Effect of cyanamide on the metabolism of ethanol and acetaldehyde and on gluconeogenesis by isolated rat hepatocytes

Previous experiments demonstrated that acetaldehyde stimulated glucose production from pyruvate, whereas gluconeogenesis from glycerol, xylitol and sorbitol was inhibited [A.I. Cederbaum and E. Dicker, Archs Biochem. Biophys. 197, 415 (1979)]. To determine the mechanism whereby acetaldehyde affects glucose production from these precursors, and to evaluate the role of acetaldehyde in the actions of ethanol, experiments with cyanamide were carried out. The oxidation of acetaldehyde by isolated rat liver cells was inhibited by cyanamide after a brief incubation period. Associated with this inhibition of acetaldehyde oxidation was an inhibition of ethanol oxidation by cyanamide and an increase in the amount of acetaldehyde which arose during the oxidation of ethanol. Ethanol oxidation was decreased because of the ineffective removal of acetaldehyde in the presence of cyanamide. Cyanamide had no effect on hepatic oxygen uptake. The increase in the β-hydroxybutyrate/acetoacetate ratio produced by acetaldehyde was completely prevented by cyanamide, whereas the slight increase in the lactate/pyruvate ratio was not prevented by cyanamide. Cyanamide partially reversed the ethanol-induced increase in the lactate/pyruvate ratio, but it completely prevented the ethanol-induced increase in the β-hydroxybutyrate/acetoacetate ratio. The ethanol-induced change in the mitochondrial redox state may, therefore, be due primarily to the mitochondrial oxidation of the acetaldehyde which arises during the oxidation of ethanol. The inhibitory effects of acetaldehyde on gluconeogenesis from glycerol, xylitol and sorbitol, as well as the stimulation of acetaldehyde of glucose production from pyruvate, were completely prevented by cyanamide. These results indicate that the effects of acetaldehyde on gluconeogenesis represent metabolic effects, rather than direct effects of acetaldehyde. Changes in the cellular NADH/NAD^? ratio as a consequence of acetaldehyde metabolism are postulated to be responsible for these actions of acetaldehyde. Ethanol stimulated glucose production from pyruvate, while inhibiting gluconeogenesis from glycerol, xylitol and sorbitol. Cyanamide, which prevented the effects of acetaldehyde on gluconeogenesis, also prevented the effects of ethanol on gluconeogenesis. This prevention by cyanamide may be suggestive for a role for acetaldehyde in the actions of ethanol on gluconeogenesis. The possibility cannot be ruled out, however, that the prevention of the effects of ethanol by cyanamide may be due to the partial inhibition of ethanol oxidation by cyanamide. These results indicate that cyanamide is an effective inhibitor of acetaldehyde oxidation by isolated liver cells and therefore can be used to determine the mechanism whereby acetaldehyde affects metabolic function. Depending on the reaction under investigation, acetaldehyde can have direct or indirect effects on cellular metabolism.     

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.     

Effect of ethanol metabolism on initiation of protein synthesis in rat hepatocytes

Rat liver parenchymal cells were incubated in the presence and absence of ethanol (80 mM). Polysomes were isolated and analysed on sucrose gradients. Ethanol was shown to (1) inhibit the incorporation of ¹⁴C-valine into proteins, (2) result in a shift in the distribution of polysomes towards smaller sizes, (3) inhibit the formation of 40S initiation complexes, and (4) diminish the concentration of glucose-6-phosphate in the hepatocytes. Addition of 4-methylpyrazole (0.5 mM) partially prevented the inhibition of protein synthesis and completely restored the polysomal distribution. It is concluded that ethanol inhibits protein synthesis partly by a mechanism linked to ethanol metabolism. This effect takes place at the level of initiation and may be mediated by a reduced gluconeogenesis.     

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.     

Effect of various metabolic inhibitors on biphenyl metabolism in isolated rat hepatocytes.

The effect of three inhibitors of mitochondrial function (menadione, rotenone and 2,4-dinitrophenol) on drug metabolism in isolated rat hepatocytes has been studied. Menadione (at 1.25 × 10^?4 M) caused almost complete inhibition of biphenyl Phase I metabolism whereas rotenone (2 × 10^?5 M) inhibited the same reaction only by 25 per cent although the subsequent conjugation of the Phase I metabolite was markedly depressed. Qualitatively similar findings were observed with hepatocytes isolated from phenobarbital-pretreated rats, and with liver microsomes isolated from control rats. 2,4-Dinitrophenol (2 × 10^?4 M) caused a marked enhancement of biphenyl Phase I metabolism but a marked inhibition of subsequent conjugation. This enhancement of Phase I metabolism was not observed in control cells with other substrates (benzo[a]pyrene, 7-ethoxycoumarin), nor in biphenyl metabolism in “induced” cells or in liver microsomes isolated from control rats. It is tentatively suggested that products of 2,4-dinitrophenol metabolism may “activate” biphenyl metabolism in intact liver cells. Furthermore, it is suggested for all three inhibitors that direct effects on the drug metabolizing enzyme systems (Phase I and Phase II) are as important as their effects on mitochondrial function in explaining their inhibition of drug metabolism. It appears that Phase II metabolism of xenobiotics is more susceptible to inhibition by metabolic inhibitors than is Phase I metabolism, probably due to depletion of the cellular ATP levels.     

Antagonism between the metabolic responses induced by epinephrine and piroxicam on isolated rat hepatocytes.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most employed therapeutic agents. They have a wide spectrum of biological effects, some of which are independent of cyclooxygenase inhibition, such as the alterations on the components of signal transduction systems. In particular, previous data from our laboratory suggested an antagonism between epinephrine and piroxicam, one of the most prescribed NSAIDs. Thus, this study deals with the epinephrine-piroxicam antagonism recorded for metabolic responses in isolated rat hepatocytes. The obtained results show that epinephrine stimulates lactate and ethanol consumption, stimulates glucose release from lactate only, and has no effect on cellular triacylglycerides content. Otherwise, in a dose-dependent basis, piroxicam stimulates lactate and ethanol consumption accompanied by an increase in triacylglycerides content, without changes in glucose release by hepatocytes. Piroxicam blocks the epinephrine-induced stimulation of glucose release from lactate, and epinephrine blocks the piroxicam-mediated increase in triacylglycerides content from lactate or ethanol. In contrast, the effects of epinephrine and piroxicam, promoting the consumption of lactate and ethanol, are not antagonized or added after the simultaneous administration of both compounds. This last result is probably related to the ability of both compounds to stimulate oxygen consumption. On isolated rat liver mitochondria, micromolar doses of piroxicam partially uncouple oxidative phosphorylation, and paradoxically stimulates an ATP-dependent mitochondrial function as citrullinogenesis. These results show for first time, on isolated rat hepatocytes, an antagonism between the metabolic responses of epinephrine and piroxicam, at the concentration found in plasma after its therapeutical administration.     

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.     

Oxidative metabolism of carbon disulfide by isolated rat hepatocytes and microsomes

The oxidative metabolism of carbon disulfide (CS2) was investigated in isolated rat hepatocytes and liver microsomes. In microsomes, CS2 metabolism was increased by phenobarbital pretreatment of the rats and decreased with pretreatment of the rats with cobaltous chloride. In both microsomes and hepatocytes, CS2 metabolism was inhibited by SKF-525A. Carbon dioxide (CO2) was the major volatile metabolite of CS2 in hepatocytes, and carbonyl sulfide (COS) was the major volatile metabolite in microsomal incubations. Addition of cytosol to microsomal incubations shifted the predominant volatile metabolite from COS to CO2 but did not change total volatile metabolite formation. Acetazolamide, a carbonic anhydrase inhibitor, significantly decreased COS metabolism but not CS2 metabolism in isolated hepatocytes or microsomes fortified with dialyzed cytosol. When [18O]H2O was included in incubations of microsomes and CS2, a substantial portion of the resulting COS was [18O] enriched, indicating that the oxygen atom was derived from water. These data are consistent with the hypothesis that CS2 is oxidized predominantly by the cytochrome P-450 containing monooxygenase system, and the product of this reaction is an unstable intermediate which reacts with water to form monothiocarbonate and reactive sulfur species. Monothiocarbonate is the hydrated form of COS. In intact hepatocytes, it is metabolized predominantly to CO2 and hydrogen sulfide. Unmetabolized monothiocarbonate can be dehydrated to COS. The majority of the reactive sulfur species and hydrogen sulfide are oxidized to nonvolatile sulfur compounds, including sulfate, but by different mechanisms.     

Toxicity, metabolism and absorption of selenite by isolated rat hepatocytes

The uptake of various levels of selenite by isolated rat hepatocytes was investigated. The LD₅₀ value of selenite was about 500 μM. The activity of lactic dehydrogenase in the medium was correlated with cell viability as determined by trypan blue exclusion. After incubation of selenite with hepatocytes, protein-bound Se was the predominant form (80–90% of the cellular Se) present. Subcellular fractionation indicated that most of the radioactivity was present in the cytosol when hepatocytes were incubated with ⁷⁵Se-selenite. The uptake of ⁷⁵Se by isolated rat hepatocytes was linear with selenite concentration up to the highest amount tested, 200 μM. Sulfite inhibited the uptake of selenite by hepatocytes.     

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.     

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.     

Influence of ethanol induction on the metabolic activation of genotoxic agents by isolated rat hepatocytes

The effects of ethanol-feeding to rats, over a 6-week period, on the activation of genotoxic compounds of different chemical classes, requiring metabolic conversion to exert their mutagenic activity, were studied in isolated rat hepatocytes. The influence of such treatment on cytochrome P-450 content and N-acetylation in isolated hepatocytes was also investigated.Benzidine (BZ), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), isoniazid (INH) and cyclophosphamide (CP) were more effectively activated to products mutagenic towards Salmonella typhimurium by hepatocytes from ethanol-pretreated rats than by hepatocytes from controls. The mutagenic potency of 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was not influenced by ethanol pretreatment. Ethanol consumption was found to be associated with increased cytochrome P-450 content and enhanced N-acetylation in the isolated hepatocytes.Our results support the hypothesis that an alteration of the hepatic drug-metabolizing system may be responsible for the ethanol-induced increase in susceptibility to certain genotoxic compounds.     

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.     

Comparative metabolism of bis(2-methoxyethyl)ether in isolated rat hepatocytes and in the intact rat: Effects of ethanol on in vitro metabolism

The metabolism of the reproductive and developmental toxicant bis(2-methoxyethyl)ether (diglyme) was studied in isolated rat hepatocytes and in the intact rat. Male Sprague-Dawley rats (190–220 g) were used in both studies. Hepatocytes, isolated by a two-step in situ collagenase perfusion of the liver, were cultured as monolayers and incubated with [¹⁴C]diglyme at 1, 10, 30, and 50 M for up to 48 h. For the in vivo study, rats were given single oral doses of [¹⁴C]diglyme at 5.1 mmol/kg body wt, and urine was collected for up to 96 h. Radioactive compounds in the culture medium or in the urine were separated by high performance liquid chromatography and quantified with an in-line radioactivity monitor. Metabolites were identified by comparison of their chromatographic retention times and their mass spectra with those of authentic compounds. The principal metabolite from hepatocytes and in the urine was (2-methoxyethoxy)acetic acid (MEAA). This metabolite accounted for approximately 36% of the radioactivity in the 48-h culture medium and about 67% of the administered dose in the 48-h urine. Other prominent metabolites common to both systems included 2-(2-methoxyethoxy)ethanol, methoxyacetic acid (MAA), 2-methoxyethanol, and diglycolic acid. The diglyme metabolite profiles from urine and from hepatocytes were qualitatively similar, demonstrating that, in the rat, hepatocytes serve as a good model system for predicting the urinary metabolites of diglyme. Moreover, MEAA was shown to be the metabolite best suited for use as a short-term biological marker of exposure to diglyme. Pretreatment of rats with ethanol resulted in a marked increase in the overall in vitro metabolism of diglyme. The major metabolic pathways for diglyme involve O-demethylation and cleavage of the central ether bond, and it is the latter pathway that leads to the formation of MAA, the metabolite associated with the reproductive and developmental toxicity of diglyme. The amounts of MAA formed in hepatocytes from ethanol-pretreated rats ranged from two to four times those formed in hepatocytes from untreated rats.Mention of Company or product names is not to be considered an endorsement by the National Institute for Occupational Safety and Health.     

Effect of inflammation on the metabolism of antipyrine, lidocaine and propranolol in isolated rat hepatocytes

A decrease of the hepatic intrinsic clearance could contribute to the increase of the plasma concentrations of alpha 1-acid glycoprotein-bound drugs such as propranolol in animals and humans with inflammation. Therefore, the influence of inflammation upon the metabolism of propranolol and another high clearance drug, lidocaine, and of the low clearance drug antipyrine, was studied in isolated rat hepatocytes. For comparative purposes, the influence of the enzyme inhibitor SKF 525A (100 mg/kg i.p.) was also evaluated. Turpentine pretreatment of the rats significantly decreased the metabolism of the three drugs by the hepatocytes; the decrease was least pronounced for propranolol. The inhibitory effect of turpentine-induced inflammation was somewhat lower than that of SKF 525A. These results are in agreement with the results found for the same drugs in the 9,000-g supernatant fraction of the rat liver and point to a marked decrease of intrinsic clearance in some types of inflammation.     

Cadmium uptake by isolated rat hepatocytes

Isolated hepatocytes have been employed to investigate the uptake of the heavy metal cadmium (¹⁰⁹Cd). A rapid initial phase of uptake was followed by a second slower phase, both of which exhibited a linear relationship between velocity of uptake and substrate concentration. The presence of potassium cyanide (1 mm) and carbonyl cyanide-m-chlorophenyl hydrazone (2 μm) did not cause any change in the uptake of Cd (200 μm). This suggests that the uptake of Cd into isolated hepatocytes is not an active process. Incubation of 10 μm Cd in the presence of zinc (50 and 130 μm) produced a decrease in the velocity of uptake of only the first phase. Pretreatment of rats with zinc, which elevates hepatic metallothionein, resulted in isolated hepatocytes which showed a greater uptake rate for the second phase when compared to hepatocytes isolated from control rats. The results therefore demonstrate that uptake of Cd by isolated rat hepatocytes is a biphasic response in which at least part of the first phase appears to be carrier mediated with no indication of involvement of a carrier in the second phase of uptake. This second phase may be related to binding of Cd to intracellular components, since elevation of hepatic metallothionein was found to increase the uptake velocity of only this phase.