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Studies in vivo using inhibitors of eicosanoid synthesis suggested that prostaglandins may play a role in mediating tumor promotion in liver by agents such as phenobarbital (PB). However, it is not clear whether any stimulation of arachidonic acid metabolism/prostaglandin formation results directly from the action of tumor promoters on hepatocytes or indirectly from effects of promoters on Kupffer cells or other non-hepatocytes. Our laboratory has been utilizing relatively pure populations of rat hepatocytes under the defined conditions of primary cultures, to investigate growth-stimulatory actions of tumor promoters, an important element in the promotion stage of carcinogenesis. It has been shown that most if not all liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with factors such as EGF, insulin, and glucocorticoid. In the present study, we sought evidence for a role of prostaglandins (PGs) in the direct growth-stimulatory actions of tumor promoters on hepatocytes. PGE(2), PGF(2 alpha), and PGD(2) cause concentration-dependent stimulation of hepatocyte DNA synthesis, while arachidonic acid was without any effect. PGE(2) and PGF(2 alpha) required the presence of dexamethasone to exert significant effects. These PGs did not further augment the stimulatory effect of EGF. In contrast, PGD(2) stimulated DNA synthesis in the presence or absence of insulin, dexamethasone, or EGF. The effect of tumor promoters on arachidonic acid metabolism, as measured by [(3)H]arachidonic acid release and PGE(2) production, was determined. The phorbol ester TPA significantly increased [(3)H]arachidonic acid release as well as PGE(2) formation in hepatocytes in line with known effects in other cell types. However, liver tumor promoters phenobarbital (PB), alpha-hexachlorocycohexane (HCH), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pregnenolone-16 alpha-carbonitrile (PCN) were without effects. Finally, inhibitors of arachidonic acid metabolism were tested for effects on the ability of TPA or liver tumor promoters to stimulate DNA synthesis by direct action on cultured hepatocytes. In all cases, lack of selective inhibition was observed. Taken together, the results show that while prostaglandins may directly stimulate DNA synthesis in hepatocytes, they are unlikely to mediate the direct growth-stimulatory actions of liver tumor promoters.

The mechanisms of tumor promotion in liver by various xenobiotics of diverse structure are not well understood. However, these tumor promoters share the ability to exert growth-stimulatory effects on hepatocytes. Our laboratory has been utilizing normal rat hepatocytes under defined conditions of primary cultures, to investigate growth-stimulatory actions of liver tumor promoters. We have shown that most, if not all, of the liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with epidermal growth factor (EGF), insulin, and glucocorticoids. In the present study, we sought evidence for the role of the Na(+)/H(+) antiporter and cytoplasmic alkalinization in the direct growth-stimulatory actions of tumor promoters on hepatocytes. Hepatocytes cultured under conditions (bicarbonate-buffered medium) where intracellular pH (pH(i)) was independent of extracellular pH (pH(e)), EGF- and insulin-stimulated rates of DNA synthesis were unaffected by modest changes in pH(e). However, under conditions (HEPES-buffered medium) where pH(i) varied in a linear fashion with pH(e), rates of EGF- and insulin-stimulated DNA synthesis were highly dependent on pH(e). Similarly, 12-O-tetradecanoylphorbol-13-acetate (TPA) and alpha-hexachlorocyclohexane (HCH)-stimulated DNA synthesis were pH(e)-dependent but were stimulatory over different pH(e) ranges, suggesting that these promoters may act by distinct mechanisms. Chemicals that are capable of inducing rapid cytoplasmic alkalinization, ammonium chloride (1 and 15 mM) and monensin (0.5 microM), were found to stimulate hepatocyte DNA synthesis. The role of the Na(+)/H(+) antiport in controlling pH(i) of hepatocytes was demonstrated by artificially acidifying 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl (BCECF)-loaded isolated hepatocytes with 20 mM sodium acetate and the use of specific inhibitors. Amiloride and its analogues inhibited pH(i) recovery from the acid load in a dose dependent manner and the relative potency of these inhibitors paralleled their K(i) values for the Na(+)/H(+) antiport. At concentrations that stimulate hepatocyte DNA synthesis, some liver tumor promoters phenobarbital (PB) and HCH, were found to cause a rapid rise pH(i) in isolated hepatocytes which was sensitive to amiloride and its analogues. Taken together, our data suggest that activation of Na(+)/H(+) antiport activity may be one mechanism whereby some liver tumor promoters stimulate hepatocytes DNA synthesis. This study has implications for the mechanisms of tumor promotion in liver carcinogenesis.

Among the chemical compounds of varied structure which possess liver tumour-promoting are steroids, such as estrogens, pregnenolone derivatives and anabolic steroids. Although the mechanism(s) of tumour promotion in liver by these xenobiotics is not well understood, it is clear that growth stimulation is one important element in their action. As a basis for better defining whether steroids stimulate growth by a common mechanism or fall into sub-groups with differing actions, the effects of 46 steroids on DNA synthesis and the expression of protooncogenes c-fos and c-myc were examined in primary cultures of normal rat hepatocytes. Tentative groupings of steroids have been identified based on apparent structural requirements for stimulation of DNA synthesis, and effects of auxiliary factors in modulating this growth stimulus. For a "progestin" group, insulin appeared to be permissive for stimulation of DNA synthesis, and presence of an ester or hydroxyl group at 17alpha-position in combination with a non-polar group at C(6) appeared to be required for stimulation. For the pregnenes, dexamethasone was stimulatory. Structural requirements include a non-polar substitution at 16alpha-position and presence of a 6alpha-methyl group. Androgens were weak or ineffective stimulators of DNA synthesis. Anabolic steroids were weak to strong stimulators and alteration to A ring structure in combination with non-polar substitution at 17alpha-position appeared to be required for the activity. With the exception of the anabolic steroid, dianabol, there do not appear to be strong correlation between ability to stimulate DNA synthesis and ability to induce protooncogene expression among the steroids. This study provides a starting point for future more detailed examination of growth-stimulatory mechanism(s) of action of steroids in the liver.

Mechanism(s) of tumour promotion in liver by estrogens and other xenobiotics such as alpha-hexachlorocyclohexane (HCH), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and phenobarbital (PB), are not well understood although it is clear that growth stimulation is one important element in their action. To help in characterizing mechanisms of growth control by these compounds, their effects on the expression of immediate-early protooncogenes c- fos and c- myc have been examined and compared with other compounds that stimulate DNA synthesis in primary cultures of normal rat hepatocytes. Expression of c- fos was undetectable in cultures not exposed to growth factors. Although neither epidermal growth factor (EGF) nor 17beta-estradiol (E(2)) alone had marked effects on c- fos mRNA, the two acted synergistically to cause clear c- fos expression, maximal 1-2 h after growth factor addition and when test agents were added on the first day in culture. Neither insulin nor dexamethasone alone induced c- fos mRNA but stimulation of c- fos expression by EGF plus estradiol occurred earlier in the presence of insulin, and was augmented by preincubation of cells with dexamethasone. EGF + E(2)-induced c- fos mRNA was completely abolished by actinomycin D, suggesting that transcription is the major mechanism for c- fos induction by E(2) + EGF. Compounds that strongly stimulate hepatocyte DNA synthesis such as norepinephrine, pyruvate, prolactin, glutethimide, monensin, ammonium chloride, and normal rat serum when in combination with EGF, all failed (when added with EGF) to affect c- fos mRNA expression. Thus, induction of c- fos expression may be a component of estradiol's growth stimulatory effect in cultured hepatocytes but this is not the case for other compounds that strongly stimulate DNA synthesis. Unlike c- fos mRNA, c- myc mRNA was detectable in hepatocyte cultures without added growth factor, was augmented within 2 h of exposure to EGF, and was further increased by adding E(2), other estrogens or a variety of other stimulators of DNA synthesis in hepatocytes. This suggests that increased c- myc expression may be a common effect of many of these agents in combination with EGF.

The mechanism(s) of tumour promotion in liver by estrogens is not well understood although growth stimulation is known to be one important element of their action. As a basis for studying mechanisms of growth control by estrogens, effects of both natural and synthetic estrogens on DNA synthesis and protooncogene c-fos mRNA expression were examined in primary cultures of normal rat hepatocytes. 17beta-Estradiol (E(2)) alone was stimulatory and exhibited dramatic synergism with epidermal growth factor (EGF) in stimulating DNA synthesis. All estrogens tested (natural, synthetic, steroidal and non-steroidal) exhibited an ability to stimulate hepatocyte DNA synthesis. This appears to correlate with their ability to induce c-fos mRNA expression. In contrast to a non-estrogenic liver tumour promoter, phenobarbital, insulin is not permissive for the growth-stimulatory action of E(2). Dexamethasone, which is required for stimulation of DNA synthesis by the non-estrogenic tumour promoter alpha-hexachlorocyclohexane and tetradecanoylphorbol acetate, completely blocked E(2)-stimulated DNA synthesis. Such differential requirements for auxiliary factors suggests that estrogen and other non-estrogenic liver tumour promoters act via distinct mechanisms in stimulating hepatocyte DNA synthesis. E(2) alone had no effect, but when in combination with EGF significantly induced c-fos mRNA expression at early times in culture (maximal at 10 h in culture). Such findings, coupled with the observations that (i) E(2) and EGF were synergistic in growth stimulation, (ii) estrogen receptor levels are higher at early times in culture and (iii) the growth-stimulatory ability of E(2) is limited to 4-24 h in culture, support the notion that in hepatocytes E(2) acts via the estrogen receptor to transactivate c-fos expression (an interaction with EGF), which ultimately culminates in enhanced DNA synthesis. Dexamethasone did not block E(2)-induced c-fos gene expression, suggesting that it acts in a pathway(s) distal to activation of fos gene expression. The possible inhibitory mechanisms of action of dexamethasone on E(2)-stimulated DNA synthesis are discussed.

Investigators have a variety of reasons for studying the factors that determine the ability of hepatocytes to grow, and for pursuing some of these studies utilising the relatively defined and easily-manipulated conditions of cell culture. There are intriguing intellectual and technical challenges in dissecting out the factors that determine patterns of hepatocyte growth, death and differentiation — the cell lineage relationships, the relevant humoral and nutritional factors and the interactions with extracellular matrix and with non-hepatocyte cell types present in the liver. Some notable issues in biology and medicine underlie efforts to address these challenges. These include a need to understand the ability of generally healthy liver to rapidly replace lost tissue via liver regeneration; to understand normal patterns of hepatocyte growth and their control and the perturbations of these processes relevant to liver carcinogenesis; and to understand factors relevant to regeneration and repair when liver is damaged by toxins (including alcohol) or by virus infection. In addition, a better understanding of hepatocyte growth (and its relationship to cell differentiation or death) could assist efforts to replace defective liver function either with transplanted hepatocytes or with extracorporeal bioartificial livers. For example, ability to manipulate growth in vitro could increase options for generating normal or genetically-modified hepatocytes for transplantation or for developing bioartificial liver devices with sufficient cell mass, appropriate tissue architecture and extended function necessary for successful application in replacing the function of failing human livers.

Acknowledgements. Preface. Isolated hepatocytes: forty years on M.N. Berry. Isolation and culture of human hepatocytes M.J. Gomez-Lechon, J.V. Castell. Isolation and culture of porcine hepatocytes H.G. Koebe, et al. Preparation and properties of isolated hepatocytes from sheep M.G. Clark, M.A. Vincent. Elasmobranch hepatocytes N. Ballatori, et al. Isolation and cultivation of teleost hepatocytes T. Braunbeck, H. Segner. Conditions for growth of hepatocytes in culture A.M. Edwards, G. Michalopoulos. Engineering of a miniature 3-D hepatic cell plate culture model A. Bader, et al. Freezing and hypothermic storage of porcine hepatocytes H.G. Koebe, et al. Storage of isolated hepatocytes A. Guillouzo, et al. Hepatocyte swelling: techniques and effects on metabolism A.J. Meijer. Permeabilisation of hepatocytes with alpha-toxin P.E. Declercq, M.I. Baes. Perifused immobilised hepatocytes for metabolic studies H. Farghali. Small hepatocytes in primary cultures T. Mitaka, F. Sato. Mechanisms of hepatocyte growth regulation by hormones and growth factors T. Christoffersen, et al. Proliferative response and metabolic effects of growth factors in human hepatocytes M.J. Gomez-Lechon, et al. Growth control and cell cycle progression in cultured hepatocytes G. Ilyin, et al. Mechanisms of active cell death in isolated hepatocytes S. Maeda. The protective role of nitric oxide in hepatocytes during responses to inflammatory mediators and induction of apoptosis C.A. Bombeck, et al. Short-term regulation by insulin of glucose metabolism in isolated and cultured hepatocytes L. Agius, et al. Studies on mitochondrial-cytoplasmic interactions in isolated hepatocytes from fasted rats J.W. Phillips, etal. The importance of hepatocytes in drug metabolism studies: an industrial perspective M.K. Bayliss, D.M. Cross. Human hepatocytes as an experimental system for the evaluation of xenobiotics A.P. Li. Constitutive expression of cytochromes P450 in rat hepatocyte culture A.J. Paine. Optimising conditions for induction of cytochrome P450 in primary hepatocyte cultures G.M. Hawksworth, I. Stupans. The use of isolated hepatocytes to study the mechanisms of action of environmental contaminants S. Reader, F. Denizeau The use of isolated hepatocytes for the study of xenobiotic hepatotoxicity J. Wu, et al. Human hepatocyte transplantation: biology and therapy R.A. Fisher, S.C. Strom. Ex vivo hepatocyte-directed gene therapy S.E. Raper, A.D. Moscioni. A review of bioartificial liver development from an artificial organ engineering perspective M.D. Smith, et al. Development and testing of a bioartificial liver J. Rozga, A.A. Demetriou. The Berlin extracorporeal liver support system B. Busse, J. Gerlach. The bioartificial liver of the Academic Medical Center at Amsterdam R.A.F.M. Chamuleau, L.M. Flendrig. Performance of a flat membrane bioreactor utilizing porcine hepatocytes cultured in an extracellular matrix L. De Bartolo, A. Bader. Index.

This study examines the effects of extracellular Ca2+ concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+ levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]o in the range 25-1,800 microM, although the peak of DNA synthesis occurred 5-10 h earlier with 1.2 mM Ca2+ than with 25 microM Ca2+. Complete removal of extracellular Ca2+ using EGTA blocked DNA synthesis if Ca2+ was removed on the second day after EGF addition but not if Ca2+ was absent only on day 1. Treatment of cultures in 1.2 mM Ca(2+)-containing media with Ca(2+)-ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters alpha-hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor-promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]o but in combination with EGF, stimulated DNA synthesis at [Ca2+]o > 0.4 mM, suggesting that Ca2+ may have some role in mediating or modulating the stimulatory effects of these agents.

Mechanism(s) of tumor promotion in liver by xenobiotics such as hexachlorocyclohexane (HCH), 1,1,1-trichloro-2,2-bis (4-chlorophenyl)ethane (DDT) and phenobarbital (PB) are not understood in detail although growth-stimulatory effects may be significant in their action. As a basis for studying mechanisms of growth control by liver tumor promoters, effects of xenobiotics on DNA synthesis have been examined in primary cultures of normal rat hepatocytes, maintained under fully-defined conditions. The xenobiotics alone were relatively ineffective but they exhibited synergism with epidermal growth factor (EGF), insulin and dexamethasone in stimulating DNA synthesis and were effective in moderate-to-low density cultures but not in confluent monolayers. Under conditions optimized for HCH or pregnenolone 16 alpha-carbonitrile (PCN) (i.e. subconfluent cultures exposed to insulin, EGF and dexamethasone), HCH, PCN, DDT or PB caused a transient stimulation of DNA synthesis, apparent after 2 days in culture. This probably reflected earlier entry of hepatocytes to S-phase. HCH was shown to increase total DNA, total numbers of nuclei and numbers of cells undergoing mitosis per culture. In optimized conditions, HCH or PCN were about additive with norepinephrine, dialyzed serum or pyruvate or with a small effect of tri-iodothyronine in stimulating DNA synthesis. Although conditions optimal for HCH or PCN were not necessarily optimal for detecting growth-stimulatory effects of other xenobiotics or steroids, these culture conditions were shown to support stimulation of DNA synthesis by a variety of known liver tumor promoters including barbiturates, estrogens, progestins, peroxisomal proliferators and bile acids. Several compounds known not to promote liver carcinogenesis failed to stimulate DNA synthesis in similar hepatocyte cultures.

The control of total δ-aminolevulinic acid (ALA) synthetase activity in mammalian liver has been studied in isolated rat liver cell suspensions. Induction of ALA synthetase by the drug, allylisopropyl-acetamide (AIA) at a rate comparable to that of drug-stimulated induction in fasted rats could be achieved in the isolated cells; induction was largely dependent on the presence of adenosine 3' : 5'-monophosphate (cyclic AMP) or its dibutyryl derivative. At optimal concentrations either AIA (1.2 mm) or dibutyryl cyclic AMP (50 µm) alone caused little induction, but in combination caused a 4- to 5-fold increase of ALA synthetase activity in isolated cells over a 6-hour period. It is suggested that the cyclic AMP dependence of induction by drugs, also demonstrated for the drug 3,5-dicarbethoxydihydrocollidine, explains glucose repression of ALA synthetase observed in vivo. Glucose had no effect on induction by AIA ± dibutyryl cyclic AMP in isolated cells. Despite reports that glucocorticoids are required for induction in vivo, addition of hydrocortisone succinate to concentrations maximally effective in inducing hepatic tyrosine aminotransferase had little effect on the increase in total ALA synthetase activity stimulated by AIA ± dibutyryl cyclic AMP in isolated cells. Induction was prevented by addition of cycloheximide or actinomycin D to cell suspensions.

Hepatocytes isolated from normal adult rats were maintained at sub-confluent density in a defined medium in primary culture. In control cultures with added epidermal growth factor and low concentrations of dexamethasone, the rate of DNA synthesis was initially low but increased after about 30 hours in culture. Addition of the xenobiotic liver tumor promoters phenobarbital, α-hexachlorocyclohexane or p,p′-dichlorodiphenyltrichloroethane to cultures after cell attachment caused concentration-dependent stimulation of DNA synthesis measured after 2 days in culture. While dexamethasone (30 nM) alone had little effect on hepatocyte DNA synthesis, the stimulatory effects of the xenobiotics required the permissive presence of the steroid.

To help clarify whether elevation of gamma-glutamyltranspeptidase (GGT) in hepatocytes is associated with particular pattern(s) of liver differentiation, adult rat hepatocytes in primary culture were maintained for up to 5 days under conditions considered to alter differentiation in liver or other cells: basal GGT activity in untreated cultures and inducibility of the enzyme by known inducers such as dexamethasone, p,p'-dichlorodiphenyltrichloroethane or pregnenolone 16 alpha-carbonitrile were measured. Basal and induced GGT activities were maximal in confluent cultures and declined with decreasing cell density, an effect probably mediated by cell contact rather than factors in the culture medium. Opposite effects of cell density on GGT and DNA synthesis suggest that increased GGT activity is not linked with growth. Epidermal growth factor (EGF) increased basal GGT and augmented the action of xenobiotic inducers in lower density cultures, giving GGT activities approaching the levels in confluent cells. EGF had no effect on confluent cultures. Activators of protein kinase C such as tetradecanoyl phorbol acetate had significant but small inducing effects on GGT. Agents including all-trans retinoic acid, butyrate and dimethylsulfoxide which are considered to favour terminal differentiation in many cell types, all partly blocked induction of GGT by dexamethasone, EGF or xenobiotics. The results are consistent with (but do not prove) the view that elevated GGT activity may be associated with liver phenotype(s) distinct from terminal differentiation but not necessarily linked with growth.

Liver cells isolated from normal and cholestyramine-treated rats were incubated as cell suspensions for up to 4 hr in a simple, defined medium. The bile acid concentration in cells plus cell medium was determined by gas-liquid chromatography. Normal hepatocytes synthesized cholic acid at an initial rate of 0.25 nmol/mg cell protein per hr, which is comparable to rates reported from in vivo methods. This rate was increased more than 4-fold when rats were fed a cholestyramine-containing diet for 7 days prior to liver cell isolation. Although cholic acid was secreted into the cell medium during the incubation, it could not be assayed reliably by the hydroxysteroid dehydrogenase assay method, contrary to the reports of Anwer et al. 1975. Biochem. Biophys. Res. Commun. 64: 603 and Gardner and Chenouda 1978. J. Lipid Res. 19: 985.

The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to investigate the effect of dexamethasone on cytochrome P-450 activity in cultured rat hepatocytes. In control hepatocytes the metabolism of AAF to its 1-, 5-, 7-, 9- and N-hydroxylated metabolites rapidly declined in culture over the first 24 hr while 3-hydroxylation remained relatively constant. These activities either remained unchanged or increased slightly during the next 48 hr in culture. The addition of dexamethasone (100 nM) to the culture medium had little effect in arresting the initial decline but by 72 hr the 7-, 5- and 3-hydroxylations increased to values 2.5, 16 and 21 times the respective 24-hr values. The inductive effect of dexamethasone on the 3- and 5-hydroxylations of AAF was maximal at 100 nM whereas the 7-hydroxylation increased linearly as a function of the dexamethasone concentration up to 1 microM. Cortisol and corticosterone and the non-glucocorticoids fluoxymesterone and methyltestosterone induced a pattern of AAF metabolism resembling that in dexamethasone-treated cultures, suggesting that a range of steroids not restricted to glucocorticoids may induce multiple cytochrome P-450 isozymes via related mechanisms. Pregnenolone 16 alpha-carbonitrile induced only the 7-hydroxylation of AAF probably reflecting induction of cytochrome P-450p. While dexamethasone was a strong inducer of the 3- and 5-hydroxylations of AAF in hepatocyte culture, assay of these activities in freshly isolated cells after in vivo treatment with dexamethasone showed a strong induction of 7-hydroxylation but only small effects on 3- and 5-hydroxylations. Indeed the profile of AAF metabolism induced in culture by dexamethasone resembles more closely the profile induced by 3-methylcholanthrene in vivo. These data suggest that factors yet to be identified strongly influence the steroid-induced pattern of cytochrome P-450 gene expression.

Rat hepatocytes maintained for up to 6 days in primary culture were used to test a variety of xenobiotics and steroids for effects on the activity of gamma-glutamyltranspeptidase (GGT) in normal cells. In control cultures GGT activity was low and increased slowly with time. When added to cultures for 5 days, a variety of xenobiotics and steroids increased GGT activity to levels 2- to 6-times those of control cultures. Induction of GGT was potentiated for most test compounds by 20-30 nM dexamethasone and diminished by nicotinamide or adenosine-3',5'-monophosphate. Effective non-genotoxic inducers included phenobarbital and some structurally related compounds, p,p'-dichlorodiphenyltrichloroethane,alpha- and gamma-hexachlorocyclohexanes, Aroclor 1254, butyl hydroxytoluene, nafenopin, various estrogens, progesterone, pregnenolone-16 alpha-carbonitrile and cyproterone acetate. A number of compounds including barbituric acid, butyl hydroxyanisole, acetaminophen, saccharin, caffeine, clofibrate and some bile acids failed to induce GGT. Except for 2-acetylaminofluorene and diethylnitrosamine, genotoxic compounds tested did not increase GGT. The results establish that a structurally diverse group of xenobiotics and steroids, many of which are considered to be liver tumour promoters, may directly enhance GGT gene expression in normal hepatocytes. Thus, a variety of compounds used in experimental studies of liver cancer induction as promoters may elevate GGT by mechanism(s) not necessarily related to carcinogenesis.

Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 microM hemin and 100 microM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 microM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40-60 microM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 microM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 microM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.

Adult rat hepatocytes maintained in primary monolayer culture with defined medium were used to characterise two effects of glucocorticoid-like steroids in regulating gamma-glutamyltranspeptidase (GGT). Low concentrations of glucocorticoids alone had little effect on GGT but synergistically enhanced induction of the enzyme by liver tumor-promoting xenobiotics such as 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)-ethane (DDT) and hexachlorocyclohexane. The enhancing effect appears to be mediated by the classical glucocorticoid hormone receptor since structural requirements and concentration-dependence for enhancement were similar to those for induction of tyrosine aminotransferase in parallel cultures. Higher concentrations (1-100 microM) of various glucocorticoids alone increased GGT activity. Most glucocorticoids induced GGT but their order of potency did not parallel that for induction of tyrosine aminotransferase under similar culture conditions. Among the most potent glucocorticoids, triamcinolone was a weak GGT inducer and cortivazol appeared to act as an antagonist of GGT induction by steroids. Some non-glucocorticoids including pregnenolone 16 alpha-carbonitrile, and some progestins, also induced but required addition of 30 nM dexamethasone for maximal effect. Some specific steroid structural features were identified which increased (presence of a 16 alpha methyl group) or impaired GGT-inducing activity. Although interpretation is complicated by differential metabolism of individual steroids in culture, the results suggest that GGT induction by pharmacological levels of steroids may be mediated, directly or indirectly, by one or more relatively specific receptors distinct from the classical glucocorticoid receptor.

Adult rat liver parenchymal cells, maintained in primary monolayer culture on floating collagen gels, were used for studies on the hormonal regulation of phosphoenolyruvate carboxykinse. In cells maintained in a defined, serum-free medium containing insulin but no other hormones, phosphoenolpyruvate carboxykinse activity expressed on the basis of cell DNA content decreased over 3 days in culture with a half-life of 9–10 h. Treatment of cells with glucagon or N6, O2′-dibutyryladenosine 3′:5′ monophosphate (Bt2-cAMP) caused an increase in enxyme activity up to 4-fold higher than in untreated cells. There was little or no response during the first 24 h of culture but addition of either glucagon or Bt2-cAMP after this period resulted in significant enzyme induction within 3 h. When cells were maintained for 2–3 days after attachment directly to palstic culture dishes rather than to collagen gels, similar inductive responses to glucagon or Bt2-cAMP were observed. Induction by glucagon was shown to be dose-dependent; a significant response was evident at 0.1 nM gucagon. Addition of epinephrine to cultured cells at concentration between 10 nM to 0.1 mM was without effect on phosphoenolpyruvate carboxykinase. Induction by Bt2-cAMP was completely prevented by the simultaneous addition of actinomycin D (0.2 μg/ml) or cordycepin (10 μg/ml) suggesting that the nucletide acts or transcription to increase phosphoenolpyruvate carboxykinase synthesis.

The proposition that changes in activity of gamma-glutamyltranspeptidase (GGT) in serum may provide a useful index of the extent of induction of liver drug-metabolizing enzymes by various drugs was examined by comparing control of GGT and monooxygenase activities in cultured hepatocytes. In rat hepatocyte monolayers maintained for up to 5 days the effects of xenobiotics and other factors on cellular GGT activity were compared with effects on a relatively broad measure of drug metabolism, the 7-ethoxycoumarin O-deethylase (ECD) activity of intact cells. A diverse group of drugs including phenobarbital and other barbiturates, diphenylhydantoin, glutethimide, aminopyrine and griseofulvin and the steroids dexamethasone and pregnenolone 16 alpha-carbonitrile were shown to induce both GGT and ECD under comparable culture conditions. Inductions of both activities were potentiated by glucocorticoids and depressed (where tested) by dibutyryl cyclic AMP. Some other hormones or nutrients modulated the activities differently. The magnitude of GGT induction by different drugs did not correlate with relative ECD induction and for several drugs the concentration-dependence of the two effects was different. Interpretation is complicated by the possible contribution of multiple forms of cytochrome P-450 to ECD activity but it seems unlikely that drugs which induce both GGT and drug metabolism do so via a common regulatory mechanism. For such drugs changes in serum GGT could provide only a crude guide to likely changes in drug metabolism. Some compounds including polycyclic hydrocarbons and warfarin induced ECD but had no associated effect on GGT in hepatocytes.

Assays of 7-ethoxycoumarin O-deethylase (ECD) activity in intact cells were used as a sensitive and convenient measure of the drug-metabolizing activity of rat hepatocytes maintained for up to 4 days in primary culture. A combination of nicotinamide or other pyridines with dexamethasone was shown to maintain ECD at or above the activity of untreated livers in vivo and to potentiate induction by xenobiotics. Inductions in vivo and in culture were quantitatively similar but differed qualitatively as judged by the proportion of ECD activity inhibitable by metyrapone. A survey of possible endogenous regulators of liver monooxygenases established that: dexamethasone and other glucocorticoids induced ECD and potentiated induction by xenobiotics, particularly phenobarbitone; other steroids including testosterone, 17 beta-estradiol and pregnenolone 16 alpha-carbonitrile caused small inductions; insulin lowered both ECD activity and the proportion of activity inhibitable by metyrapone; dibutyryl cyclic AMP or glucagon lowered ECD; and high concentrations of aminolevulinate partly repressed induction by xenobiotics. Based on these findings, hepatocyte culture conditions which maintain ECD activity and inducibility at or above in vivo levels are defined.