Adult Male Rat Hepatocytes Research Articles

Histological study of adult male albino rats’ hepatocytes after formaldehyde administration and the possible protective role of dill oil

Background There have been numerous studies on formaldehyde-induced health problems including the hepatocytes. It has been estimated that the general population is exposed to food and products of daily use that contain formaldehyde in a concentration that is presumed to be safe. Meanwhile, many herbs have been used for the treatment of liver diseases. Dill, for instance, has been potently used for its various biologically active constituents. Aim The present study was performed to investigate, histologically and biochemically, the safety of 0.2% formaldehyde on the hepatocytes of adult male rats. The study was further extended to determine the possible protective role of dill oil. Materials and methods The study was carried out on 20 rats (120–150 g), which were randomly divided into three groups. Group I (the control group) included 10 rats and was further subdivided into two equal subgroups: subgroup Ia and subgroup Ib. Each rat received distilled water (1 ml/kg body weight) and dill oil orally (1 ml/kg body weight), respectively. Group II included five rats, and each received 1 ml of 0.2% formaldehyde. Group III included five rats that received similar dose as group II accompanied by the same dose of dill oil as subgroup Ib. All treatments were given daily by means of gavage for 8 weeks. All animals were sacrificed 24 h after the last administration. Liver functions were assessed biochemically and histologically using both light and electron microscopes. Results The study depicted that serum levels of hepatic transaminases were significantly elevated in group II compared with group I. However, in group III, they were nearly similar to that reported in group I. This was accompanied by noticeable histological changes in group II. Coadministration of dill oil showed considerable preservation of the histology of hepatocytes. Conclusion A volume of 0.2% formaldehyde caused changes in the hepatocytes that could be prevented by dill oil coadministration.

Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes.

The induction of cytochrome P450 (CYP450) and Phase II conjugating enzymes by prototypical hepatic enzyme inducers was studied in adult male rat hepatocytes. Hepatocytes were suspended and cultured in diluted Matrigel in a basal serum-free Dulbecco's modified Eagle medium and exposed to the prototypical liver enzyme inducers, 3-methylcholanthrene, phenobarbital, hydrocortisone, and clofibrate for 48 h. Total RNA and microsomes were isolated and prepared, respectively, at 72 h. The expression of CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A1, CYP3A2, CYP4A1, fatty acyl-CoA oxidase, uridine diphosphate-glucuronosyltransferase, glutathione-S-transferase, and sulfotransferase was determined at the mRNA level with reverse transcriptase polymerase chain reaction (RT-PCR). The expression of CYP1A1, CYP2B1, CYP2C11, CYP2E1, and CYP4A1 was also measured at the apoprotein level by Western immunoblotting. Using these culture and expression analysis techniques, we have found that the expression of these metabolic enzymes can be maintained in culture for up to 7 d at the mRNA and apoprotein levels. In addition, hepatocytes were found to respond to chemical enzyme inducers with marked increases in enzyme expression at either the mRNA or protein level and in a concentration-related fashion. Cells were responsive to enzyme induction as early as 24 h after initial plating. The results obtained from this investigation indicate that the presence of diluted Matrigel (at a concentration of 0.35 mg/ml), the use of low concentrations of insulin (1 microM), hydrocortisone (0.1 microM), and serum-free culture medium can maintain the differentiated phenotype and responsiveness of cultured hepatocytes to chemical-induced metabolic enzyme expression. Under the conditions used in this study, enzyme induction in adult male rat hepatocytes shows close agreement with enzyme induction observed in the livers of rats exposed to these or similar prototypical enzyme inducers. Rat hepatocytes cultured in the presence of diluted Matrigel coupled with enzyme mRNA expression analysis with RT-PCR are proven to be a valuable and important in vitro toxicological approach to assess the chemical-induced changes in expression of liver CYP450 and Phase II conjugating enzymes.

Hormonal regulation of microsomal flavin-containing monooxygenase activity by sex steroids and growth hormone in co-cultured adult male rat hepatocytes

To investigate the hormonal control of the expression of flavin-containing monooxygenase (FMO; EC 1.14.13.8) under defined in vitro conditions, adult male rat hepatocytes were isolated by collagenase perfusion and co-cultured with rat liver epithelial cells of primitive biliary origin. The direct effect of 17β-estradiol, testosterone, 5α-dihydrotestosterone (5α-DHT) and human growth hormone (hGH) on FMO activity was studied using this in vitro model. Optimal, non-cytotoxic hormonal concentrations were determined by measuring the lactate dehydrogenase (LDH) index. In addition, the microsomal protein content of the cultured hepatocytes was determined as a function of culture time. The female sex hormone 17β-estradiol caused a significant decrease in FMO as a function of culture time. After 14 days of exposure, FMO activity decreased by 56%. Neither of the male sex hormones or human growth hormone had an effect on FMO activity. These results in co-cultured male rat hepatocytes support in vivo observation that 17β-estradiol is a potent hormone involved in the negative regulation of the expression of FMO in male rat liver.

Effect of Thyroid Hormones onFlavin-containing Monooxygenase Activity in Co-cultured Adult Rat Hepatocytes

The regulation of flavin-containing monooxygenase (FMO) by thyroid hormones was examined under well defined in vitro conditions using adult male rat hepatocytes co-cultured with rat liver epithelial cells of primitive biliary origin. Serum free medium was used to avoid interferences from foetal bovine serum. The effect of thyroxine (T4) and its major metabolite l-triiodothyronine (T3) on FMO activity was estimated spectrophotometrically by measuring the rate of methimazole oxygenation. The highest non-cytotoxic doses of T3 and T4 that could be used in co-cultures were determined by measuring both lactate dehydrogenase leakage into the medium and microsomal protein content of the hepatocytes as a function of culture time. In addition, hormonal responsiveness of the in vitro system used was confirmed by malic enzyme activity measurements. Administration of 10 μ m T3 or T4 was found to cause a significant decrease in FMO activity and content, suggesting a suppressive role of both hormones on the regulation of FMO activity in male rats.

Effect of pyruvate on glutathione s-transferase expression in primary cultures of rat hepatocytes

Recently, primary rat hepatocyte cultures supplemented with 30 m m pyruvate and various hormones have been proposed as suitable long-term in vitro models for xenobiotic metabolism studies. In this study, the effect of 30 m m pyruvate on the cytosolic phase II biotransformation enzyme glutathione S-transferase (GST, EC 2.5.1.18) has been investigated. Adult male rat hepatocytes were brought into primary culture in serum free Williams' medium E with and without supplementation of 30 m m pyruvate. Total, Mu and Alpha class GST activities as well as GST subunit patterns were determined on the day of the isolation and as a function of culture time. Cell morphology and albumin secretion were also examined. After 7 days of culture, the morphology of pyruvate-treated cultures was still intact and bile canaliculi were clearly visible, whereas control cells without pyruvate had deteriorated and lost their normal morphology. The albumin secretion rate was higher in pyruvate-supplemented than in non-supplemented cultures. Total, Mu and Alpha class GST activities were well maintained in the presence of pyruvate; conversely, without pyruvate, all GST isoenzyme activities were significantly decreased. These findings were confirmed at the protein level. However, when hepatocytes received medium containing pyruvate for 7 days, the Pi class subunit 7, normally not present in adult liver cells, was expressed at a much higher level than was the case in untreated hepatocyte cultures. This study clearly shows that GST activities and GST proteins, as well as cell morphology and albumin secretion, are better maintained in primary monolayer cultures of adult rat hepatocytes supplemented with 30 m m pyruvate than in control cultures. However, it was also found that pyruvate is a very strong inducer of GST Pi. Consequently, the percentage GST subunit pattern of cultured rat hepatocytes is significantly affected by pyruvate supplementation, which may have consequences for in vitro xenobiotic metabolism studies.

Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone

The response of an SV40-immortalized hepatocyte cell line (CWSV-1) derived from adult male rat hepatocytes to human growth hormone (hGH) was investigated. CWSV-1 cells, which have been characterized extensively, retain certain differentiated functions of normal liver (Woodworth and Isom, Mol Cell Biol 7: 3740–3748, 1987). This cell line consists of tightly associated polygonal, mononucleated cells that grow as monolayers. These cells showed no significant morphological changes with the addition of hGH. Northern blot analysis showed that continuous treatment of the CWSV-1 cells with hGH induced the expression of insulin-like growth factor I (IGF-I) and 5α-reductase RNAs. In addition, continuous exposure to hGH resulted in the induction of expression of the growth hormone receptor/growth hormone binding protein (GHR/GHBP) genes. This study indicates that the CWSV-1 cells may serve as a valuable in vitro model system for studying the signaling pathway of GH.

Absence of enantioselectivity in the pharmacodynamics of P450 2B induction by 5‐ethyl‐5‐phenylhydantoin in the male rat liver or in cultured rat hepatocytes

To explore the enantioselectivity of ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of cytochrome P450 2B (CYP2B) induction by racemic 5-ethyl-5-phenylhydantoin and its two enantiomers were investigated in the male F344/NCr rat and in cultured adult male rat hepatocytes. Steady-state serum drug concentrations, measured following 14 days of administration of the compounds in the diet (0-1320 ppm, n = 3 rats per group), were used as an approximation of intrahepatocellular drug concentration. The serum xenobiotic concentrations associated with half-maximal hepatic CYP2B induction were 5-10 microM, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values in the hepatocyte culture experiments were 8-12 microM, based on measurement of total cellular RNA coding for CYP2B1. In both the in vivo and the hepatocyte culture experiments, the potencies for CYP2B induction were essentially equivalent for the racemate and the individual enantiomers of 5-ethyl-5-phenylhydantoin. In the case of this compound, there would appear to be no enantioselectivity for CYP2B induction. This finding may be interpreted as evidence against receptor mediation in the induction of CYP2B activity, although it is also possible that a receptor is involved that does not exhibit enantioselectivity.

GLUT-1 and GLUT-2 mRNA, protein, and glucose transporter activity in cultured fetal and adult hepatocytes.

To understand glycogenesis in the fetal hepatocyte, we examined glucose transport in cultured fetal and adult male rat hepatocytes. GLUT-1 mRNA was detected in fetal hepatocytes at isolation but in adult hepatocytes only after culture. GLUT-1 mRNA was more abundant in fetal than in adult hepatocytes (P < 0.005). GLUT-1 protein paralleled its message. GLUT-2 mRNA was more abundant in adult than in fetal hepatocytes (P < 0.05), and abundance did not change during culture, but GLUT-2 protein was discordantly regulated. There was more GLUT-2 protein in fetal hepatocytes at 45 h (P < 0.025). An Eadie-Hofstee plot of 3-O-methylglucose transport appeared to have two linear components. One component was presumed to be GLUT-1 [variable Michaelis constant (Km) approximating 6-8 mM, maximal uptake rate (Vmax) for fetal vs. adult hepatocytes 106 vs. 35 nmol.min-1.mg protein-1], and a second was presumed to be GLUT-2 (Km of 23 mM, Vmax for fetal vs. adult hepatocytes 198 vs. 92 nmol.min-1.mg protein-1). Early phosphorylation of 2-deoxyglucose was greater in fetal than in adult hepatocytes, but transport was always greater than phosphorylation. Increased expression of both GLUT-1 and GLUT-2 by fetal hepatocytes permits greater glucose uptake and positions the fetal rat hepatocyte for efficient glycogenesis at low plasma glucose concentration.

Alpha-1 adrenergic receptor number and receptor density in isolated hepatocytes from foetal, juvenile and adult rats

Alpha-1 adrenergic receptor number was defined by [ 3H]-prazosin binding in crude membrane preparations of hepatocytes and in intact hepatocytes isolated from foetal (day 22 of gestation), juvenile (12 days old), adult female and adult male (90–150 days old) rats and compared with the alpha-1 adrenergic response (measured by epinephrine stimulated glucose liberation in presence of the beta-antagonist propranolol). The alpha-1 receptor number (expressed as fmol bound [ 3H]-prazosin/mg membrane protein or as receptor number/cell) increases in an age-dependent fashion reaching the highest values in hepatocytes of adult female and male rats. Statistically significant differences could be found between foetal, juvenile and adult rat hepatocytes. No differences in [ 3H]-prazosin binding were observed between hepatocytes of adult female and adult male rats. The receptor density (expressed as receptor number/μm 2 cell surface), however was found to be equal in juvenile and adult rats. There are no differences of alpha-1 adrenergic response in juvenile, adult female and adult male rat hepatocytes, whereas the values in foetal hepatocytes were significantly lower. So the biological response is closely correlated with the receptor density and not with the receptor number per cell.

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and β-adrenergic receptors in cultured hepatocytes: Dependence on protein and RNA synthesis and suppression by isoproterenol

Adult male rat hepatocytes, which normally respond poorly to β-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the β-adrenoceptor ligand [ 125I]cyanopindolol. The emergence of β-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydortestosterone to the cultures, singly or in combination, did not prevent the augmented β-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [ 125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in β-adrenoceptor binding.

Metabolism, cytotoxicity, and genotoxicity of the pyrrolizidine alkaloid senecionine in primary cultures of rat hepatocytes

Senecionine, a pyrrolizidine alkaloid isolated from Senecio vulgaris, was a potent toxicant to primary cultures of adult male rat hepatocytes. A nontoxic dose of [ 14C]senecionine (1.9–2.4 nmol/10 6 cells) was rapidly taken up from the culture medium by hepatocytes and converted to more polar metabolites recovered in the culture medium. Concomitantly some metabolites formed stable covalent bonds with cellular macromolecules. After a 24-hr incubation, 88% of the dose was water soluble while only 10% could be extracted with chloroform; 1.3% of the dose was bound to trichloroacetic acid-precipitated material and could not be extracted with organic solvents. Senecionine was a potent cytotoxin as evidenced by the leakage of lactate dehydrogenase from the hepatocytes into the surrounding culture medium and the loss of cells from the collagen substratum. A linear dose-response relationship for cytotoxicity was obtained for doses ranging from 2.6 to 128 nmol per 10 6 cells. Senecionine was also genotoxic as suggested by the evidence of covalent binding and the stimulation of DNA repair. The lowest dose that stimulated DNA repair was 16 nmol/10 6 cells. Based on these data, one would predict that senecionine is not only a potent hepatotoxin but is also likely to be carcinogenic.

A random arrangement of albumin-containing hepatocytes seen with histo-immunologic methods. I. Verification of the artifact.

The cellular and subcellular localization of albumin in hepatocytes of adult male rats was established with immunofluorescence and immunoperoxidase techniques. Livers were fixed while either filled or devoid of blood. In some rats, prior treatment with cycloheximide was used to deplete the albumin content of hepatocytes. Immunofluorescence of blood-free livers from untreated rats showed that all hepatocytes contained albumin. However, using the peroxidase method, the amount of immunoprecipitate in cisternae of the rough endoplasmic reticulum was so slight that specific localization of albumin was impossible. Yet in all cases, a positive reaction for the presence of albumin was seen on ribosomes attached to the endoplasmic reticulum. In contrast, immunofluorescence of blood-filled livers from untreated rats and those previously injected with cycloheximide showed that only a few scattered hepatocytes were positive for albumin. In these cases, subcellular localization of albumin was obvious because the immunoprecipitate was found in heavy concentration, buy only in the cytosol compartment.

Inhibition by insulin of the formation of autophagic vacuoles in rat liver. A morphometric approach to the kinetics of intracellular degradation by autophagy.

Electron microscopic morphometry has demonstrated a rapid decrease in the fractional volume of autophagic vacuoles (AV) in hepatocytes of adult male rats after the intraperitoneal administration of insulin (5 U/kg of body weight). Except for a significant decrease in glycogen to about one-half its initial value, no major changes in the composition of the remaining cytoplasm, or in the average volume of the single hepatocyte, were seen. The decrease found in the AVs is attributed to an inhibition of the formation of new AVs-probably the morphologic counterpart of the well-known anticatabolic effects of insulin. The decay of the fractional volume of the AVs appeared to follow first-order kinetics. Thus, the termination of the "life" of an AV by destruction of its contents may not depend directly on the "age" of the AV. The average half-life of the AVs amounted to approximately 9 min. Similar values were found for the different types of AVs, except for those containing glycogen. The half-life of these AVs was approximately 18 min. From the half-life values and from the "segregated fractions" at time zero, which were different for the different cytoplasmic components, rates of removal from the cytoplasm by autophagy were calculated. Expressed as "percent per day", the following rates were found: whole cytoplasm, 2.3; mitochondria, 3.9; microbodies, 8.9; and glycogen, 1.1. The results indicate that autophagy, to some extent, is selective and plays an important, but not an exclusive, role in intracellular turnover.