Anthony M. Edwards

7-ethoxycoumarin deethylase activity as a convenient measure of liver drug metabolizing enzymes: Regulation in cultured rat 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.

Control of hepatocyte DNA synthesis by intracellular pH and its role in the action of 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 (pHi) was independent of extracellular pH (pHe), EGF‐ and insulin‐stimulated rates of DNA synthesis were unaffected by modest changes in pHe. However, under conditions (HEPES‐buffered medium) where pHi varied in a linear fashion with pHe, rates of EGF‐ and insulin‐stimulated DNA synthesis were highly dependent on pHe. Similarly, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and α‐hexachlorocyclohexane (HCH)‐stimulated DNA synthesis were pHe‐dependent but were stimulatory over different pHe 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 μM), were found to stimulate hepatocyte DNA synthesis. The role of the Na+/H+ antiport in controlling pHi 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 pHi recovery from the acid load in a dose dependent manner and the relative potency of these inhibitors paralleled their Ki 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 pHi 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.

Stimulation of DNA synthesis by tumor promoters in primary rat hepatocytes is not mediated by arachidonic acid metabolites

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. PGE2, PGF2α, and PGD2 cause concentration‐dependent stimulation of hepatocyte DNA synthesis, while arachidonic acid was without any effect. PGE2 and PGF2α required the presence of dexamethasone to exert significant effects. These PGs did not further augment the stimulatory effect of EGF. In contrast, PGD2 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 [3H]arachidonic acid release and PGE2 production, was determined. The phorbol ester TPA significantly increased [3H]arachidonic acid release as well as PGE2 formation in hepatocytes in line with known effects in other cell types. However, liver tumor promoters phenobarbital (PB), α‐hexachlorocycohexane (HCH), 1,1‐bis(p‐chlorophenyl)‐2,2,2‐trichloroethane (DDT), and pregnenolone‐16α‐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.

Conditions for growth of hepatocytes in culture

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.

Effect of dexamethasone on cytochrome P-450 mediated metabolism of 2-acetylaminofluorene in cultured rat hepatocytes.

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.

Two distinct mechanisms for regulation of γ-glutamyl transpeptidase in cultured rat hepatocytes by glucocorticoid-like steroids

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.

Relationship between inductions of monooxygenase activity and γ-glutamyltranspeptidase in rat hepatocyte primary cultures

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.

Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes.

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.