Human hepatocytes as a tool for studying toxicity and drug metabolism

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.     

Age-related toxicity of paracetamol in mouse hepatocytes

Hepatocytes from postnatal and adult mice were isolated by perfusion of the liver with a collagenase-containing bicarbonate buffer. These were allowed to attach to collagen-coated tissue culture dishes and were then examined for their susceptibility to paracetamol toxicity. After an 8 hr incubation in either 0.1 or 1.0 mM paracetamol, the extent of lactate dehydrogenase leakage and depletion of glutathione were similar in hepatocytes from young (1-, 2- and 3-week-old) mice when compared to adult mice. The covalent binding of [14C]-paracetamol to protein was greater in the hepatocytes from young mice. The results indicate that while the amount of reactive metabolites free to react with cellular constituents is greater in hepatocytes from young mice, the amount of damage produced was not different than that found in those from adults.     

tert-Butyl hydroperoxide-induced injury in isolated rat hepatocytes: a model for studying anti-hepatotoxic crude drugs

Tert-butyl hydroperoxide induces in freshly isolated rat hepatocytes malonaldehyde formation and lacticodehydrogenase and aspartate amino-transferase leakage. This model, when adapted to crude extracts, permits the demonstration of both anti-lipoperoxidant and antihepatotoxic activity of reference products like quercetin and silymarin and plant extracts like Rosmarinus officinalis and Eschscholzia californica.     

Freshly prepared rat hepatocytes used in screening the toxicity of blue-green algal blooms

The acute toxicity of extracts of blue-green algae was tested in freshly prepared rat hepatocytes in suspension. The results were compared with the traditional in vivo mouse bioassay. Sixty samples of natural algal blooms from freshwater lakes in Norway, Sweden, and Finland and 14 samples cultured in the laboratory were tested. The mouse bioassay revealed hepatotoxins in a large number of the algae, while neurotoxins were not found. Acute hepatotoxicity in vitro was scored by measurement of leakage of the enzyme lactate dehydrogenase (LDH) from damaged cells and of morphological changes of the cells. The correlation coefficients between mouse toxicity and LDH, mouse toxicity and morphological cell damage, and between LDH and morphological cell damage were 0.812, 0.735, and 0.882, respectively. Consequently, the rat hepatocyte toxicity test seems to be well suited for screening blooms of blue-green algae for the presence of hepatotoxins.     

A role for the glutathione peroxidase/reductase enzyme system in the protection from paracetamol toxicity in isolated mouse hepatocytes

The role of the glutathione peroxidase/reductase (GSH-Px/GSSG-Rd) enzyme system in protection from paracetamol toxicity was investigated in isolated mouse hepatocytes in primary culture. The effect of inhibitors of these enzymes on the toxicity of paracetamol and on t-butylhydroperoxide (t-BOOH), used as a positive control, was determined. 1,3-Bis(chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GSSG-Rd, and goldthioglucose (GTG) used to inhibit GSH-Px. Both these inhibitors increased cell membrane damage in response to oxidative stress initiated by t-BOOH. However, they also increased the susceptibility of hepatocytes to paracetamol toxicity, indicating that a component of paracetamol's toxic effect involves formation of species that are detoxified by the GSH-Px/GSSG-Rd enzymes. To further examine the role of these enzymes, age-related differences in their activity were exploited. Hepatocytes from two-week-old mice were less susceptible to both t-BOOH and paracetamol toxicity than were those from adult mice. This corresponds to higher activity of cytosolic GSH-Px/GSSG-Rd in this age group. However, after inhibition of GSSG-Rd with BCNU, hepatocytes from these postnatal mice were more susceptible to paracetamol toxicity. This suggests that the higher activity of GSH-Px/GSSG-Rd in hepatocytes from two-week-old mice is responsible for their reduced susceptibility to paracetamol toxicity. The data indicate that the GSH-Px/GSSG-Rd enzymes contribute to protection from paracetamol toxicity and suggest that formation of peroxides contributes to this drug's hepatotoxic effects.     

The effectiveness of N-acetylcysteine in isolated hepatocytes,against the toxicity of paracetamol,acrolein, and paraquat

The protective effect of N-acetylcysteine against the toxicity of paracetamol, acrolein, and paraquat was investigated using isolated hepatocytes as the experimental system. N-acetylcysteine protects against paracetamol toxicity by acting as a precursor for intracellular glutathione. N-acetylcysteine protects against acrolein toxicity by providing a source of sulfhydryl groups, and is effective without prior conversion. Paraquat toxicity can be decreased by coincubating the cells with N-acetylcysteine, but the mechanism for the protective effect is not as clear in this instance. It is probable that N-acetylcysteine protects against paraquat toxicity by helping to maintain intracellular glutathinone levels.     

Acrylamide toxicity in isolated rat hepatocytes

Acrylamide (ACR) is an important industrial chemical used primarily in the production of polymers and co-polymers. Acrylamide is mainly neurotoxic to experimental animals as well as humans and has also been shown to be mutagenic and carcinogenic. The present study was designed to investigate the toxicity of ACR on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and were incubated with different concentrations of ACR (0.1, 1, 10 m ) for 2 hours. Cell viability by trypan blue exclusion and leakage of the enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) were determined. Reduced glutathione (GSH), glutathione S-transferase (GST) activity were also measured. A significant decrease in the cell viability was observed after exposure to 10 m ACR for 30 min, while 1 m ACR caused a significant decrease in the viability after 60 min. ALT leakage was parallel to the cell viability. AST leakage was significantly increased at 30 min of incubation with 10 m ACR, whereas 2 hours of incubation was required for the leakage of AST from rats hepatocytes with 1 m ACR. 10 m ACR decreased significantly GSH as early as 30 min, while GSH level was decreased at 60 min after exposure to 1 m ACR. Also, the GST activity increased with increasing the dose of ACR. Cytochrome P450 concentration was decreased after exposure to 10 m ACR. The effect of ACR on cell viability, ALT and AST leakage, GSH and GST activity was time and dose dependent.     

Copper toxicity in isolated rat hepatocytes

In relative excess, copper is a cytotoxic metal. The injury may be related to the process of lipid peroxidation. Isolated hepatocytes provide a suitable system for an examination of this aspect of copper toxicity. Furthermore, interactions between copper and agents that protect against its toxic effects in vivo can be examined at a cellular level by use of isolated hepatocytes. Therefore, isolated rat hepatocytes were incubated with varying cupric chloride concentrations (5–200 μm) for up to 90 min. The copper caused a concentration and time-related decrease in cell viability as assessed by loss of intracellular potassium ion (K⁺) and aspartate aminotransferase (AST). An increase in lipid peroxidation and a decrease in reduced glutathione were also observed in response to copper. Of several potentially interactant compounds tested, only chromic chloride, diethyldithiocarbamate, and penicillamine were found to reduce the loss of K⁺. Ammonium molybdate alone and in combination with sodium sulfate were able to markedly decrease the release of AST from the hepatocytes. While the antioxidants, butylated hydroxyanisole and N,N′-diphenyl-p-phenylenediamine decreased the lipid peroxidation attributable to copper, they had no protective effects against loss of cell viability. This suggests that lipid peroxidation is not the cause of the injurious effects of this metal in isolated rat hepatocytes.     

Primary hepatocytes as a model to analyze species-specific toxicity and drug metabolism

BACKGROUND: Compound failures have been emerging in later stages of pharmaceutical drug development and are in many cases not detected until the administration to humans in clinical trials or even after approval. Among the most frequent adverse effects are drug-induced liver injury generated by species-specific susceptibilities (e.g., in xenobiotic metabolism and/or the occurrence of idiosyncratic drug hepatotoxicity). OBJECTIVES: Detecting or predicting unfavorable drug effects on the liver as early as possible in drug development is crucial in making the drug development process more efficient and the application of new drugs to humans in clinical studies and medical use safer. Methods: To achieve this goal, primary hepatocyte cultures from various species, including humans, are analyzed for morphological, functional and gene expression alterations after compound treatment. RESULTS/CONCLUSION: Primary hepatocyte cultures appear to be a promising tool for the detection of general or liver toxicity and the evaluation of species-specific drug effects.     

Vasocystostomy: a model for studying male reproductive toxicity in the rat

A nonsacrificial rat model has been developed which permits the easy collection and measurement of spermatozoa. The ductus deferens is anastomosed to the bladder (Vasocystostomy) and urinary sperm is collected daily. The correlation between testicular histology and sperm counts indicates that this model is reliable. Using this model we demonstrate inhibition of sperm production by fluoroacetate.     

Benoxaprofen induced toxicity in isolated rat hepatocytes

The toxicity of benoxaprofen, a non-steroidal anti-inflammatory compound was investigated using rat hepatic microsomal and isolated hepatocyte suspensions. In microsomes, benoxaprofen produced a Type I binding spectra and competitively inhibited (k_i 380 μM) the oxidative metabolism of aminopyrine. Marked toxicity was observed following incubation of benoxaprofen with isolated hepatocytes from either untreated, phenobarbitone (PB) or 3-methylcholanthrene (3-MC) pretreated male rats. In untreated hepatocytes increases in the intracellular lactate/pyruvate (L/P) ratio and alanine aminotransferase (ALT) release were related to the benoxaprofen concentration and duration of incubation. Alterations in L/P ratio preceded the release of cytosolic ALT and at 4 h a well defined dose-response relationship existed between the benoxaprofen concentration and the observed increases in the L/P ratio and ALT release. Pretreatment of animals with either PB or 3-MC did not affect the temporal nature nor the magnitude of the hepatocyte response to benoxaprofen. In addition, inhibitors of cytochrome P-450 isozymes (SKF-525A, metyrapone and -napthoflavone) were ineffective with regard to modifying the observed toxicity. The results of this study suggest that hepatic cytochrome P-450 mediated metabolism may not be implicated in the toxicity of benoxaprofen in isolated hepatocytes. However, alterations in the cellular redox state and evidence of plasma membrane bleb formation suggest that benoxaprofen may uncouple oxidative phosphorylation and disturb intracellular calcium ion homeostasis.     

Mechanisms of toxicity of naphthoquinones to isolated hepatocytes

The possible mechanisms of naphthoquinone-induced toxicity to isolated hepatocytes were investigated using three structurally-related naphthoquinones, 1,4-naphthoquinone (1,4-NQ), 2-methyl-1,4-naphthoquinone (2-Me-1,4-NQ) and 2,3-dimethyl-1, 4-naphthoquinone (2,3-diMe-1,4-NQ). 1,4-NQ was more toxic than 2-Me-1,4-NQ whereas 2,3-diMe-1,4-NQ did not cause cell death at the solubility-limited concentrations used. All three naphthoquinones extensively depleted intracellular glutathione (GSH). However, the depletion of GSH induced by 1,4-NQ and 2-Me-1,4-NQ prior to cell death was more rapid and extensive than that induced by the nontoxic 2,3-diMe-1,4-NQ. Further studies demonstrated that 2,3-diMe-1,4-NQ was cytotoxic in the presence of dicoumarol, a compound which also potentiates the cytotoxicity of 1,4-NQ and 2-Me-1,4-NQ. To investigate the differential cytotoxicity of these three naphthoquinones, their relative capacities to redox cycle and to bind covalently to cellular nucleophiles were assessed. Redox cycling was investigated using rat liver microsomes where the order of potency for quinone-stimulated redox cycling was 1,4-NQ approximately 2-Me-1,4-NQ much greater than 2,3-diMe-1,4-NQ as indicated by nonstoichiometric amounts of NADPH oxidation and O2 consumption. NADPH-cytochrome P-450 reductase was implicated as the enzyme primarily responsible for naphthoquinone-stimulated redox cycling. The reactivity of the naphthoquinones with glutathione and, by implication, with other cellular nucleophiles was 1,4-NQ greater than 2-Me-1,4-NQ much greater than greater than 2,3-diMe-1,4-NQ. Overall, these studies indicate that 2,3-diMe-1,4-NQ is not cytotoxic (except in the presence of dicoumarol) and this lack of toxicity may be related either to its lesser capacity to redox cycle and/or its inability to react directly with cellular nucleophiles.     

Hamster hepatocytes in culture as a model for acetaminophen toxicity: Studies with inhibitors of drug metabolism

A hamster hepatocyte system was developed for use in studying the toxicity of acetaminophen (APAP). The cells were isolated and placed in culture conditions in petri dishes containing a film of collagen. Hepatocytes, after attachment to collagen, were exposed for various periods of time to different concentrations of APAP. Hepatocytes exposed to APAP exhibited concentration- and time-dependent GSH depletion followed by cytoplasmic enzyme leakage and an increase in malondialdehyde content (TBA-reactive material). These effects were reduced by the drug metabolism inhibitors metyrapone, piperonyl butoxide, and dithiocarb. Removal of APAP and its unbound metabolites from cells prior to 1.5 hr followed by culture in drug-free medium resulted in no observable damage to the cells over a 24-hr period. Removal of drug after longer exposure times followed by culture in fresh medium resulted in eventual cell damage. This finding showed that deleterious changes caused by APAP occurred over a 1.5-hr period after which eventual hepatocyte damage could not be reversed by removal of the drug. Further experiments showed that metyrapone and dithiocarb had some protective effect when added after APAP had been completely removed from damaged cells. This result indicates that these agents have a protective action separate from, and in addition to, their ability to inhibit APAP oxidation via cytochromes P-450     

Zebrafish as a model for studying the developmental neurotoxicity of propofol

Anesthetics can cause widespread apoptotic neurodegeneration and adverse effects on synaptogenesis during early postnatal life. Synaptogenesis correlates with several proteins, including myelin basic protein (MBP). However, little is known about the adverse effects of exposure to propofol on MBP, particularly during embryonic development. Our goal was to use zebrafish to explore the effect of propofol on embryonic development, apoptosis and MBP expression. Zebrafish embryos were exposed to propofol at defined doses and stages from 6 to 48 h postfertilization by immersion. The survival rate, hatchability, aberration rate, cell apoptosis and gene expression were analyzed at defined stages. Analysis revealed that doses of 1, 2 and 3 µg ml^–1 propofol were reasonable anesthetic concentrations for zebrafish embryos. These doses of propofol caused a significant decrease in hatchability and an increase in aberration rate. Moreover, 6 days postfertilization (dpf) larvae are anesthetized by immersion into water containing 1, 2 or 3 µg ml^–1 of propofol. The number of apoptotic cells in the head of propofol‐treated 36 h postfertilization embryos were significantly increased, and the expression of caspases‐3, ‐8 and ‐9 were upregulated. Apoptosis was also induced in the brain of 3 dpf larvae exposed to propofol. However, propofol caused a decrease in mbp gene and protein (dose‐dependent) expression levels in the central nervous system of 3 dpf zebrafish. These data show that embryonic exposure to propofol is neurotoxic, causing increased apoptosis and decreased MBP expression. We believe zebrafish can be used as a novel model to explore the mechanisms of propofol neurotoxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

Acute toxicity of hexavalent chromium in isolated teleost hepatocytes

Acute toxic effects of hexavalent chromium [Cr(VI)], a widely recognised carcinogenic, mutagenic and redox active metal, were investigated in isolated hepatocytes of goldfish (Carassius auratus). Exposure to 250 microM Cr(VI) induced a significant decrease of cell viability from 94% in controls to 88% and 84% after 30 min and 4 h of exposure, respectively. Cr-toxicity was associated with a concentration-dependent stimulation of the formation of reactive oxygen species (ROS). As one potential source of ROS formation we identified the lysosomal Fe(2+) pool, since the ferric ion chelator deferoxamin inhibited ROS formation by approximately 15%. Lysosomal membranes remained nevertheless intact during Cr-exposure, as determined from neutral red retention in this compartment. Another significant source of ROS appear to be the mitochondria, where a presumably uncoupled increase of respiration by 20-30% was triggered by the metal. Inhibition of mitochondrial respiration by cyanide caused an approximately 40% decrease of Cr-induced ROS-formation, whereas the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazine was without effect. Cellular Ca(2+) homeostasis was not disturbed by Cr(VI) and thus played no role in this scenario. Overall, our data show that Cr(VI) is acutely toxic to goldfish hepatocytes, and its toxicity is associated with the induction of radical stress, presumably involving lysosomes and mitochondria as important sources of ROS formation.     

Chemical structure and toxicity of diuretics in isolated hepatocytes

The effects of several diuretics, including tienilic acid and indacrinone, on isolated rat heaptocytes were examined. Addition of tienilic acid and indacrinone at 1 mM to a suspension of freshly isolated cells caused dose-dependent loss of cell viability as judged by the LDH-latency test. Survey of 19 structurally related compounds revealed that the extent of cell injury and chemical structure were correlated, and an intense adverse effect was attributed to the 2-thienylcarbonyl moiety. Several other factors influencing cell viability are also disclosed. Further study revealed that tienilic acid and indacrinone were toxic to the primary culture of hepatocytes at a lower dose than that a freshly isolated hepatocytes. Thus, an isolated hepatocyte system can be used to select compounds displaying low hepatotoxicity, as for example is needed when screening diuretics.     

The toxicity of disulphides to isolated hepatocytes and mitochondria

The disulfide metabolites of thiono-sulfur drugs were found to be about 50 to 100 times more toxic to isolated rat hepatocytes than the corresponding parent drugs. The order of decreasing cytotoxicity for the disulfide metabolites was disulfiram greater than propylthiouracil disulfide greater than formamidine disulfide greater than phenylthiourea disulfide greater than thiobenzamide disulfide greater than cystamine. Depletion of intracellular GSH levels preceded cytotoxicity. GSH could be restored and cytotoxicity averted by adding the thiol reducing dithiothreitol. Depletion of GSH with diethylmaleate potentiated the toxicity of disulfides 3 to 4-fold confirming the protective role of GSH in disulfide toxicity. The toxicity of disulfiram was increased 4-fold in cells pretreated with ATP (0.8 mM) to effect a transient increase in cytosolic Ca2+ suggesting an impairment of Ca2+ homeostasis by the toxicant. Disulfiram (200 microM) rapidly depleted hepatocyte ATP levels within 15 minutes which suggests that ATP production is inhibited. The disulfide effectiveness at causing mitochondrial Ca2+ release was similar to their effectiveness at inducing hepatocyte cytotoxicity. These results suggest that hepatocyte toxicity is the result of oxidative inactivation of membrane protein thiols that regulate intracellular Ca2+ homeostasis.     

Freshly isolated or cryopreserved human hepatocytes in primary culture: Influence of drug metabolism on hepatotoxicity

The use of human hepatocytes in drug metabolism studies overcomes the difficulty in extrapolating from animal data. As drug metabolites are often involved in pharmacological activity and toxicity, it is essential to determine these compounds early in the development of a drug. We previously demonstrated that mitoxantrone metabolism exhibits a great interspecies variability: the two main metabolites in humans being dicarboxylic acid (the major metabolite) and monocarboxylic acid. In this paper we analyse their respective cytotoxicities with two endpoint tests: neutral red uptake (cell viability) and MTT reduction (cell metabolism) after a 24- or 48-hr exposure. Results demonstrated less or no toxicity of mono- and dicarboxylic acid derivatives compared with mitoxantrone. In vitro studies using human hepatocytes are scarce because of the limited availability of human livers. After developing a new method for cryopreservation of human hepatocytes, we evaluated them before and after cryopreservation using erythromycin as a test molecule. The cell viability test was more reproducible than the cell metabolism test after 38 hr of cell seeding.     

Effect of diethylether on the formation of paracetamol sulphate and glucuronide in isolated rat hepatocytes

Diethylether has previously been shown to inhibit several pathways of drug metabolism, including conjugation of paracetamol in isolated rat hepatocytes. Since overall paracetamol conjugation consists of pathways of different subcellular localization (cytosolar sulphation and microsomal glucuronidation) the response of both pathways to diethylether was tested. The elimination of paracetamol (160 mumol/l, initial concentration) and the formation of paracetamol sulphate and glucuronide were measured (high-performance liquid chromatography) in suspensions of isolated rat hepatocytes from fasted and fed animals over 1 h in the absence and presence of diethylether (30 mmol/l). Approximately 90% of the paracetamol elimination was by sulphation and nearly 10% by glucuronidation both in the controls and in the presence of ether. The overall disposition of paracetamol and the formation of sulphate were both reduced by about 50% in the presence of ether compared to the controls while the formation of glucuronide was reduced by 70%. The results were not influenced by the nutritional state of the animals before sacrifice. It is concluded that the inhibitory effect of ether on total paracetamol metabolism was mainly caused by reduced sulphation. Since microsomal glucuronidation was also inhibited by ether, both cytosolar and microsomal enzyme systems were sensitive to diethylether.