Treatment Of Hepatocyte Cultures Research Articles

Cross-talk between Aryl Hydrocarbon Receptor and Mitogen-Activated Protein Kinase Signaling Pathway in Liver Cancer through c-raf Transcriptional Regulation

c-raf is a serine-threonine kinase and a downstream effector of ras signaling. This kinase plays an essential role in cell proliferation, differentiation, and apoptosis. In the past, we reported induction of c-raf gene expression in rat liver cancer on treatment with a mixture of aryl hydrocarbon receptor (AhR) agonists. This prompted our interest in investigating the role of AhR in the transcriptional regulation of c-raf. Initially, we cloned the rat c-raf promoter and sequenced the genomic DNA and cDNA by Southern blotting and capillary electrophoresis. Then, a genetic algorithm was applied to search for putative AhR-binding sites. DNA-binding activity of AhR was confirmed by electromobility shift assay. We also studied c-raf gene expression in rat hepatoma cell lines with functional and/or devoid AhR and in primary human and rat hepatocyte cultures. Overall, we identified five and three AhR-binding sites in the human and rat c-raf gene, respectively. Treatment of hepatocyte cultures with the AhR antagonist resveratrol reduced DNA binding of AhR. Only rat hepatoma cells with functional AhR responded to 1 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment with >10-fold c-raf mRNA induction. Treatment of human and rat hepatocyte cultures with various AhR-activating chemicals resulted in induction of c-raf gene expression, albeit at different levels. Taken collectively, we show AhR to be a master regulator of c-raf and propose cross-talk between AhR and the mitogen-activated protein kinase signaling pathway in chemically induced hepatocarcinogenesis.

Regulation of rat hepatic hydroxysteroid sulfotransferase (SULT2-40/41) gene expression by glucocorticoids: evidence for a dual mechanism of transcriptional control.

Glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2-40/41) gene transcription was investigated in primary cultured rat hepatocytes transiently transfected with a series of SULT2-40/41 5'-flanking region-luciferase reporter constructs, with emphasis on examining the functional role of an inverted repeat-0 nuclear receptor motif (IR0). Treatment of transfected cultures with any of four glucocorticoids activated luciferase expression from a construct containing 1938 base pairs (bp) of the SULT2-40/41 gene 5'-flanking sequence, whereas deletion of bp -227 to -158 (containing the IR0 motif) largely abolished the effect. On closer analysis, treatment of hepatocyte cultures with either of the potent glucocorticoids dexamethasone [strong cytochrome P-450 3A (CYP3A) inducer] or triamcinolone acetonide (weak CYP3A inducer) produced dose-dependent increases in luciferase activity when hepatocytes were transiently transfected with a construct containing as little as 158 bp of 5'-flanking sequence or containing a mutated IR0 motif. The dexamethasone dose-dependent increase in luciferase activity continued through a dose of 10(-6) M when the transfected construct contained the IR0 motif, but was maximal at 10(-7) M when the transfected construct lacked the IR0 motif. In contrast, triamcinolone acetonide-induced luciferase activity was maximal at a dose of 10(-7) M, irrespective of the presence or absence of the IR0 motif. Coincubation of transfected hepatocytes with 10(-8) M dexamethasone and the antiglucocorticoid RU486 inhibited luciferase expression. Luciferase induction by the prototypical CYP3A inducer pregnenolone 16alpha-carbonitrile was restricted to constructs containing the IR0 motif. These data suggest that glucocorticoid-inducible SULT2-40/41 gene expression occurs through a dual mechanism, whose components possibly involve the glucocorticoid receptor and the pregnane X receptor.

Neomycin inhibits secretion of apolipoprotein[a] by increasing retention on the hepatocyte cell surface

Neomycin therapy reduces plasma levels of low density lipoprotein and lipoprotein[a] (Lp[a]). To determine whether neomycin directly alters the biogenesis of Lp[a], we have examined the effect of neomycin on apolipoprotein[a] (apo[a]) synthesis and secretion in primary cultures of baboon hepatocytes. Using this system, we have previously shown that apo[a] is synthesized as a lower molecular weight precursor that upon maturation becomes associated with the cell surface before release into the culture medium. Treatment of hepatocytes with 10 mM neomycin reduced levels of apo[a] in the culture medium by as much as 12-fold. Although a portion of the reduced secretion could be accounted for by a reduction in total protein synthesis, the greatest effect of neomycin on apo[a] secretion was to decrease the release of mature apo[a] from the hepatocyte cell surface into the culture medium. Treatment of hepatocyte cultures with trypsin confirmed that mature apo[a] in neomycin-treated cells was still transported to the cell surface. Examination of related antibiotics demonstrated that inhibition of apo[a] secretion is a general property shared by the deoxystreptamine antibiotics. The mechanism by which neomycin affects the apo[a]-cell surface interaction is not known, but neomycin is known to perturb cell surface membranes, inhibit the interaction of some ligands with their cell surface receptors, and inhibit the metabolism of phosphatidylinositol 4,5 biphosphate. These studies suggest that cell surface association of apo[a] may play a role in Lp[a] biogenesis in vivo.

11 beta-hydroxysteroid dehydrogenase is an exclusive 11 beta- reductase in primary cultures of rat hepatocytes: effect of physicochemical and hormonal manipulations.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the conversion of corticosterone to inert 11-dehydrocorticosterone, thus regulating glucocorticoid access to intracellular receptors. This type 1 isoform (11 beta HSD-1) is a bidirectional NADPH(H)-dependent enzyme in vitro and is highly expressed in liver, where it is regulated by glucocorticoids, thyroid hormones, estrogen, and GH in vivo. In humans in vivo, enzyme inhibition alters glucose homeostasis, an effect thought to be mediated in the liver. However, detailed investigation of the biology of 11 beta HSD-1 in liver, its function, regulation, and indeed even reaction direction, has been hampered by the lack of clonal hepatic cell lines that express 11 beta HSR-1. Studies of nonhepatic cell lines have suggested that 11 beta HSD-1 is directly regulated by hormones, and transfection of nonhepatic cell lines has sown that reaction direction varies between cell types, possibly reflecting intracellular cosubstrate (NADP+/NADPH) ratios or PH. To investigate reaction direction and gene regulation of 11 beta HSD-1 in hepatocytes, we defined conditions for primary culture of adult rat hepatocytes that maintain high 11 beta HSR-1 messenger RNA expression. In intact primary hepatocytes over a wide range of steroid concentrations (2.5-250 nM), 11 beta-reduction was the predominant reaction direction [33.5 +/- 0.5% conversion of 11-dehydrocorticosterone (25 nM) to corticosterone after 30 min], with undetectable 11 beta-dehydrogenation. However, homogenates of hepatocyte cultures showed plentiful 11 beta-dehydrogenase activity. Treatment of hepatocyte cultures with the metabolic inhibitors sodium azide (5 nM) and KCN (1 nM) altered cellular NADP+/NADPH ratios from 0.244 +/- 0.042 in controls to 0.020 +/- 0.001 and 0.152 +/- 0.009, respectively, but had no effect on 11 beta-reductase or 11 beta- dehydrogenase activity. High concentrations of KCN (10 mM) modestly increased 11 beta-reductase activity (32.4 +/- 1.7% to 48.8 +/- 0.5%, whereas 11 beta-dehydrogenation remained at the limit of detection. Manipulation of culture medium pH (6.2-8.0) had no effect on enzyme activity. Dexamethasone (10-7 M) induced hepatocyte 11 beta-reductase activity from 23.4 +/- 0.7% to only weakly affects reaction direction. Glucocorticoid and insulin regulation of hepatic 11 beta HSD-1 is directly mediated, but other hormonal controls are either lost in culture or mediated indirectly. This primary hepatocyte culture system will allow investigation of the control of 11 beta-reductase activity and its implications for glucocorticoid-regulated hepatic functions.

Long-term inhibitory effect of cAMP on β-adrenoceptor acquisition and nonselective attenuation of adenylyl cyclase in hepatocytes

Long-term effects of cAMP on the surface expression of β-adrenoceptors and adenylyl cyclase activity were investigated in primary cultures of rat hepatocytes. β-Adrenoceptor density and catecholamine-responsive adenylyl cyclase activity increased during culturing in a biphasic manner, with a plateau of 10–20 h duration occurring approximately 10 h after plating. Treatment of hepatocyte cultures with 8-bromo-cAMP during the plateau period did not affect the density of β-adrenoceptors. In contrast, addition of 8-bromo-cAMP, 8-chlorophenylthio-cAMP, forskolin or glucagon during a period of active recruitment of surface β-adrenoceptors resulted in a suppression of the acquisition of β-adrenoceptors. In both experimental situations there was a partial decrease in hormone-stimulated and basal adenylyl cyclase activity. The results suggest that cAMP exerts at least two types of long-term regulation of adenylyl cyclase in hepatocytes: a suppressive effect on β-adrenoceptor acquisition, and a partial, nonselective decrease in adenylyl cyclase activity not involving β-adrenoceptor down-regulation.

Regulation of Phenobarbital-Inducible Cytochrome-P450 2B1/2 mRNA by Lovastatin and Oxysterols in Primary Cultures of Adult Rat Hepatocytes

Lovastatin (LOVA) is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase widely used in clinical practice. We treated primary cultures of adult rat hepatocytes, maintained in a minimal, serum-free medium on Matrigel, a reconstituted basement membrane, with this drug, and found that the amounts of P450 2B2 mRNA detected on Northern blots were increased at the same doses (10−5 to 3 × 10−5 M) required for induction of HMG-CoA reductase mRNA, a gene known to be under oxysterol regulatory control. LOVA treatment produced selective effects increasing also the mRNA levels for P450s 2C6, 2C7, 3A1, and 4A1 but not for 1A1, 2A1/2, or NADPH-cytochrome P450 oxidoreductase. LOVA treatment increased the induction of 2B1/2 mRNA in cells cotreated with either phenobarbital (PB; 10−4 M) or clotrimazole (CTZ; 10−5 M), or of 3A1 mRNA in cells cotreated with PB (2 × 10−3 M), but not dexamethasone (10−5 M). LOVA treatment did not potentiate the induction of 1A1 or 4A1 mRNA in cells cotreated with β-naphthoflavone (10−5 M) or ciprofibrate (10−4 M), respectively. In contrast to the potentiation of 2B1/2 mRNA induction produced by treatments with LOVA in combination with PB or CTZ, cotreatment of hepatocytes with PB and CTZ did not result in increased induction relative to that seen in cells treated with either agent alone. Treatment of hepatocyte cultures with either mevalonate (3 × 10−4 to 3 × 10−3 M), the immediate product of HMG-CoA reductase, or 25-hydroxycholesterol (10−6 to 10−5 M), a model oxysterol, resulted in dose-dependent suppression of 2B1/2 mRNA induction in cells treated with PB-like inducers. Taken together, our results demonstrate that LOVA is a unique inducer of P450 mRNA in cultured rat hepatocytes and implicate oxysterols as potential intracellular modulators of 2B1/2 induction. We conclude that endogenous metabolic factors including those related to cholesterol biosynthesis are critical in induction of liver cytochromes P450 2B1 and 2B2 by PB and "PB-like" agents.

Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes.

Because the metabolic environment can alter gene expression in cultured cells, we examined the effects of change of medium on the levels of several cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on Matrigel. The amounts of P450 1A2, 2B1/2, or 3A1 mRNA were unaffected by changing the medium. In contrast, P450 1A1 mRNA levels were increased 1 to 2 h after media change, reached maximum levels by 6 h, and declined to baseline by 24 h. Supplementing day-old media with components of the medium revealed that only addition of amino acids resulted in 1A1 mRNA induction. From the results of direct additions and omissions, we showed that tryptophan, but not histidine, was largely responsible for the 1A1 mRNA induction. Moreover, mild photoactivation of the tryptophan resulted in a substantially increased magnitude of 1A1 mRNA induction. The time course for 1A1 mRNA induction by treatment with photoactivated tryptophan was identical to that observed after medium change. Treatment of hepatocyte cultures with beta-naphthoflavone, which is metabolized by 1A1, also resulted in a transient 1A1 mRNA induction time-course profile over a 24-h period, whereas treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which is relatively stable to metabolic transformation, produced sustained elevations of 1A1 mRNA, suggesting that the transient response to tryptophan also may involve metabolism of the inducer. Our results extend previous data showing that oxidized products of tryptophan induce 1A1, and suggest that the transient nature of the induction may be due to elimination of the activated tryptophan molecule.

Induction of cytochrome P-450 by dimethyl sulfoxide in primary cultures of adult rat hepatocytes

Treatment of hepatocyte cultures with dimethyl sulfoxide (DMSO) induced P-450IIE1-specific aniline 4-hydroxylase activity and P-450IA1-specific ethoxyresorufin O-deethylase activity at a concentration of 0.1% (v/v). The P-450IIB-specific pentoxyresorufin O-dealkylase activity was induced only at the 2% (v/v) level. Dot blot analysis of the total cellular RNA and cycloheximide treatment of the culture suggested that induction of ethoxyresorufin O-deethylase activity by DMSO may be due to the increase of de novo synthesis of the P-450IA1 protein, not to accumulation of mRNA in the hepatocyte culture.

Inhibition of Microcystin-Induced Release of Cyclooxygenase Products from Rat Hepatocytes by Anti-Inflammatory Steroids

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.

Inhibition of mouse hepatocyte intercellular communication by paraquat-generated oxygen free radicals

Intercellular communication through gap junctions functions in electrical synapsing, homeostasis, hormonal response, embryogenesis, and growth control. Many neurotoxicants, teratogens, and carcinogens are capable of inhibiting gap junctional intercellular communication and this effect may be related to their toxic activity. In addition, many of these toxic agents are capable of stimulating oxygen free radical production in cells. The purpose of this study was to determine if oxygen free radicals at noncytotoxic levels could inhibit intercellular communication in primary cultured mouse hepatocytes. Intercellular communication was evaluated in 24-hr-old cultures of male B6C3F1 Cr1BR mouse hepatocytes by microinjection of fluorescent Lucifer Yellow CH dye and visualization of dye spread to adjacent hepatocytes (dye-coupling). Dye-coupling was rapidly established in freshly plated primary cultured hepatocytes reaching a level of over 90% after 24 hr of culture. After 24 hr, dye-coupling paralleled hepatocyte survival. Treatment of hepatocyte cultures with noncytotoxic concentrations of paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride; PQ) (3–5 m m), hydrogen peroxide (2–5 m m), glucose oxidase (0.1 U/ml), or xanthine oxidase (0.2 U/ml plus 1 m m xanthine) for exposure durations of 2–8 hr resulted in concentration-dependent decreases in dye-coupling. Addition of the antioxidants DPPD ( N,N-diphenyl- p-phenylenediamine; 25 μ m) and vitamin E ( d,l-α-tocopherol acetate; 100 μ m) decreased the inhibitory effect of PQ on dye-coupling. In contrast, addition of the catalase inhibitor 3-amino-1,2,4-triazole or the glutathione depletor diethylmaleate to PQ-treated cultures potentiated PQ-induced inhibition of dye-coupling. PQ stimulated NADPH-dependent mouse liver microsomal superoxide radical production. Thus, one effect of prooxidant compounds appears to be the inhibition of IC. This effect may be important in the sublethal toxicity of oxygen radical generating compounds.

The substituted pyridines metyrapone and nicotinamide are inducers of 5-aminolevulinate synthase and cytochrome P-450 in hepatocyte culture

The effects of metyrapone and nicotinamide, two substituted pyridines, were studied in cultured chick embryo hepatocytes, a system characterized by preserved inducibility of cytochrome P-450 hemoproteins. Both metyrapone and nicotinamide caused a dose-dependent increase in cytochrome P-450 concentration. Their inducing potencies differed by one to two orders of magnitude and correlated with the known difference in the binding affinity of these two pyridines to cytochrome P-450. The increase of cytochrome P-450 concentration after metyrapone and nicotinamide was additive to the induction of cytochrome P-450 by phenobarbital and β-naphthoflavone and was abolished by cycloheximide. Treatment of hepatocyte cultures with metyrapone resulted in an increase in a microsomal protein with an apparent mol. wt of 52,000. In addition, induction of cytochrome P-450 by the substituted pyridines was associated with enhanced 5-aminolevulinate synthase, the rate-limiting enzyme of heme biosynthesis. These data suggest that in cultured chick embryo hepatocytes the substituted pyridines metyrapone and nicotinamide induce cytochrome P-450.