Differential induction of gamma-glutamyl transpeptidase in primary cultures of rat and mouse hepatocytes parallels induction during hepatocarcinogenesis

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.     

Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.     

The dual role of 2-acetylaminofluorene in hepatocarcinogenesis: specific targets for initiation and promotion

The main feature separating a living pacemaker from other oscillators is the fact that the former has not only the oscillatory property but also slow and fast changes of the membrane potential corresponding to the sub- and suprathreshold regions. We propose a simple mathematical model called mRIC and show that the model exhibited the essential feature of pacemakers generating spikes at constant time intervals. The behavior of the model driven by periodic pulse trains is analysed using the phase transition curve.     

Light and electron microscopic analysis of liver sinusoids during hepatocarcinogenesis with 2-acetylaminofluorene in rats

To clarify the sequential changes and morphological differences of the sinusoidal structures between hepatocellular carcinoma (HCC) and hepatocellular adenoma (HA), we examined morphological changes of sinusoidal cells and related structures such as basement membrane during hepatocarcinogenesis in the rat. During continuous feeding of carcinogenic diets containing 2-acetylaminofluorene to rats, HA appeared at the 8th week in the peripheral area and then extended toward the centrolobular area. The appearance of HCC was recognized at the 27th week. In the HA lesion, the morphology of sinusoidal cells and related structures was basically the same as that of normal liver except for a slight thickening of the basement membrane and a decreased amount of vitamin A-lipid droplets of stellate cells. In HCC, the fenestrations of endothelial cells disappeared and the basement membrane became continuous, thick and often multilayered. Stellate cells contained almost no vitamin A-lipid droplets and were associated with abundant collagen fibers. Kupffer cells and pit cells were not seen inside the sinusoid. All these features of the sinusoids in HCC resembled the morphological characteristics of the capillary. The present study has revealed that HCC possesses sinusoid structures distinct from those of HA. This suggests that HCC may not derive directly from HA but may develop newly within the HA.     

Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol

BACKGROUND & AIMS: We reported previously that patients with alcoholic liver disease (ALD) have circulating immunoglobulins reacting with cytochrome P4502E1 (CYP2E1) complexed with hydroxyethyl free radicals. The aim of this study was to investigate whether hydroxyethyl radical adducts are present on the plasma membranes of ethanol-treated hepatocytes and their role in antibody-dependent cytotoxicity. METHODS: Immunofluorescence confocal laser microscopy, Western blotting, and antibody-dependent cell-mediated cytotoxicity assay were used. RESULTS: Isolated rat hepatocytes incubated in vitro with ethanol or obtained from ethanol-treated animals showed strong surface fluorescence when exposed to rabbit anti-hydroxyethyl radical serum or sera from patients with ALD. No surface fluorescence was evident on control hepatocytes or after scavenging hydroxyethyl radicals with 4-pyridyl-1-oxide-t-butyl nitrone. The presence of CYP2E1-hydroxyethyl radical adducts on hepatocyte plasma membranes was shown by Western blot and by immunofluorescence using double staining for human and rabbit anti-CYP2E1 immunoglobulin G. Cytotoxicity was observed in ethanol-treated hepatocytes incubated with immunoglobulin G from patients with ALD and normal human blood mononuclear cells. This effect was blocked by preabsorbing the sera with human albumin complexed with hydroxyethyl radicals, which also eliminated the antibody reaction with the plasma membranes. CONCLUSIONS: Hydroxyethyl radicals bound to CYP2E1 on hepatocyte plasma membranes can target immune reactions triggered by alcohol abuse.     

Atypical beta-adrenergic receptors in rat liver: evidence for transient expression during aging

This study was designed to determine whether functional beta 3-adrenergic receptors exist in the rat liver and whether there are alterations in the beta 3-adrenergic response with age in a manner similar to that which occurs for beta 2-adrenergic receptors. The beta 3 stimulation of adenylyl cyclase activity was assessed using the novel beta 3-specific agonist, CGP-12177A. Adenylyl cyclase activation by CGP-12177A was seen only in the adults. Isoproterenol-stimulated adenylyl cyclase activity was high in the neonates, declined by 45% in the adults, and was high again in the senescent rats. ICI 118551, a beta 2-selective antagonist, attenuated the isoproterenol-stimulated activity by two-thirds but had no effect on the CGP-12177A-stimulated activity. These data demonstrated the presence of the beta 3-adrenergic receptor in the rat liver, although only at the adult stage of development. In addition, these data confirm earlier findings that beta 2-adrenergic activation of adenylyl cyclase is biphasic with age and indicate that the emergence of the beta 3-stimulated activity coincides with the attenuation of beta 2-stimulated activity.     

Regulation of the expression of follistatin in rat hepatocytes

The effect of lipopolysaccharide (LPS) treatment on noradrenergic responses elicited by electrical field stimulation (EFS) was investigated in the rabbit anococcygeus muscle. In the absence of LPS, EFS-induced contractions were enhanced and nitrergic relaxations were inhibited by NG-nitro-L-arginine (L-NOARG) but not by NG-monomethyl-L-arginine (L-NMMA). Administration of L-NMMA prior to L-NOARG inhibited the enhancement of EFS-induced contractions by L-NOARG and reversed the inhibitory effect of L-NOARG on nitrergic relaxations. Treatment with LPS induced a time-dependent loss of phenylephrine-induced tone which was inhibited by cycloheximide, dexamethasone, L-NMMA, or L-NOARG. Treatment of the anococcygeus muscle with LPS also resulted in a time-dependent loss in the magnitude of EFS-induced contractions and an increase in the delay of onset of contractions. These effects were reversible by pretreatment with cycloheximide or by treatment with L-NMMA. These results suggest that LPS induces a loss of tone and of noradrenergic responses through expression of the inducible NO synthase in the rabbit anococcygeus muscle. L-NMMA blocks these effects but does not affect nitrergic transmission, while L-NOARG is active against both.     

Sulfotransferase gene expression in primary cultures of rat hepatocytes

Hepatocyte cultures have been used in pharmacotoxicological studies, and sulfotransferases (ST) are important drug-metabolizing enzymes in liver. The expression of sulfotransferases in hepatocyte cultures has not been examined systematically. In the present study, the mRNA levels of different sulfotransferases in male and female rat hepatocytes were examined by northern-blot analyses. Various culture conditions such as different matrices (collagen, matrigel, collagen sandwich, or co-culture with epithelial cells), medium (Way-mouth's MB 752/1 and Modified Chee's Medium) and glucocorticoid supplementation (dexamethasone, 0.1 microM) were compared. Phenol ST (ST1A1) mRNA levels decreased to about 50% of initial mRNA levels within 10 hr of culture. At 96 hr, ST1A1 mRNA levels were approximately 20% of initial values when cultured on collagen, matrigel or co-culture. The two media did not differ in ability to maintain ST1A1 mRNA levels in the absence of dexamethasone (DEX); however, DEX addition to either medium resulted in ST1A1 mRNA levels greater than 100% of the initial mRNA levels at 96 hr, with the greatest increase observed using the matrigel substratum and Chee's medium. In the absence of DEX, the mRNA levels of N-hydroxy-2-acetylaminoflurene sulfortransferase (ST1C1), estrogen sulfotransferase (ST1E2) and hydroxysteroid sulfotransferase (ST-20/21, ST-40/41, ST-60) fell to approximately 20% of their initial levels within 24 hr, and to less than 5% at 96 hr. The loss of expression of these sulfotransferases was observed with all culture conditions. Addition of DEX to the media resulted in ST-40/41 and ST-60 mRNA expression at 20 and 35% of their initial values, respectively, in cultures maintained on matrigel and Chee's medium at 96 hr. These data suggest that sulfotransferases lose their constitutive expression in hepatocyte culture, but retain their inducibility.     

Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver

It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of rats with fluvastatin had no effect on hepatic levels of CYP3A1/2 mRNA or immunoreactive protein. However, treatment with 50 mg/kg/day fluvastatin induced CYP1A1 mRNA and protein. The effects of fluvastatin treatment on P450 expression seen in primary cultured rat hepatocytes thus largely recapitulated the effects seen in vivo. The differences in effects among the HMG-CoA reductase inhibitors suggest that simple inhibition of HMG-CoA reductase cannot explain all of the effects of these drugs on P450 expression.     

Spontaneous chemiluminescence production, lipid peroxidation, and covalent binding in rat hepatocytes exposed to acetaminophen

Spontaneous chemiluminescence associated with the cell injury was observed in the isolated rat hepatocyte suspension during acetaminophen (APAP) metabolism, indicating the occurrence of oxidative stress. APAP apparently affected the hepatocytes in various manners. APAP, at low concentrations (1-2 mM), damaged the hepatocytes due to lipid peroxidation provoked during APAP metabolism, while at high concentrations (5-50 mM), APAP protected the hepatocytes due to a chemical antioxidant effect of the unmetabolized APAP that remained in the medium because of the saturation of APAP metabolism. The covalent binding of APAP to the hepatocytes increased with APAP concentration up to 50 mM without loss of cell viability. When an overdose of APAP was administered to rats, the APAP plasma concentration was around 1-3 mM, which corresponded to the concentration range where lipid peroxidation occurred in the isolated hepatocytes. Thus, it seems likely that lipid peroxidation contributes to the APAP-induced hepatotoxicity in the early stage of the toxic process.     

Glycogen phosphorylase: developmental expression in rat liver

We studied the superficial abdominal reflexes of 83 normal men, using as stimuli a train of electrical pulses or a needle scratch. Electrical stimulation delivered to the midline of the abdominal wall evoked, almost symmetrically on both sides, two reflex discharges: an early response having an oligophasic wave form, and a late response of polyphasic wave form. The threshold of the early response significantly exceeded that of the late response. With repetitive stimulation, the late response generally revealed habituation. Electrical stimulation of the unilateral abdominal wall evoked two responses on the stimulated side, whereas it evoked only the late response on the contralateral side. A needle scratch on the unilateral abdominal wall evoked one reflex discharge with a long latency and a polyphasic wave form. This response occurred generally on the stimulated side and became habituated to repeated scratching. These observations suggest that the superficial abdominal reflexes elicited by electrical stimulation are composed of two reflex discharges with a different reflex arc. They appear to closely resemble the blink reflex. The response elicited by needle scratching is thought to correspond to the late response of the electrically elicited abdominal reflexes.     

Presence of physiologically stimulated RET in adult rat brain: induction of RET expression during nerve regeneration

The product of the RET proto-oncogene is a protein belonging to the receptor-like tyrosine kinase superfamily. RET is expressed in several neural crest-derived cell lineages and has been implicated in the correct development of the peripheral nervous system. To gain further insight into RET function, we investigated the presence of active RET in adult rat tissues. We show, by immunoblotting, that the products of the RET proto-oncogene (p155ret) are present in specific regions of adult rat brain, including the cerebellum, striatum, brainstem, hypothalamus, hippocampus, and olfactory bulb. Moreover, in the cerebellum, p155ret is phosphorylated in tyrosine residues, thus indicating that this brain structure contains p155ret in an activated state. Finally, the presence of RET in motoneurons prompted us to analyze the effects of hypoglossal nerve section on its expression. We observed a dramatic increase in p155ret in the motoneuron nuclei, thus suggesting that RET tyrosine kinase plays a role in the neuronal response to axotomy and/or during nerve regeneration.     

Caenorhabditis elegans lin-25: cellular focus, protein expression and requirement for sur-2 during induction of vulval fates

Induction of vulval fates in the C. elegans hermaphrodite is mediated by a signal transduction pathway involving Ras and MAP kinase. Previous genetic analysis has suggested that two potential targets of this pathway in the vulva precursor cells are two novel proteins, LIN-25 and SUR-2. In this report, we describe further studies of lin-25. The results of a genetic mosaic analysis together with those of experiments in which lin-25 was expressed under the control of an heterologous promoter suggest that the major focus of lin-25 during vulva induction is the vulva precursor cells themselves. We have generated antisera to LIN-25 and used these to analyse the pattern of protein expression. LIN-25 is present in all six precursor cells prior to and during vulva induction but later becomes restricted to cells of the vulval lineages. Mutations in genes in the Ras/MAP kinase pathway do not affect the pattern of expression but the accumulation of LIN-25 is reduced in the absence of sur-2. Overexpression of LIN-25 does not rescue sur-2 mutant defects suggesting that LIN-25 and SUR-2 may function together. LIN-25 is also expressed in the lateral hypodermis. Overexpression of LIN-25 disrupts lateral hypodermal cell fusion, suggesting that lin-25 may play a role in regulating cell fusions in C. elegans.     

Decreased expression and activity of P-glycoprotein in rat liver during acute inflammation

PURPOSE: Drug disposition is often altered in inflammatory disease. Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked. The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs. Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp. METHODS: AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration. Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay. P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes. RESULTS: Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50-70% and caused a significant 45-65% reduction in the P-gp mediated efflux of R123. Diminished mRNA levels of all three MDR isoforms were seen. LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver. Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific. CONCLUSIONS: Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver. This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation.