Steroid metabolism in isolated rat hepatocytes

In vivo predictability of results obtained from studies of drug metabolism using isolated rat hepatocytes is questionable, mainly because of modeling difficulties due to the simultaneously occurring substrate transferring processes. In the present study, an attempt was made at simplifying the models used to describe the kinetics of biotransformation by enzymes enclosed in a cellular environment. Viability assessment of the cell preparation indicated that the cell membrane was intact and functional. Six corticosteroids were used in these studies. Simplifying assumptions concerning uptake and protein binding were confirmed by running independent experiments. Progress curves of unchanged steroid disappearance from the cell suspending medium at different initial concentrations were used to either confirm applicability or detect deviations from simple Micha?lis-Menten behavior and were fitted to the appropriate kinetic models by means of nonlinear least-squares regression analysis. As an example, corticosterone extraction ratio obtained in this study compared well with literature values from intact rats. A linear correlation was found between the logarithm of the apparent first order rate constant (Vm/Km) obtained at low substrate concentrations and the logarithm of the oil/water partition coefficients of 17 alpha-hydroxyprogesterone, corticosterone and hydrocortisone.     

Metabolism of 15N-labelled N-nitrosodimethylamine and N-nitroso-N-methylaniline by isolated rat hepatocytes

The N-demethylation of ¹⁵N-labeled N-nitrosodimethylamine (DMN) and N-nitroso-N-methylaniline (NMA) by isolated rat hepatic cells has been investigated. The values obtained in this system for molecular nitrogen formed during metabolism, compared with substrate consumed, were DMN 47%, NMA 23%, and N-nitroso-N-methylurea (NMU) 105%. The results for DMN are roughly halfway between those previously determined with rat liver S-9 fraction in vitro (33%) and in vivo (67%). For NMA, the hepatocyte data are closer to those obtained from S-9 in vitro (19%), rather than the in vivo (52%). No mixed nitrogen (¹⁵N¹⁴N) or labeled nitrogen oxides were found.     

Kinetics of benzylpenicillin metabolism in isolated rat hepatocytes

The metabolism of benzylpenicillin (PCG) in isolated rat hepatocytes was investigated. The evidence of metabolizing activity for PCG in hepatic cells was obtained as follows; the disappearance rate of PCG from the incubation medium followed Michaelis-Menten kinetics and was dependent on the cellular protein concentration, while PCG did not disappear when it was incubated with cells denaturated by heat. The rate of disappearance of PCG was reduced significantly in the presence of the structural analogue of PCG such as phenoxymethylpenicillin in the incubation medium. The major metabolite of PCG was identified, by high performance liquid chromatographic analysis, to be penicilloic acid (PA) of PCG. A kinetic model describing the intra- and extra-cellular concentrations of PCG and PA was developed. The proposed model fitted well the time course of changes in the concentration of PCG and PA. The clearance of the uptake of PCG by isolated hepatocytes was evaluated to be about 23-times greater than that of metabolism of PCG.     

Investigation of praziquantel metabolism in isolated rat hepatocytes

Isolated rat hepatocytes were used to elucidate the metabolism of praziquantel (PZQ). Our studies were designed to investigate mainly qualitative differences in the biotransformation of PZQ enantiomers. Additionally, the main metabolites cis- and trans-4-hydroxypraziquantel were determined semiquantitatively. For this purpose, racemic PZQ and both enantiomers were incubated with isolated rat hepatocytes. The incubation mixtures were investigated using high-performance liquid chromatography/mass spectrometry. Hepatocytes prepared from male Wistar rats were incubated in Krebs-Ringer buffer at 37 degrees C for 4 h. Aliquots were withdrawn hourly throughout 4 h of incubation. We found that hepatocytes converted both enantiomers of PZQ to the major metabolites cis- and trans-4-hydroxypraziquantel. Additional metabolites were detected after incubating the S-(+)-enantiomer. These minor metabolites were identified by means of their mass/charge ratio as monohydroxypraziquantel metabolites of different, unknown structure.     

Glipentide and glucose metabolism in isolated rat hepatocytes

Glipentide, a second generation sulfonylurea, raised the cellular concentration of fructose 2,6-bisphosphate in isolated rat hepatocytes. Parallel to accumulating this regulatory metabolite, glipentide inhibited basal gluconeogenesis and increased the rate of L-lactate production, as well as the metabolic flux through the 6-phosphofructo 1-kinase reaction. Tolbutamide elicited similar metabolic effects to those reported for glipentide, although the latter sulfonylurea was about 10 times more potent. The biochemical mechanism by which sulfonylureas promote the accumulation of fructose 2,6-bisphosphate in hepatocytes seems to be related to a significant increase of the hexose 6-phosphate pool (glucose 6-phosphate plus fructose 6-phosphate), together with the activation of 6-phosphofructo 2-kinase and inactivation of fructose 2,6-bisphosphatase, enzyme activities responsible, respectively, for the synthesis and degradation of fructose 2,6-bisphosphate.     

The metabolism of cyclophosphamide by isolated rat hepatocytes

The metabolism of cyclophosphamide was studied in vitro using isolated rat hepatocytes and mass spectrometry. The major product of primary oxidative metabolism in hepatocytes from phenobarbital treated rats was 4-hydroxycyclophosphamide, isolated as the O-ethyl derivatives, but dechloroethylation was also a substantial pathway. 4-Hydroxycyclophosphamide was converted mainly into carboxy phosphamide and the formation of 4-ketocyclophosphamide was a minor pathway. Evidence is presented that under certain conditions a substantial amount of an O-glucuronide of 4-hydroxycyclophosphamide was formed. The pattern of metabolism in hepatocytes otherwise resembled qualitatively that observed previously in vitro using subcellular fractions and in vivo, but quantitative differences were found. The metabolism of cyclophosphamide by hepatocytes resembles more closely that in vivo than does the metabolism in subcellular fractions, and hepatocytes should be the preferred in vitro system for studying the metabolism of anti-tumour agents.     

Heterogeneous distribution of drug metabolism in elutriated rat hepatocytes

Centrifugal elutriation was used to separate isolated rat hepatocytes into five fractions containing cells of different sizes. These fractions were compared with regard to cell size, morphology and function. Analyzed by flow cytometry, the small cells were found to be enriched in fraction 1 and the large cells in fraction 5. Evaluation by light and electron microscopy indicated that the fractions contained single hepatocytes of normal structure. The cytochrome P-450 content and the 7-ethoxycoumarin O-deethylase activity were assessed in hepatocytes from untreated animals, those treated with phenobarbital, and those treated with phenobarbital plus allylisopropylacetamide. In both untreated and phenobarbital-treated animals, cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity rose significantly from fraction 1 to fraction 5. The P-450 content gradually rose up to 2-fold. The enzyme activity rose 5-fold, and it increased steeply between fractions 2 and 3. The cytochrome P-450 content in phenobarbital-plus-allylisopropylacetamide-treated animals was decreased in all fractions but more extensively in fraction 5 than in fraction 1.     

Acute effects of halothane and enflurane on drug metabolism and protein synthesis in isolated rat hepatocytes

The metabolism of sulphanilamide, antipyrine and paracetamol was studied in the absence and presence of the anaesthetics halothane and enflurane at three different concentrations (0.5, 1.0 and 2.0 mM) in isolated hepatocytes from the rat. Cell viability and protein synthesis were monitored to evaluate toxic effects. A strong concentration related inhibition of antipyrine oxidation (40-70%) and paracetamol conjugation (20-40%) was caused by both halothane and enflurane. Acetylation of sulphanilamide was not inhibited, however, as a slight augmentation was noticed. A significant dose related decrease of cell viability (3-13%) was caused by both anaesthetics. Dose dependent inhibition of the synthesis of stationary cell proteins (15-60%) and the synthesis/secretion of medium proteins (35-85%) was caused by halothane. Similar but slightly less pronounced effects were caused by enflurane. The present findings show that volatile anaesthetics may have general effects as well as different degrees of specific effects on both membrane bound enzyme and soluble enzyme activities.     

Comparative drug metabolism of diazepam in hepatocytes isolated from man, rat, monkey and dog

Diazepam (DZ) was used as a substrate in drug metabolism studies to characterise the differences in metabolite profiles in hepatocytes isolated from four species: Wistar rat, cynomolgus monkey, beagle dog and man. Hepatocytes were incubated with DZ (20 microM) for 180 min at 3 hr post isolation in culture, and the disappearance of parent compound and appearance of its metabolites determined. DZ disappearance was found to be monoexponential in rat, monkey and human cells, but that DZ disappearance in dog hepatocytes was best described by a two compartment process. There were considerable differences in both the rates of formation and the profiles of metabolites produced from DZ in each species. Drug metabolism of DZ was determined in five human hepatocyte preparations. The rates of formation of both the major metabolites, temezepam (TEM) and nordiazepam (NOR) were highly variable between samples, and oxazepam (OX) was detected in only three of the preparations. There was no evidence of further metabolism of these metabolites, and the profiles were comparable with in vivo findings. In a single case, human hepatocytes were cultured for five days, and DZ was used as substrate to characterise the changes in drug metabolising activities. There was a rapid loss in the production of OX in the initial 24 hr, and a complete loss of 3-hydroxylation activities in the succeeding 120 hr. N-demethylation activities, however, were well maintained, and the appearance of NOR declined to 47% of initial rate. The hepatocytes of all species were found to produce NOR and TEM as metabolites; NOR representing the principal metabolite in the dog, monkey and human cells. In the dog, TEM was found only as a minor metabolite. OX was a significant metabolite in the monkey and a minor metabolite in the dog and human hepatocytes, but was not detected in rat cultures. The principal metabolite in rat cells was 4'-hydroxy diazepam, which was rapidly further metabolised to its glucuronide. The drug metabolising activities of the hepatocyte cultures towards DZ were comparable with the drug metabolism of DZ found in vivo in each species. These findings substantiate hepatocytes as an in vitro model of hepatic metabolism.     

The characterization of perfluorosuccinate as an inhibitor of gluconeogenesis in isolated rat hepatocytes

The effects on metabolism of the fluorinated dicarboxylic acid, perfluorosuccinate, were examined in hepatocytes from fasted rats. Perfluorosuccinate (5 mM) inhibited gluconeogenesis from lactate by 80% and from pyruvate by 40%. Significant inhibition (up to 30%) occurred at a concentration of perfluorosuccinate of 50 microM. Cellular ATP levels were not affected by perfluorosuccinate, nor was the rate of formation of ketone bodies from palmitate, although the ratio [3-hydroxybutyrate]/[acetoacetate] was increased up to 5-fold relative to the control. An increased concentration of cellular L-malate was measured in the presence of perfluorosuccinate but this did not reflect inhibition of malate transport between the mitochondrial and cytoplasmic compartments. In addition, ethanol oxidation by hepatocytes was inhibited 25% by 1 mM perfluorosuccinate. Ureogenesis from ammonia was relatively insensitive to inhibition by perfluorosuccinate. In cytoplasmic extracts of rat liver, the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were inhibited 40-50% and 23%, respectively, by 1 mM perfluorosuccinate. The observed metabolic effects of perfluorosuccinate are consistent with inhibition of the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase within the cytoplasm.     

Changes in methionine metabolism induced by D-galactosamine in isolated rat hepatocytes

We studied several steps of methionine metabolism in isolated rat hepatocytes both with and without the presence of a hepatotoxic agent (D-galactosamine). By use of selective labelling either on methyl or on carboxyl groups, we showed that intracellular methionine is used preferentially for the methylation of phospholipids (42%) and nucleic acids (31%) via S-adenosylmethionine. In the presence of D-galactosamine, the incorporation of L-(14CH3) methionine into macromolecules is significantly inhibited (greater than 50%). This inhibition is associated with a decrease of S-adenosylmethionine and an increase of methionine in the injured cells. These results suggest that hepatotoxicity of galactosamine may be due in part to an inhibition of the methylation of nucleic acids and phospholipids. Consequently, we hypothesize that hypermethioninemia associated with human liver disease could be due, at least partly, to a defect in synthesis and/or utilization of S-adenosylmethionine by hepatocytes.     

The effect of insulin on steroid metabolism in isolated rat hepatocytes

Insulin administration has previously been shown to reverse the effects of chemically-induced and spontaneous diabetes on hepatic drug and steroid metabolism in the rat. The complex network of the intact hormonal system of the body and its physiological feedback mechanisms makes it difficult to ascribe the effects seen to any particular hormones. The present study investigated the effect of insulin on hepatic steroid metabolism in the absence of other hormonal influences by using isolated rat liver cells. Insulin (10(-9) M) produced two peaks of increased enzyme activity in the hepatocytes (at 1/2 hr and 24 hr). Dose-response curves at 1/2 hr and 24 hr insulin preincubation suggest that these two peaks are probably generated by different mechanisms. The absence of any significant changes in cytochrome P-450 content after 1/2, 1 and 2 hr of insulin treatment indicates that the increase in steroid metabolizing enzyme activities is not due to an increase in de-novo enzyme synthesis. Our observations provide further evidence for the role played by insulin in the regulation of hepatic steroid and drug metabolism in the rat.     

Effect of epinephrine on ethanol metabolism by isolated rat hepatocytes

The effect of epinephrine on ethanol metabolism was determined in isolated rat hepatocytes. Epinephrine (10 microM) enhanced an initial rapid rate of ethanol elimination observed in the first 5 min. Thereafter, between 5 and 90 min, the rate of ethanol elimination was slower and not affected by epinephrine. Epinephrine resulted in higher acetaldehyde concentrations at 2 min, but not thereafter. Acetaldehyde production in the presence and absence of epinephrine was inhibited by 4-methylpyrazole, by a low free extracellular calcium concentration, and by the alpha 1-adrenergic blocker prazosin. Ethanol alone and epinephrine alone increased oxygen consumption, but the effects were not additive. The ethanol-induced decreases in the cytosolic NAD-/NADH and NADP++NADPH ratios and in the mitochondrial NAD+/NADH ratio were delayed by the presence of epinephrine. An accelerated initial alcohol dehydrogenase activity sufficient to account for the rapid initial rate of ethanol elimination shown with epinephrine was demonstrated by coupling ethanol oxidation with lactaldehyde reduction, a system which increases the rate of dissociation of NADH from the enzyme and its oxidation back to NAD+. The findings in this study indicate that an increased reoxidation of NADH during ethanol oxidation by alcohol dehydrogenase is the basis for the rapid transient increase in ethanol elimination produced by epinephrine.     

Paracetamol metabolism and toxicity in isolated hepatocytes from rat and mouse

Hepatocytes isolated from mouse and rat catalyzed the formation of glucuronide, sulphate, glutathione and cysteine conjugates of paracetamol. These metabolites were separated by high pressure liquid chromatography. 1. Sulphation had higher affinity for paracetamol than glucuronidation in hepatocytes from both mouse and rat, whereas glucuronidation had higher capacity. The maximal rate of glucuronidation was similar in hepatocytes from both species, the rate of sulphation was, however, several-fold less in hepatocytes from mouse. 2. Formation of the glutathione conjugate was directly correlated with loss of intracellular glutathione (GSH). The rate of glutathione conjugate formation increased about three times in rat hepatocytes after phenobarbital treatment. This induced rate was, however, only half of that in hepatocytes from control mouse. In both species the reaction was saturated only at very high paracetamol concentrations. The rate of formation of the cysteine conjugate was very low compared to the other reactions. 3. Only hepatocytes isolated from mouse lost integrity, measured as increased permeability of the cell membranes, upon incubation in the presence of paracetamol.     

Morphine metabolism in isolated rat hepatocytes and its implications for hepatotoxicity

Isolated rat hepatocytes metabolized morphine to its glucuronide conjugate, morphinone-glutathione conjugate, normorphine and morphinone. Addition of morphine to the isolated hepatocytes induced a marked decrease in the level of glutathione in the cells and resulted in cell death. The formation of glutathione conjugate was correlated well with the loss of intracellular glutathione. The cytotoxicity of morphinone was higher than that of morphine. Naloxone and normorphine showed no cytotoxic effect on the cells. Naloxone inhibited the formation of morphinone-glutathione conjugate and prevented the morphine-induced cytotoxicity. Naloxone also blocked morphine-induced liver damage in vivo. In contrast, the morphinone-induced hepatotoxicity was not prevented by naloxone. It is concluded that morphine has a hepatotoxic effect, that the morphine-induced hepatotoxicity is due to its metabolic activation, and that naloxone acts as an inhibitor of an enzyme converting morphine to morphinone.     

Application of isolated hepatocytes to studies of drug metabolism in large food animals

A definitive hazard assessment of xenobiotics translocated through food animals into edible products such as meat or milk requires a complete analysis of metabolism in food animals. However, large animal metabolism studies present many experimental difficulties. None of several in vitro alternatives such as subcellular fractions has been established as an acceptable predictor of in vivo metabolism. The feasibility of using isolated hepatocytes to predict the metabolism of xenobiotics, both quantitatively and qualitatively, in large ruminant animals (e.g. cattle) is being studied in our laboratory. A procedure was developed for isolating hepatocytes aseptically from the caudate process of the liver which was obtained surgically from 100-125 kg calves. A modified two-step vascular perfusion procedure provides hepatocyte suspensions that are typically greater than or equal to 85% viable and greater than or equal to 1 X 10(7) viable hepatocytes/g of liver (wet wt). Xenobiotic metabolism has been evaluated in suspensions and primary cultures using aldrin epoxidation, ethoxycoumarin O-deethylation, and 7-hydroxycoumarin glucuronidation and sulfation. Metabolic activities are relatively short-lived in suspensions less than or equal to 4 h, but quite stable up to 10 h when cultured on collagen-coated plates in chemically defined medium. Bovine hepatocytes behave similarly in culture to rodent hepatocytes. Although primary culturing of hepatocytes is more difficult than suspensions, primarily due to the asepsis requirements, it is the method of choice for xenobiotic metabolism determinations in isolated hepatocytes of cattle.     

Application of isolated hepatocytes to studies of drug metabolism in large food animals

1. A definitive hazard assessment of xenobiotics translocated through food animals into edible products such as meat or milk requires a complete analysis of metabolism in food animals. However, large animal metabolism studies present many experimental difficulties. None of several in vitro alternatives such as subcellular fractions has been established as an acceptable predictor of in vivo metabolism.

2. The feasibility of using isolated hepatocytes to predict the metabolism of xenobiotics, both quantitatively and qualitatively, in large ruminant animals (e.g. cattle) is being studied in our laboratory. A procedure was developed for isolating hepatocytes aseptically from the caudate process of the liver which was obtained surgically from 100–125 kg calves. A modified two-step vascular perfusion procedure provides hepatocyte suspensions that are typically ?85% viable and ?1 × 10⁷ viable hepatocytes/g of liver (wet wt). Xenobiotic metabolism has been evaluated in suspensions and primary cultures using aldrin epoxidation, ethoxycoumarin O-deethylation, and 7-hydroxycoumarin glucuronidation and sulfation. Metabolic activities are relatively short-lived in suspensions ?4h, but quite stable up to 10h when cultured on collagen-coated plates in chemically defined medium. Bovine hepatocytes behave similarly in culture to rodent hepatocytes. Although primary culturing of hepatocytes is more difficult than suspensions, primarily due to the asepsis requirements, it is the method of choice for xenobiotic metabolism determinations in isolated hepatocytes of cattle.