Drug metabolism in hepatocyte sandwich cultures of rats and humans

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 micromol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.     

Visual neglect in pretrigeminal cats with lateral suprasylvian lesions

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme present in the peripheral tissues of the rat, including the liver, and is mediated by a number of factors in animal in vivo studies. However, the effect of peptide hormones and glucocorticoids on the activity of 11 beta-HSD in isolated rat hepatocytes is not clear. To investigate these effects, we determined 11 beta-HSD activity in a primary culture of rat hepatocytes by adding various concentrations of growth hormone, insulin and dexamethasone (Dex). 11 beta-HSD activity increased significantly after treatment with Dex (10(-9)M-10(-6)M) for 48h. Dex (100nM) treated hepatocytes, incubated for 12h to 48h, resulted in a significant two-to four-fold rise in 11 beta-HSD activity compared to control (p < 0.01), which was in contrast to GH (10(-9)M-10(-6)M) and insulin (10(-8)M-10(-5)M), which inhibited 11 beta-HSD activity (p < 0.05). These results suggest that the 11 beta-HSD of rat hepatocytes is under multifactorial regulation; Dex stimulates and GH and insulin inhibit 11 beta-HSD activity in primary cultures of rat hepatocytes.     

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.     

Fixed combination verapamil SR/trandolapril

Urapidil is a peripheral postsynaptic alpha 1-adrenoceptor antagonist with central agonistic action at serotonin 5-HT1A receptors. It reduces blood pressure by decreasing peripheral vascular resistance. Oral urapidil decreases blood pressure in patients with mild to moderate essential hypertension and associated risk factors such as hyperlipidaemia or type 2 (non-insulin-dependent) diabetes mellitus with no effect on heart rate. The antihypertensive efficacy of urapidil is similar to that of most comparators in patients with mild to moderate essential or secondary hypertension and no concomitant risk factors. However, the antihypertensive efficacy of urapidil was lower than that of hydrochlorothiazide in a well designed trial. Lipid levels and glucose metabolism are not adversely affected and may improve with urapidil in patients with lipid or glucose abnormalities. Urapidil can be safely combined with other antihypertensive agents such as hydrochlorothiazide and nifedipine and improves blood pressure control in previous nonresponders to monotherapy. Intravenous urapidil reduces blood pressure in patients with pre-eclampsia or hypertension in pregnancy and in patients with hypertensive crises or peri- or postoperative hypertension. The decrease in blood pressure is similar to that observed after nifedipine, enalaprilat, sodium nitroprusside and dihydralazine, greater than that of ketanserin according to 1 larger study, and greater than that of sublingual nitroglycerin in 1 trial in patients with nonsurgical hypertensive crises and pulmonary oedema. However, more patients responded to treatment with urapidil than with enalaprilat or nifedipine. Heart rate is less likely to be altered by urapidil than with some comparator drugs. Urapidil appears to be well tolerated, with most adverse events being mild and transient. The incidence of adverse events with urapidil is similar to that with prazosin, metoprolol, atenolol, sodium nitroprusside and hydrochlorothiazide and less than that with nifedipine and clonidine. Urapidil may not be as well tolerated as captopril and, in 1 study, more urapidil than nitrendipine recipients discontinued treatment because of adverse events. Conclusions: urapidil reduces blood pressure without altering heart rate. The oral formulation is an effective choice in patients with hypertension and concomitant dyslipidaemia or type 2 diabetes mellitus, in whom the drug does not adversely affect and may improve lipid profiles and glucose metabolism. The intravenous formulation is effective in controlling various hypertensive crises and hypertension associated with pregnancy or surgery and is similar to or better than other first-line agents used in these conditions. Thus, urapidil may be a useful alternative to currently available antihypertensive agents.     

Lipid balance in HepG2 cells: active synthesis and impaired mobilization

The human hepatoma cell line HepG2 in culture medium synthesized fatty acids de novo (144 +/- 9 nmol fatty acid/mg protein per 24 h) at a rate similar to that observed in freshly prepared rat hepatocytes (192 +/- 8 nmol/mg per 24 h) and in primary cultures of rat hepatocytes (165.4 +/- 29.3 nmol/mg per 24 h). In HepG2 cells, fatty acid synthesis was inhibited by extracellular oleate (0.75 mM), and, to a lesser extent, by glucagon (10(-7) M). Insulin (7.8 x 10(-8) M) had a mild stimulatory effect. Fatty acid synthesis was not influenced by lipogenic precursors (lactate plus pyruvate), substances which, in rat hepatocytes, had pronounced stimulatory effects. Fatty acid synthesis rates were also unchanged in the presence of prostaglandin E2 (PGE2). In general, compared to rat hepatocytes, fatty acid synthesis in HepG2 cells was less sensitive to manipulation of the culture medium in vitro. HepG2 cells had a high capacity for triacylglycerol synthesis from extracellular oleate (469 +/- 43 nmol triacylglycerol/mg protein per 24 h) but phospholipid synthesis was relatively low (15.8 +/- 0.4% of total glycerolipids). Very little of the above newly synthesized triacylglycerol was secreted as lipoprotein (4.62 +/- 0.88 nmol triacylglycerol/mg protein per 24 h) resulting in a large intracellular accumulation of triacylglycerol. This was exacerbated by the absence of any detectable ketogenesis. The secretion of triacylglycerol produced from de novo synthesized fatty acids was also very low in HepG2 compared to that observed in primary cultures of rat hapatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biotransformation of the ergot derivative CQA 206-291 in human, dog, and rat liver slice cultures and prediction of in vivo plasma clearance

Liver slice cultures from humans, dogs, and rats were used to investigate the biotransformation of the dopaminergic ergot agonist CQA 206-291 and to predict pharmacokinetic values for hepatic intrinsic clearance and plasma clearance. CQA 206-291 was extensively metabolized in the liver slice cultures and in vivo. The HPLC metabolite patterns from the liver slice cultures were similar for all three species, indicating the occurrence of the same metabolic pathways for CQA 206-291 biotransformation. The rate of formation of CQ 32-084, a pharmacologically active N-deethylated metabolite, exceeded that of metabolite d, a primary metabolite, by 1.4 fold in human liver slices, and by 1.7 fold in rat liver slices. In dog liver slice cultures, metabolite d formation exceeded CQ 32-084 formation by 1.3 fold and was formed at a statistically significantly greater rate (3 fold) than in either human or rat liver slices. The metabolism of ergots like CQA 206-291 by human fetal liver was also demonstrated in this study. However, the prominent metabolite from fetal and adult human liver microsomes was metabolite d with minor amounts of CQ 32-089 being formed. A major route of excretion for the metabolites of CQA 206-291 is the kidney, yet the kidney does not contribute to the metabolism of CQA 206-291. Kidney slices derived from humans, rats, and dogs did not metabolize CQA 206-291 within 24 hr. CQA 206-291 intrinsic clearance was derived from the half-life of parent drug disappearance in the liver slice and hepatocyte cultures, and from the ratio of Vmax/Km of human and rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative metabolism of the antiviral dimer 3'-azido-3'-deoxythymidine-P-2',3'-dideoxyinosine and the monomers zidovudine and didanosine by rat, monkey, and human hepatocytes

AZT-P-ddI is an antiviral heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (AZT) and one molecule of 2',3'-dideoxyinosine (ddI) linked through their 5' positions by a phosphate bond. The metabolic fate of the dimer was studied with isolated rat, monkey, and human hepatocytes and was compared with that of its component monomers AZT and ddI. Upon incubation of double-labeled [14C]AZT-P-[3H]ddI in freshly isolated rat hepatocytes in suspension at a final concentration of 10 microM, the dimer was taken up intact by cells and then rapidly cleaved to AZT, AZT monophosphate, ddI, and ddI monophosphate. AZT and ddI so formed were then subject to their respective catabolisms. High-performance liquid chromatography analyses of the extracellular medium and cell extracts revealed the presence of unchanged dimer, AZT, 3'-azido-3'-deoxy-5'-beta-D-glucopyranosylthymidine (GAZT), 3'-amino-3'-deoxythymidine (AMT), ddI, and a previously unrecognized derivative of the dideoxyribose moiety of ddI, designated ddI-M. Trace extracellular but substantial intracellular levels of the glucuronide derivative of AMT (3'-amino-3'-deoxy-5'-beta-D-glucopyranosylthymidine [GAMT]) were also detected. Moreover, the extent of the formation of AMT, GAZT, and ddI-M from the dimer was markedly lower than that with AZT and ddI alone by the hepatocytes. With hepatocytes in primary culture obtained from rat, monkey, and human, large interspecies variations in the metabolism of AZT-P-ddI were observed. While GAZT and ddI-M, metabolites of AZT and ddI, respectively, as well as AZT 5'-monophosphate (MP) and ddI-MP were detected in the extracellular media of all species, AMT and GAMT were produced only by rat and monkey hepatocytes. No such metabolites were formed by human hepatocytes. The metabolic fate of the dimer by human hepatocytes was consistent with in vivo data recently obtained from human immunodeficiency virus-infected patients.     

The cell surface of Trypanosoma musculi bloodstream forms. II. Lectin and immunologic studies

Report is given on a metabolic investigation with non-radioactive and 14C-labelled methiothepin(1-[10',11'-dihydro-8'-(methylthio)-dibenzo mean value of b,f thiepin-10'yl]-4-methyl-piperazine) in rat, dog, and man. After i.p. and oral administration of the drug to the rat, the metabolites of methiothepin were excreted fecally. In the same species, a considerable biliary secretion of the compounds has been demonstrated. In dog and in man, excreted metabolites have been found both in urine and feces after oral application of the drug. The biotransformation of methiothepin within the species investigated proceeds via hydroxylation, sulfoxidation, O-methylation, N-demethylation, N-oxidation and formation of conjugates. The large number of metabolites is due to the various sites of action within the molecule, that are accessible to in vivo oxidation. Of a large number of isolated positionally isomeric compounds, merely the basal structures could be clarified. Possibly the mode of biotransformation to which methiothepin is subjected in the organism, proves determinant for the way of excretion. In the rat, all metabolites are hydroxylated and reach the intestinal tract as conjugates with the bile. In dog and man, however, non-hydroxylated, sulfoxidized metabolites were likewise found, which were excreted mainly renally in both species.     

Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture

The success of oltipraz (OPZ) [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] as a chemoprotective agent against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat is thought to depend principally on its ability to enhance detoxication by inducing phase II enzymes, especially glutathione transferases. However, in primary cultures of human hepatocytes, we recently demonstrated that OPZ also has an important inhibitory effect on the major cytochromes P450 (CYPs) of human hepatic AFB1 metabolism. This has prompted a detailed study of the effect of OPZ on some CYPs involved in metabolism of AFB1 in the rat. Primary cultures of rat hepatocytes behaved similarly to human hepatocytes and responded to OPZ by inhibition of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities mainly associated, respectively, with CYP1A and CYP2B. A time-course shows that this inhibition is largely reversible, with EROD and PROD activities reaching a minimum at 12 h and tending towards control values within 24 h. As is to be expected, the incubation of isolated microsomes with OPZ also inhibits CYP1A and 2B. The effect of OPZ on CYP1A is not a phenomenon limited to cells in culture, but also occurs in vivo. Using the whole animal, we were able to demonstrate that OPZ also transiently inhibited CYP1A activity in a rat given caffeine, by measuring the amounts of methylxanthines found in the serum. However, microsomes isolated from rats, that had been treated with OPZ in vivo, show no such inhibition, presumably because, since OPZ is a reversible inhibitor, it dissociates and is lost during the course of conventional procedures of microsomal preparation. This explains some earlier failures in studies of isolated microsomes to observe the inhibition of CYPs by OPZ. In addition to inhibiting their enzymatic activity, OPZ is also an inducer of CYP1A and 2B as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more. It is concluded that the mechanism of chemoprotection by OPZ, of toxic chemical metabolism in the rat, is complex and involves competitive inhibition of activation succeeded by induction of the enzymes of both activation and detoxication.     

In vitro toxicity of zamifenacin (UK-76,654) and metabolites in primary hepatocyte cultures

1. We compared the sensitivities of primary hepatocytes from rat, dog and monkey to zamifenacin and two major metabolites, the methylenedioxy ring-opened catechol, UK-80,178 and its methylated product, UK-82,201. Toxicity was determined both via neutral red uptake and enzyme leakage data. 2. Canine hepatocytes were most sensitive to the cytotoxic effects of zamifenacin during 24-h exposure. Significant decreases in medium concentrations of zamifenacin in the presence of primary hepatocytes verified cellular uptake during the initial 2-h incubation. All three cell types were much more sensitive to UK-82,201 than to the catechol metabolite or parent drug. 3. The rapid onset of cytotoxicity indicated by elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers in the medium after UK-82,201 exposure, the delayed but substantial cytotoxic response to the parent drug which was suggestive of biotransformation to a reactive moiety, in vivo and in vitro drug metabolism results and subacute toxicology data suggest that dog may more effectively transform zamifenacin into UK-82,201, which is relatively hepatotoxic. 4. Because the catechol was generally less toxic than the O-methylated product, species that eliminate zamifenacin primarily as the catechol or its conjugate may be less affected by the potential hepatotoxicity of the methylated product. Our studies show that dog is the most sensitive species due to metabolism of the common catechol metabolite. The low incidence of potential hepatotoxicity in the clinic points to rare but important differences in the metabolism of Zamifencin. We conclude that the findings in dog were not predictive of subsequent effects in man.     

Dichloroacetic acid induction of peroxisome proliferation in cultured hepatocytes

Trichloroethylene is a widespread industrial solvent and one of the most common environmental contaminants. Trichloroethylene causes hepatocarcinoma in the B6C3F1 mouse in a dose-dependent manner. Trichloroethylene's hepatocarcinogenicity is thought to be mediated through its metabolites trichloroacetate and dichloroacetate. Although the mechanism of action is not well understood, hepatic tumors are thought to arise as a result of excessive peroxisome-dependent active oxygen production or secondary to enhanced cell replication. The peroxisome proliferative activity of trichloroacetate has been replicated in cultured rodent hepatocytes, while that of dichloroacetate has not been demonstrated. The present experiments were designed to characterize the peroxisome proliferative response to dichloroacetate in hepatocyte cultures from male B6C3F1 mice and male Long Evans rats. The cultured hepatocytes were treated after attachment with 0.1, 0.5, 1.0, 2.0, or 4.0 mM dichloroacetate for 72 hours. Peroxisome proliferation was assessed by measuring palmitoyl-CoA oxidation and by immunoquantitation of peroxisomal bifunctional enzyme. Palmitoyl CoA oxidation increased in a concentration-dependent manner, with maximal induction of 5.5- and 5-fold in mouse and rat hepatocytes, respectively, after treatment with 2.0 mM dichloroacetate. Peroxisomal bifunctional enzyme protein levels also increased in a concentration-dependent manner in both rat and mouse hepatocytes in response to dichloroacetate exposure. These results indicate that the peroxisomal response observed in vivo in response to dichloroacetate administration can be reproduced in primary cultures of rat and mouse hepatocytes treated with dichloroacetate. Further studies using this model system will help elucidate mechanisms of dichloroacetate-induced hepatocarcinogenesis.     

Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.     

In vivo electrochemical studies of dopamine clearance in subregions of rat nucleus accumbens: differential properties of the core and shell

The effects of the rodent hepatocarcinogens clofibric acid and diprofibrate on the activity of the peroxisomal fatty acyl-CoA oxidase, DNA synthesis, and apoptosis were compared in cultured rat and human hepatocytes. Rat hepatocytes expressed a 10-fold greater level of the peroxisomal fatty acyl-CoA oxidase compared to human hepatocytes. At the highest concentration (1.0 mM), both drugs induced a two- to threefold increase in this enzyme activity in both rat and human hepatocytes. Ciprofibrate (0.1 and 0.2 mM) caused a twofold increase in DNA synthesis in rat hepatocytes, whereas clofibric acid had no effect on DNA synthesis in these cells. In contrast, increasing concentrations of both clofibric acid and ciprofibrate produced inhibition of DNA synthesis in human hepatocytes. By using the terminal transferase dUTP-biotin nick end labeling technique, it was observed that 0.1 and 0.2 mM clofibric acid and ciprofibrate suppressed transforming growth factor-beta (TGF beta)-induced apoptosis by 50% in rat hepatocytes, but they had no effect on TGF beta-induced apoptosis in human hepatocytes. Although clofibric acid and ciprofibrate diminished TGF beta-induced apoptosis, they had no effect on the basal apoptotic levels in the rat hepatocyte cultures. However, both drugs significantly increased the percent of apoptotic cells in the human hepatocyte cultures. It is concluded that primary rat and human hepatocyte cultures respond differently to peroxisome proliferators. The differences in effects on DNA synthesis and apoptosis support the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatocarcinogenesis.     

Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that overexpress transforming growth factor alpha 1

Transforming growth factor alpha (TGF-alpha) is a polypeptide closely associated with hepatocyte proliferation in vivo and in vitro. In order to investigate the mechanisms by which TGF-alpha contributes to hepatocyte replication and transformation, we isolated hepatocytes from mice bearing a human TGF-alpha transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-alpha mRNA and peptide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72-96 h in culture before senescing, similar to nontransgenic hepatocytes supplemented with epidermal growth factor. Constitutive expression of TGF-alpha rendered the transgenic hepatocytes unresponsive to further growth stimulation by exogenous TGF-alpha, as well as other mitogens such as epidermal growth factor and hepatocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGF beta 1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epidermal growth factor-supplemented normal hepatocytes to replicate repeatedly and survive for > or = 2 months in primary culture while maintaining differentiated traits. From these long-term primary cultures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively). Both lines continued to express differentiated adult hepatocytic markers such as albumin, alpha-1-antitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also expressed mRNAs for the oncofetal markers alpha-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the transgenic TAMH hepatocyte line was quasi-tetraploid, strongly expressed human TGF-alpha mRNA, and was highly tumorigenic in nude mice. Well-differentiated hepatocellular carcinomas developed in nude mice given injections of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-alpha transgenic mice with regard to histology and strong expression of mouse and human TGF-alpha, insulin-like growth factor II, and alpha-fetoprotein mRNAs. Our data show that TGF-alpha overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations must occur for carcinogenesis. Inappropriate expression of insulin-like growth factor II may constitute one of these steps. The TGF-alpha transgenic mouse hepatocyte line TAMH appears to undergo transformation in a similar manner to that of hepatocytes overexpressing TGF-alpha in vivo, and should serve as an ideal system in which to study hepatocarcinogenesis.     

Seroprevalence of measles- and rubella-specific antibodies among military recruits, Canada, 1991

OBJECTIVE: To determine the plasma and cerebrospinal fluid (CSF) levels of urapidil after i.v. administration and the effect on CSF serotonin and 5-hydroxyindoleacetic acid (5-HIAA) concentrations. DESIGN: Open, single-dose study. SETTING: Post-surgery following neurosurgical removal of the hypophysis (n = 5) or aneurysm clipping (n = 1). PATIENTS: 6 patients, aged 32-71 years, with intact blood-brain barrier (BBB); 1 patient was studied twice. INTERVENTIONS: Single dose of 25 mg urapidil i.v. as prophylaxis of BP increase during extubation or as treatment of hypertensive episodes. MEASUREMENTS AND RESULTS: Urapidil, serotonin and 5-HIAA were measured by HPLC in CSF during 8 h after urapidil administration. Urapidil was detected in CSF as soon as 5 min after injection in 3 patients. The concentration ratio of plasma/CSF after the distribution phase was about 5:1. No significant effect on serotonin and 5-HIAA in CSF was seen. CONCLUSION: After administration of a therapeutic dose, urapidil permeates the BBB and may interact with central 5-HT1A-receptors.     

The influence of medium composition on the maintenance of cytochrome P-450, glutathione content and urea synthesis: a comparison of rat and sheep primary hepatocyte cultures

Rat and sheep primary hepatocytes have been cultured in four different medium formulations: Williams' E, Chee's, Medium 199 and Modified Earle's. The total cytochrome P450 content, intracellular concentration of reduced glutathione, rate of urea synthesis and total protein content of cultures of cells from both species in each medium have been determined. Modified Earle's and Chee's medium proved to be the most favourable formulations for the culture of rat hepatocytes. After 48 h, cells cultured in Modified Earle's had significantly more cytochrome P450 and a significantly greater rate of urea synthesis than cells in any other medium. After 6 days in culture the difference in cytochrome P450 levels between rat hepatocytes in Chee's medium and those in Modified Earle's medium was abrogated. The difference in the rate of urea synthesis between rat hepatocytes cultured in each of these two media was shown to be more dependent on the medium in which the cells were maintained during the period of urea synthesis measurement than on the medium in which the cells had been previously cultured. Sheep hepatocytes cultured in Chee's medium ruptured and died within 24 h. Apart from this, sheep cells were less sensitive to changes in medium formulation than were rat hepatocytes. The initial plating efficiency was lower in sheep cells. Total cytochrome P450 content was the most discriminatory of the four parameters for evaluating the status of rat hepatocyte cultures. However, urea synthesis may be the most useful parameter for assessment of hepatocyte function in hybrid liver devices such as bioartificial liver support systems where access to the cells during operation of the device is restricted.     

Reductive activation of benzazolo[3,2-a]-quinolinium chlorides

An in vitro technique for the determination of radiopharmaceutical metabolism has been developed using isolated hepatocytes. Radioiodinated N-isopropyl-p-iodoamphetamine (IMP; iofetamine, USP) was employed a model tracer in these studies because its labeled metabolites are well-characterized. Hepatocytes isolated from the rat and human produced labeled metabolites in vitro for up to 4 h in a manner similar to that reported for humans in vivo. Identical metabolites were generated by all cell types investigated, but the rate of metabolism differed (rat > human; female > male; fresh > frozen). The utility of this methodology for the preclinical evaluation of potential radiopharmaceuticals is described.     

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

The effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.     

Effects of insulin-like growth factor I and II on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We compared the effects of insulin-like growth factor I (IGF-I) and II (IGF-II) on DNA synthesis and proliferation and investigated various signal transduction mechanisms involved in insulin-like growth factor-induced mitogenesis in primary cultures of adult rat hepatocytes. IGF-I stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 75 ng/ml within 4 h of culture. These effects were sensitive to the IGF-I concentration and cell density. Hepatocyte proliferation induced by IGF-I was potentiated by metaproterenol (10(-6) M) as well as by 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA; 10(-8) M) and was inhibited by U-73122 (1-(-[[17beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-+ ++1Hpyrrol-2,5-dione)), genistein, wortmannin, PD98059 (2'-amino-3'-methoxyflavone) and rapamycin. The IGF-I effect was independent of pertussis toxin (100 ng/ml). IGF-II also dose dependently stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 0.75 ng/ml within 4 h of culture. However, these effects were not dependent on the initial plating density. The stimulatory effects of IGF-II were potentiated by UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (10(-5) M) and inhibited by phenylephrine, PMA, metaproterenol, 8-bromo-cAMP, PD98059, rapamycin, and pertussis toxin. The IGF-II effects were not affected by genistein, U-73122, and wortmannin. These results suggest that IGF-I and IGF-II rapidly stimulate the DNA synthesis and proliferation of adult rat hepatocytes by separate mechanisms.     

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.