and can only be used for short incubation times. When intact cells are used, gene expression, metabolic pathways, cofactors/enzymes and plasma membrane are largely preserved, but fully differentiated cells such as primary cultured hepatocytes need to be used, since hepatoma cell lines have only very low and partial CYP expression. CYP-engineered cells or their microsomes (‘supersomes’) have made the identification of the CYPs involved in the metabolism of a drug candidate straightforward and easier.Inhibition of CYP is an undesirable feature for a drug candidate, and needs to be addressed by examining whether the drug candidate inhibits the metabolism of other compounds or whether other compounds inhibit the metabolism of the drug candidate. Such experiments can be conducted both with microsomes and in cells. The major limitation of microsomes is that inhibition parameters may not accurately reflect the situation in vivo, since the contribution of drug transport is not considered. The best picture of a potential drug-drug interaction can be obtained in metabolically competent hepatocytes.Screening of CYP inducers cannot be done in microsomes. It requires the use of a cellular system fully capable of transcribing and translating CYP genes, and can be monitored in vitro as an increase in enzyme mRNA or activity. Human hepatocytes in primary culture respond well to enzyme inducers during the first few days; this ability is lost thereafter. Rat hepatocytes are much less stable and soon become unresponsive to inducers. Hepatoma cell lines respond poorly to inducers, although the induction of a few isoenzymes has been reported. Primary cultured hepatocytes are still the unique in vitro model that allows global examination of the inductive potential of a drug.

Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method

Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. The method has been validated according to FDA guidelines, and it generally provides good results in terms of intra- and interday precision (less than 8.6% and 16.0%, respectively), accuracy (relative error measurement between –11.9% and 8.6%), and linearity (R2 > 0.996 and dynamic ranges between two and four orders of magnitude), with limits of quantification between 2.5 and 20 nM. The new analytical approach was applied to determine BA concentrations in human, rat, and mouse serum and in liver tissue. Our comparative study confirmed and extended previous reports, showing marked interspecies differences in circulating and hepatic BA composition. The targeted analysis revealed the presence of unexpected minoritary BAs, such as tauro-alpha-Muricholic acid in human serum, thus allowing us to obtain a thorough profiling of human samples. Its great sensitivity, low sample requirements (25 µl of serum, 5 mg of tissue), and comprehensive capacity to profile a considerable number of BAs make the present method a good choice to study BA metabolism in physiological and pathological situations, particularly in toxicological studies.

Potential Impact of Steatosis on Cytochrome P450 Enzymes of Human Hepatocytes Isolated from Fatty Liver Grafts

Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of “cellular steatosis” by incubation of cultured hepatocytes with increasing concentrations (0.25–3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45–65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.

Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models

BACKGROUND/AIMS The pig is considered the best donor of hepatocytes for bioartificial liver devices, but little is known about the metabolic capability of pig hepatocytes. Therefore, we have evaluated drug metabolizing activities in pig hepatocytes and liver microsomes and compared the results with those of man and other animal hepatic cellular models that are potential sources of cells for bioreactors, such as rat, rabbit and dog hepatocytes and hepatoma cell lines. METHODS Total cytochrome P450 levels, six phase 1 activities representative of the most relevant cytochrome P450 enzymes (7-ethoxycoumarin O-deethylase, 7-ethoxy-, 7-methoxy- and 7-benzoxyresorufin O-dealkylases, coumarin 7-hydroxylase and p-nitrophenol hydroxylase), two phase 2 activities (glutathione S-transferase and UDP-glucuronyltransferase) and CYP-dependent regioselective testosterone metabolism were evaluated in in vitro models of different species. RESULTS The pattern of specific cytochrome P450 activities and the metabolic profile of testosterone in intact hepatocytes were essentially the same as those measured in liver microsomes. Relatively low ethoxy-, methoxy-, and benzoxyresorufin O-dealkylation rates were found in pig liver microsomes and hepatocytes as compared to hepatic in vitro human models. However, in contrast with the other species studied, stereoselective testosterone oxidation profiles were practically identical in human and pig models. Finally, the metabolic capability of hepatoma cell lines was very limited in comparison with that of hepatocytes. CONCLUSIONS Pig hepatocytes are able to maintain in culture the phase 1 and phase 2 activities found in liver microsomes. The high metabolic similarities found between pig and human hepatocytes lend support to the use of pig hepatocytes in bioartificial liver devices.

Development of a Multiparametric Cell-based Protocol to Screen and Classify the Hepatotoxicity Potential of Drugs

Hepatotoxicity is a major reason for drug nonapprovals and withdrawals. The multiparametric analysis of xenobiotic toxicity at the single cells level using flow cytometry and cellular imaging-based approaches, such as high-content screening (HCS) technology, could play a key role in the detection of toxicity and the classification of compounds based on patterns of cellular injury. This study aimed to develop and validate a practical, reproducible, in vitro multiparametric cell-based protocol to assess those drugs that are potentially hepatotoxic to humans and to suggest their mechanisms of action. The assay was applied to HepG2 human cell line cultured in 96-well plates and exposed to 78 different compounds for 3 and 24 h at a range of concentrations (1-1000μM). After treatments, cells were simultaneously loaded with five fluorescent dyes showing optical compatibility and were then analyzed with the High-Content Screening Station Scan^R (Olympus). By using the new technology of HCS cell parameters associated with nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium concentration, and oxidative stress, indicative of prelethal cytotoxic effects and representative of different mechanisms of toxicity, were measured at the single cells level, which allows high-throughput screening. This strategy appears to identify early and late events in the hepatotoxic process and also suggests the mechanism(s) implicated in the toxicity of compounds to thereby classify them according to their degree of injury (no injury, low, moderate, and high injury).

A Comprehensive Untargeted Metabonomic Analysis of Human Steatotic Liver Tissue by RP and HILIC Chromatography Coupled to Mass Spectrometry Reveals Important Metabolic Alterations

Steatosis, or excessive accumulation of lipids in the liver, is a generally accepted previous step to the development of more severe conditions like nonalcoholic steatohepatitis, fibrosis, and cirrhosis. We aimed to characterize the metabolic profile that defines simple steatosis in human tissue and to identify potential disturbances in the hepatic metabolism that could favor the switch to progressive liver damage. A total of 46 samples, 23 from steatotic and 23 from nonsteatotic human livers, were analyzed following a holistic LC-MS-based metabonomic analysis that combines RP and HILIC chromatographic separations. Multivariate statistical data analysis satisfactorily classified samples and revealed steatosis-associated biomarkers. Increased levels of bile acids and phospholipid degradation products, and decreased levels of antioxidant species, were found in steatotic livers, indicating disturbances in lipid and bile acid homeostasis and mitochondrial dysfunction. Changes in hypoxanthine, creatinine, glutamate, glutamine, or γ-glutamyl-dipeptides concentrations, suggestive of alterations in energy metabolism and amino acid metabolism and transport, were also found. The results show that the proposed analytical strategy is suitable to achieve a comprehensive metabolic profile of steatotic human liver tissue and provide new insights into the metabolic alterations occurring in fatty liver that could contribute to its predisposition to damage evolution.

Drug metabolizing enzymes in rat hepatocytes co-cultured with cell lines

SummaryWe have developed new co-cultures of continuous cell lines 3T3 (clone A31) and C3H/10T1/2 (colone 8) with hepatocytes as an alternative to co-cultures with nonconinuous epithelial cells. In this biological system we studied in detail the expression of the hepatic biotransformation system. After 7 d in culture, total cytochrome P-450 content and the monooxygenase activities aryl hydrocarbon hydroxylase and 7-ethoxycoumarino-deethylase still maintained about 30% of their initial value, whereas in pure cultured hepatocytes these activities were undetectable. A significant response to induction by methylcholanthrene and phenobarbital of monooxygenase activities was observed in co-cultures for 7 d. NADPH-cytochrome c reductase activity remained unchanged for at least 7 d in co-cultured hepatocytes, whereas in pure cultures this activity was reduced to about 75% of the initial value after only 24 h. Finally, the activity of the conjugating enzymes UDP-Gt and GSH-t was maintained at nearly the initial levels during the complete period of study. The easy handling of continuous cell lines and the maintenance of the biotransformation system of hepatocytes in co-culture make this approach simpler and easier to standardize.

Competency of different cell models to predict human hepatotoxic drugs

Introduction: The liver is the most important target for drug-induced toxicity. This vulnerability results from functional liver features and its role in the metabolic elimination of most drugs. Drug-induced liver injury is a significant leading cause of acute, chronic liver disease and an important safety issue when developing new drugs. Areas covered: This review describes the advantages and limitations of hepatic cell-based models for early safety risk assessment during drug development. These models include hepatocytes cultured as monolayer, collagen-sandwich; emerging complex 3D configuration; liver-derived cell lines; stem cell-derived hepatocytes. Expert opinion: In vitro toxicity assays performed in hepatocytes or hepatoma cell lines can potentially provide rapid and cost–effective early feedback to identify toxic candidates for compound prioritization. However, their capacity to predict hepatotoxicity depends critically on cells’ functional performance. In an attempt to improve and prolong functional properties of cultured cells, different strategies to recreate the in vivo hepatocyte environment have been explored. 3D cultures, co-cultures of hepatocytes with other cell types and microfluidic devices seem highly promising for toxicological studies. Moreover, hepatocytes derived from human pluripotent stem cells are emerging cell-based systems that may provide a stable source of hepatocytes to reliably screen metabolism and toxicity of candidate compounds.

Effect of xenobiotics on monooxygenase activities in cultured human hepatocytes

The activity of human cytochrome P450 monooxygenases, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase can be increased by 3-methylcholanthrene, phenobarbital and ethanol in human hepatocytes maintained in primary culture. Total cytochrome P450 content increased two-fold after 48 hr of incubation with methylcholanthrene or phenobarbital and 1.5-fold after incubation with ethanol. The three chemicals elicited different effects on cytochrome P450 dependent activities. Addition of 3-methylcholanthrene caused a time- and concentration-dependent increase in both monooxygenase activities, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase, while phenobarbital and ethanol increased 7-ethoxycoumarin O-deethylase activity but had no effect on aryl hydrocarbon hydroxylase. Dexamethasone per se had little or no effect on either monooxygenase activities, but potentiated the effect of the three chemicals on 7-ethoxycoumarin O-deethylase.

Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers

SummaryThe stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation).