In vitro Metabolism of Propoxyphene in Rat Liver: Reaction of a Carbinol Metabolite with Acetaldehyde

Abstract: The metabolism of the analgesic drug propoxyphene (α-d-propoxyphene) has been investigated in the rat liver 9,000Xg supernatant fraction. The incubations were analyzed by HPLC. The major metabolite was norpropoxyphene carbinol, obtained through demethylation and ester hydrolysis. The demethylated metabolite of propoxyphene, norpropoxyphene, was also detected. Addition of acetaldehyde to the incubation mixture decreased the metabolism of propoxyphene. Reactions between norpropoxyphene carbinol and acetaldehyde resulted in a fast disappearence of the carbinol and the formation of a reaction product, the significance of which is discussed.     

Cytotoxic Effects of N-Hydroxyparacetamol in Suspensions of Isolated Rat Hepatocytes

Abstract: The cytotoxicity of N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of the hepatotoxic and nephrotoxic analgesic paracetamol, was studied in suspensions of hepatocytes isolated by collagen-perfusion of livers of male rats. Incubation of cells with 0.25–2.0 mM N-OH-pHAA led after 3–5 hours to increased cell permeability measured by increased trypan blue uptake, increased NADH penetration or leakage of prelabelled ⁵¹Cr. N-OH-pHAA rapidly depleted cellular glutathione, 16% of initial levels were seen after 30 min. incubation. ³H-N-OH-pHAA bound covalently to cellular proteins in a time- and concentration-dependent manner, considerably higher binding rates were seen with boiled cells compared to intact cells. Pretreatment of animals with the cytochrome P-450 inducer phenobarbital did not affect N-OH-pHAA cytotoxicity or covalent binding, whereas the cytochrome P-450 inhibitor metyrapone inhibited both cytotoxicity and binding. Lipid peroxidation in hepatocytes could be seen as a late event after a limited range of N-OH-pHAA concentrations. In contrast, lipid peroxidation was an early event in cells exposed to carbon tetrachloride. A minimal exposure time of 30 min. of the hepatocytes to N-OH-pHAA was sufficient to elicit cellular damage occurring after 3–5 hours.     

Modulation of N-Hydroxyparacetamol Cytotoxicity in Suspensions of Isolated Rat Hepatocytes

Abstract: The effect of inhibitors of toxicity of N-hydroxyparacetamol(N-OH-pHAA), a postulated proximate metabolite of paracetamol, was studied in isolated rat hepatocytes. Additions of ascorbate, menadione, thiol-containing amino acids and glutathione (GSH) led to an increased stability of N-OH-pHAA, reduced the covalent binding of N-OH-pHAA to cellular protein and decreased GSH depletion caused by N-OH-pHAA. Two to three hours elapsed after a 30 min. exposure of the cells to N-OH-pHAA before the cells responded with increased cell permeability. Ascorbate, acetylcysteine, GSH and promethazine were capable of inhibiting this second phase of N-OH-pHAA cytotoxicity in addition to their effects during the initial exposure phase. In contrast, the anti-oxidant tocopherole and phenacetin were only effective during the second phase. Increasing the incubation medium pH during the second phase of N-OH-pHAA mediated cellular damage resulted in decreases in cytotoxicity. Lipid peroxidation, as measured by accumulation of thiobarbituric acid reactive metabolites, did not seem to be directly correlated with cytotoxicity, since cysteamine or higher concentrations of N-OH-pHAA inhibited lipid peroxidation without decreasing cellular damage.     

Effects of Ethanol on Ethylmorphine Metabolism in Isolated Rat Hepatocytes: Characterization by Means of a Multicompartmental Model

Abstract: Hepatic cytochrome P-450 enzymes mediate at least two important biotransformation pathways of codeine and ethylmorphine starting with either N-demethylation or O-dealkylation, producing polar metabolites which are then subsequently glucuronidated. The present study was designed to characterise the acute effects of ethanol on the metabolism of ethylmorphine and to compare it with the effects on codeine in suspensions of freshly isolated rat hepatocytes. Isolated rat hepatocytes from male Wistar rats were prepared by a collagenase perfusion method. Ethylmorphine, codeine and their metabolites were quantified by HPLC with UV detection. The total ethylmorphine elimination rate was reduced by 12% at 5 mM and 38% at 100 mM ethanol. The corresponding percentages for codeine were 16 and 43%. In the presence of ethanol the concentrations of several intermediate and end products of ethylmorphine and codeine changed markedly from the control situation. The experimental data were applied to a mathematical compartmental linear model to estimate the influence of ethanol on the separate reaction rates in the two main metabolic pathways. The ratios between reaction rate constants in the ethylmorphine experiments at 100 and 0 mM ethanol were 0.65 for ethylmorphine→norethyl-morphine, 0.63 for norethylmorphine→normorphine, 0.56 for ethylmorphine→morphine, 0.49 for morphine→normor-phine, 0.31 for normorphine→normorphine-3-glucuronide and 0.49 for morphine→morphine-3-glucuronide. Almost similar effects of ethanol on codeine metabolism were found. In additional experiments, norethylmorphine or norcodeine (50 μM) was incubated with 5 mM to 100 mM of ethanol and the metabolism of both norethylmorphine and norcodeine was found to be inhibited by ethanol in a concentration-dependent manner. The glucuronidation of morphine and normorphine added in separate experiments was also inhibited by ethanol, from 22 to 36% for morphine-3-glucuronide and 30 to 60% for normorphine-3-glucuronide, respectively, in the presence of 5 mM to 100 mM of ethanol. It was concluded that all steps in the metabolism of ethylmorphine (and codeine) leading to the end products morphine-3-glucuronide and normorphine-3-glucuronide were inhibited by ethanol, and that the glucuronidation processes were the ones most affected by ethanol.     

Drug Metabolism Activities of Isolated Rat Hepatocytes in Monolayer Culture

Abstract: The levels of cytochrome P-450 in hepatocytes cultured as monolayers for 22 hrs in Dulbecco's modified Eagle medium supplemented with serum and insulin was reduced to approximately 40% of initial values of freshly isolated hepatocytes. In correspondance with this the activities of the cytochrome P-450 monooxygenases aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) and ethylmorphine (EM) N-demethylase were reduced to 40 and 22% of their initial activities, respectively. Modifying the culture medium through omission of cysteine and cystine, and adding dexamethazone and delta-amino levulinic acid, increased the content of cytochrome P-450 to 59 % and EM N-demethylase to 46 % of initial values, but was without effect on AHH activity. However, further modifications by adding high concentrations of asparagine and leucine increased AHH activity to 62% of initial values, but did not further enhance the total content of cytochrome P-450 or the EM N-demethylase activity. The activities of cytochrome P-450 reductase, flavin containing monooxygenase, epoxide hydrolase and glutathione S-transferase decreased less (to about 70–80% of initial values) than cytochrome P-450 associated monooxygenase activities, whereas UDP-glucuronyl transferase decreased to about 50% of initial values. In contrast to what was observed regarding cytochrome P-450 and associated monooxygenase activities, modification of the incubation conditions did not affect the non-cytochrome P-450 enzymatic activities.     

Combined Effects of Ethanol and pH-Change on Protein Synthesis in Isolated Rat Hepatocytes

Abstract: Rat liver parenchymal cells were isolated and incubated for 80 min. in buffered salt solutions. ¹⁴C-valine incorporation in the presence of a high concentration of unlabelled valine (4.2 mM) was taken as a measure of protein synthesis. The pH dependence of the synthesis of cell and medium proteins was studied at pH values varying from 6.6 to 7.9. The results showed that maximum protein synthesis occurred close to the physiological pH value. Protein synthesis was reduced at both lower and higher pH-values. Protein synthesis was inhibited by the addition of ethanol (30 mM). The relative inhibition caused by ethanol got more pronounced as the pH of the hepatocyte suspensions was lowered. The metabolism of ethanol resulted in lowering of the pH in cell suspensions. It is suggested that the depression of protein synthesis by ethanol could be mediated by the fall in pH due to ethanol metabolism.     

The Interaction Between Manganese and Ethanol in Rats

Abstract The effects of manganese and ethanol interaction on some chemical constituents of the liver and serum of rats were investigated in order to assess the influence of these substances in inducing susceptibility to manganese poisoning. Manganese and ethanol alone or in combination were administered to the rats as drinking solutions for a period of 30 days. Both the chemicals had a synergistic effect in altering the activity of SDH and ATPase in the liver of rats. The combined treatment also produced significant increase in the activity of adenosine deaminase and α-amylase in the liver and serum respectively. Furthermore, the accumulation of manganese in the liver and the increase in the calcium content of the serum were significantly greater after combined ethanol and manganese administration – than either of them alone. These alterations indicate that the toxic effects of manganese are enhanced when the metal and ethanol interact in the biological system.     

Metabolism of 14C-Antipyrine in Suspensions of Isolated Rat Liver Cells

Abstract Suspensions of liver cells isolated from perfused rat livers were incubated with antipyrine-N-methyl-¹⁴C. Antipyrine was eliminated by first-order kinetics during incubations for 3 hours with primary suspensions (parenchymal cells + non-parenchymal cells) and suspensions of purified parenchymal cells. Antipyrine concentrations were unchanged when incubated with suspensions of non-parenchymal cells, dead cells or medium only. At the end of incubation period, 4-OH-antipyrine and 3-CH₂OH-antipyrine were detected mainly as the glucuronide or sulphate conjugates, and evidence for the N-demethylation of antipyrine was also obtained. Half-lives for elimination of antipyrine in primary cell suspensions were not significantly different from the half-lives measured in parenchymal cell suspensions. This finding together with the lack of metabolism of antipyrine found in non-parenchymal cell suspensions suggest that oxidation and conjugation of antipyrine is mainly confined to the parenchymal cells. There was significant inhibition of antipyrine metabolism in primary suspensions by phenylbutazone (1.6 × 10^-3 M), dexamethasone (2 × 10^-4 M) and ethanol (1.3 × 10^-2 M, 0.75 %₀). We suggest that the use of primary suspensions of isolated rat liver cells provide a rapid and simple method for the study of factors influencing drug metabolism in the liver.     

Comparative Metabolism of Methyl Parathion in Intact and Subcellular Fractions of Isolated Rat Hepatocytes

Metabolism of the widely used insecticide methyl parathion byisolated hepatocytes and various subcellular fractions was comparedto determine the effects of cellular integrity on the metabolicprofile observed. A reverse-phase ion-pair high-performanceliquid chromatographic method was developed to separate andquantify methyl parathion and six of its hepatic biotransformationproducts: methyl paraoxon; desmethyl parathion; desmethyl paraoxon;p-nitrophenol; p-nitrophenyl glucuronide; and p-nitrophenylsulfate. Most compounds exhibited linear responses and limitsof detection below 1 nmol. The chromatographic method was usedto determine metabolic profiles of methyl parathion in isolatedrat hepatocytes, sonicated hepatocytes, postmitochondrial fraction,microsomes, and cytosol. Isolated hepatocytes produced significantlymore desmethyl parathion and p-nitrophenyl sulfate than thesubcellular preparations, demonstrating that cellular integritysignificantly affects the quantitative metabolic profile observed.     

Acute Exposure to Ethanol in Pregnancy-Assessment of Cardiovascular Effects in the Isolated Perfused Rat Heart

Abstract

Numerous pregnant women are exposed to ethanol. Given the marked cardiovascular changes induced by pregnancy and the known cardiac toxicity of ethanol, we conducted this study to explore the effects of pregnancy on the cardiac toxicity of ethanol. Isolated, perfused rat hearts obtained from pregnant and nonpregnant Sprague-Dawley rats were exposed to increasing doses of ethanol (0.1, 0.2, 0.4, 0.6%). Heart rate, changes in left ventricular pressure over time (dP/dt), left ventricular systolic pressure, and coronary artery flow rate were measured. Ethanol induced profound cardiac depression in all parameters. Pregnancy neither exacerbated nor attenuated this cardiac toxicity. In the isolated perfused rat heart model, pregnancy does not affect the cardiac toxicity of acute ethanol exposure.     

Inhibition of Paranitroanisole and Antipyrine Monooxygenation in Isolated Rat Hepatocytes by Compounds Interacting with Mitochondrially Related Carbohydrate Metabolism

Abstract: The monooxygenation of paranitroanisole (PNA) and antipyrine (AP) were measured in isolated rat hepatocytes incubated with compounds interacting with mitochondrially related carbohydrate metabolism. Phenylpyruvate, an inhibitor of pyruvate carboxylase, reduced the rate of PNA and AP metabolism to about 60 and 20%, respectively, in hepatocytes both from fasted and fed rats. Inhibition of amino acid transaminase with aminooxyacetate, decreased the metabolism of both PNA and AP to 60–70% of control values in hepatocytes from fasted rats, whereas this effect was not seen in fed rats. n-Butylmalonate, an inhibitor of mitochondrial malate/phosphate exchange, had only minimal effects on PNA and AP monooxygenation in both the nutritional states. The simultaneous presence of glyoxylate and pyruvate, known to inhibit the NADPH specific isocitrate dehydrogenase, reduced the metabolism of both PNA and AP in hepatocytes from fasted rats to about 60 and 35% of control values respectively, while the effect was not so marked in hepatocytes from fed rats. The metabolism both of PNA and of AP in hepatocytes from fasted rats was reduced to 50–60% of control values with the addition of NH₄Cl. This effect could be blocked either by incubating the hepatocytes with pyruvate or by using hepatocytes isolated from fed rats. The addition of various carbon intermediates generally reduced the effect of the inhibitors used. Phenobarbital-treatment did not change the effects observed with cells from uninduced animals. The inhibitors did not alter PNA or AP metabolism in microsomal incubations, and therefore most likely reduced the monooxygenation in intact cells by affecting NADPH generation pathways.     

Effect of Rat Serum Lipoproteins on mRNA Levels and Amiodarone Metabolism by Cultured Primary Rat Hepatocytes

Hyperlipidemia can significantly increase amiodarone (AM) in vivo liver uptake and decrease its velocity of microsomal metabolism. Here, hepatocytes isolated from normolipidemic (NL) and hyperlipidemic rats were incubated with AM in the presence or absence of diluted NL or hyperlipidemic serum. The serum was added either as preincubation before drug, or concurrently with drug; incubations without rat serum were used as controls. The hepatocyte levels of mRNA for several proteins and enzymes were also measured. Disappearance of AM was seen up to 72 h. There was little difference between hepatocytes from NL or hyperlipidemic animals in intrinsic clearance (CL_int) of AM. The effect of hyperlipidemic rat serum, either before or with AM, was profound, causing a significant reduction in the CL_int. Reductions were seen in mRNA for cytochrome P450 1A1, 3A2, and 2D1, some transporters, and low‐density lipoprotein receptors after exposure of hepatocytes to lipoprotein‐rich sera. In conclusion, exposure of isolated hepatocytes to hyperlipidemic serum caused decreases in AM CL_int and lower mRNA levels for some proteins involved in the uptake and metabolism of AM. When coincubated with serum, an additional effect of increased binding to lipoproteins seemed to further contribute to a reduced CL of AM.     

Placental and Foetal Metabolism of Acetaldehyde in Rat I. Contents of Ethanol and Acetaldehyde in Placenta and Foetus of the Pregnant Rat during Ethanol Oxidation

Abstract: Following the administration of ethanol to pregnant rats, the ethanol content of the maternal aortic blood was compared with that of the intact placenta and whole foetuses. The results support previous observations that ethanol is present in the placenta and foetus in about the same amount as in the maternal circulation. In contrast, only 25 % of the acetaldehyde content present in the maternal aortic blood could be found in the intact placenta and no acetaldehyde was found in the intact foetal tissue. These findings suggest that during maternal ethanol oxidation there is no accumulation of acetaldehyde in the foetal tissue.     

Metabolic Effects of Prolonged Ethanol Administration in Rats Treated with Clofibrate

Abstract Rats receiving clofibrate (ethyl–α–p–chlorophenoxyisobutyrate) were given ethanol by stomach–tube, 6–9 g per kg body weight per day, for 3 weeks. The toxicity of ethanol was not greater in these animals than in the controls. No signs of ketoacidosis appeared and, in contrast to the controls, the clofibratetreated animals did not give a hyperglycaemic response after a single large dose of ethanol. Neither Mallory bodies nor signs of inflammation or fatty infiltration were ever seen in livers of clofibrate–treated rats; however, but both histochemical and biochemical techniques revealed a slight although insignificant increase in liver total lipid content in the ethanol–treated controls. The possibility of preventing adverse effects of alcohol in man by clofibrate treatment is discussed.     

鹿茸醇提物对离体脂肪组织分解的影响

目的 研究鹿茸醇提物对离体脂肪分解的影响及其促进脂肪分解的活性因子。方法 以梅花鹿鹿茸为原料,分别取鹿茸蜡片、粉片、血片,以75%乙醇为溶剂,提取3次后离心,合并上清液,减压回收乙醇,冷冻干燥得到鹿茸各部位的醇提物,考察鹿茸各部位醇提物对离体脂肪组织分解的作用和关系,通过高效液相色谱法测定鹿茸各部位中次黄嘌呤的含量。结果 次黄嘌呤与脂肪分解呈线性关系,推测其促进脂肪分解的活性因子可能为次黄嘌呤。次黄嘌呤、鹿茸蜡片、粉片和血片醇提物在盐酸肾上腺素存在条件下,脂肪分解率分别为472.00%,183.20%,145.56%和115.84%;无盐酸肾上腺素条件下,次黄嘌呤、鹿茸蜡片和粉片醇提物促进脂肪分解率分别为220.00%,104.74%和112.80%,血片无促进脂肪分解的作用。蜡片、粉片、血片中次黄嘌呤的含量分别为0.386,0.268,0.162 mg.g 1。结论 蜡片促进离体脂肪分解作用最强,粉片次之,血片最弱。次黄嘌呤可能是促进脂肪分解的主要活性因子。     

In Vitro Interaction of Heavy Metals with Ouabain Receptors in Rat Brain Microsomes

ABSTRACT

This study investigates the influence of heavy metals on ouabain-binding in presence of thiol (sulfhydryl) compounds. The data on in vitro effects of mercury (Hg), lead (Pb) and cadmium (Cd) showed significant inhibition of ³H-ouabain binding to microsomal membrane in a concentration-dependent manner. Maximum inhibition of ³H-ouabain binding was observed at 1 μM for Hg and 100 μM each for Pb and Cd. Preincubation with monothiol (L-cysteine or glutathione) or dithiol (dithiothreitol) protected inhibition of ³H-ouabain binding to the membranes by Hg or Pb. Dithiol but not monothiols partially protected Cd-inhibition. The present data confirm that the heavy metals interact with ouabain receptors in a manner similar to SH-blocking agents and protection of metal-inhibited ³H-ouabain binding by thiol compounds is metal specific.     

Effect of Purified Saponin Mixture from Astragalus corniculatus on Toxicity Models in Isolated Rat Hepatocytes

A purified saponin mixture (PSM) isolated from Astragalus corniculatus Bieb. (Fabaceae) was investigated for its protective effect in two models of toxicity, carbon tetrachloride (CCl₄) and tert-butyl hydroperoxide (t-BuOOH), using isolated rat hepatocytes. CCl₄ undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (CCl₃) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. Oxidative damage is widely recognized as being involved in the development of many pathological conditions. In our experiment, t-BuOOH was used as a model of oxidative stress. The hepatocytes were incubated with the PSM alone (0.01–100 μ M) and along with CCl₄ (86 μ M) and t-BuOOH (75 μ M). As a sign of cytotoxicity, cell viability was used. CCl₄ and t-BuOOH significantly decreased hepatocyte viability. Our data indicate that PSM showed lower toxic effects compared to CCl₄ and t-BuOOH and in combination exerted statistically significant protection of cell viability against the toxic agents.     

Interaction between Drug Acetylation and Ethanol,Acetate, Pyruvate,Citrate, and L(minus;)Carnitine in Isolated Rat Liver Parenchymal Cells

Abstract: The actylation of sulfanilamide and procainamide in suspensions of isolated rat parenchymal cells was studied in absence and presence of ethanol (33 mM), acetate (0.5–5 mM), citrate (4 mM), pyruvate (4 mM), and L(minus;)carnitine (2 mM). Ethanol treatment enhanced the sulfanilamide acetylation whereas the acetylation of procainamide was considered to be unchanged. Acetate (1–5 mM), citrate, and pyruvate treatment enhanced the acetylation of both sulfanilamide and procainamide. Acetate (4 mM) increased both K_m and V_max of both sulfanilamide and procainamide acetylation. Combined treatment with L(minus;)carnitine and either acetate, pyruvate, or citrate enhanced the acetylation rate of sulfanilamide more than acetate, pyruvate, or citrate, respectively alone. In cell suspensions treated with L(minus;)carnitine and acetate or pyruvate, the acetylation kinetics of sulfanilamide changed from zero-to apparent first-order. With procainamide as test drug, a further increase of the acetylation rate was found when L(minus;)carnitine was added to citrate or pyruvate. Acetyl-CoA increased the rate of sulfanilamide acetylation in rat liver homogenates in a dose dependent manner.     

高浓度大鼠肝实质细胞与非实质细胞共培养的细胞功能研究

目的:高浓度肝实质细胞与非实质细胞原代共培养,研究其功能活性。方法:应用原位胶原酶灌流法分离大鼠肝细胞,获得有活性的肝细胞,并应用高浓度实质和非实质肝细胞共培养的方法原代培养（共培养组）,并以微囊肝细胞培养为对照组（微囊组）进行了比较。结果:两组均维持白蛋白分泌、尿素合成功能7天;共培养组的白蛋白分泌与微囊组一样为下降趋势,共培养组从（0.870±0.102）降至（0.492±0.040）g·L-1·10-6cells·24h-1,微囊组从（1.147±0.099）降至（0.375±0.012）g·L-1·10-6cells·24h-1;共培养组的肝细胞第4天后维持在一个较稳定的水平,而微囊组肝细胞前3天明显高于共培养组,而后两天显著低于共培养组（P<0.05）。共培养组的尿素合成功能,由（4.50±0.56）降至（4.37±0.19）μmol·L-1·10-6cells·90min-1,微囊组由（5.42±0.81）降至（3.60±0.33）μmol·L-1·10-6cells·90min-1,前3天微囊组高于共培养组,后2天共培养组明显高于微囊组（P<0.05）。共培养2～7天肝细胞的对氨基苯甲酸（PABA）浓度稳定在 7.2～9.9mg/L·10-6cells·24h-1。结论:高浓度肝实质细胞与非实质细胞共培养,可使肝细胞的特异性功能维持7d,而且较微囊肝细胞更适合应用于中空纤维舱型生物人工肝。     

Effect of GABA Analogues on Blood Pressure and Central GABA Metabolism in the Rat

Abstract: Gamma aminobutyric acid (GABA) and different GABA analogues were examined for their cardiovascular actions and their influence on striatal dopamine (DA) levels and GABA accumulation after aminooxyacetic acid (AOAA). Gamma hydroxybutyric acid (GHBA) and baclofen caused hypertension and tachycardia after systemic as well as intracerebroventricular administration, while the opposite was true for GABA and muscimol. The hypertension after GHBA and baclofen was not reduced by picrotoxin or bicuculline and was not influenced by varying GABA levels by 3-mercaptopropionic acid (3-MPA)or AOAA. GHBA and muscimol but not baclofen reduced GABA accumulation induced by AOAA. Picrotoxin in a subconvulsive dose increased GABA accumulation and antagonized the inhibition after GHBA or muscimol. Bicuculline and a moderate dose of picrotoxin tended to decrease GABA accumulation by themselves and if anything augmented the effects of GHBA and muscimol. GHBA and baclofen but not muscimol in combination with AOAA increased DA levels, which was not prevented by picrotoxin or bicuculline. We conclude that the cardiovascular actions of GHBA and baclofen are probably not mediated by mechanisms identical to those of muscimol or exogenous GABA. In view of the biochemical results their actions would however be compatible with a concept of different GABA receptors.