Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes

Maintenance of microsomal cytochrome P-450 content by cultured rat hepatocytes has proven an elusive goal. It is reported here that exogenous heme maintains cytochrome P-450 content of cultured rat hepatocytes at high levels during the first 72 h of incubation. The maintenance studies have been expanded to demonstrate the in vitro induction of cytochrome P-450 by phenobarbital treatment. The induction of P-450 in vitro by phenobarbital required the trace element, selenium, in the presence of exogenous heme. The present findings suggest that selenium, and other trace elements, may have an essential role in the formation of holocytochrome P-450 in vitro.     

Induction of cytochrome P-450c in hematopoietic cells of fetal liver

The transplacental inductive effect of beta-naphthoflavone (beta NF) on cytochrome P-450 isozymes was studied in separate hematopoietic and hepatocyte cells from fetal rat liver. Two fractions of dispersed fetal liver cells were isolated by Ficoll-Paque centrifugation and shown by histologic examination to be enriched in erythroblasts and hepatocytes, respectively. beta NF treatment increased ethoxyresorufin-O-deethylase activity 250-fold in both erythroblast and hepatocyte cell fractions. Polyacrylamide gel electrophoresis and immunostaining techniques showed the induction of cytochrome P-450c, but not P-450d, in erythroblast and hepatocyte fractions.     

Comparison of cytochrome P-450 levels in adult rat liver, postnatal rat liver, and primary cultures of postnatal rat hepatocytes

A system of primary cultures of postnatal rat hepatocytes has been developed to serve as an experimental model for drug metabolism and toxicity investigations. The purpose of this study was to examine the reported loss of cytochrome P-450 of hepatocytes when placed in culture and to compare activity in culture to intact liver and freshly isolated hepatocytes. A medium enriched with several hormones and a system of floating filters as a substratum for cell attachment were investigated as methods to reduce the expected loss of cytochrome P-450. When compared to initial values of cytochrome P-450 in whole liver and isolated hepatocytes, these methods failed to prevent the reduction of cytochrome P-450 in culture. However, our results compare favorably with other values reported in the literature.     

Induction of Cytochrome P-450s and Expression of Liver-specific Genes in Rat Primary Hepatocytes Cultured on Different Extracellular Matrices

Freshly isolated hepatocytes were cultured on an EHS-gel prepared from EHS-tumor, poly-N-p-vinylbenzyl-d-lactonamide (PVLA), and type I collagen (TIC). Hepatocytes on EHS-gel showed a spherical shape and much more strongly maintained the inducible expression of cytochrome P-450 genes which were lost on PVLA and TIC. Further, the expression of liver-specific genes were maintained on EHS gel at the highest level, and then higher on PVLA than TIC.     

Effects of ethanol and clofibrate on expression of cytochrome P-450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes

Cultured and cocultured rat hepatocytes were used to study the effects of ethanol and clofibrate on cytochrome P-450 (P-450) enzymes and epoxide hydrolase. We showed that in the presence of ethanol, clofibrate or both compounds, rat hepatocytes were able to express, after 3 days of pure culture, quantitatively and qualitatively reasonable levels of most cytochrome P-450 enzymes and epoxide hydrolase, compared to freshly isolated hepatocytes. However, ethanol induced the P-450IA subfamily, and clofibrate the P-450-IVA subfamily. In cocultures, after 6 days, most P-450 enzymes were still expressed while P-450IIC11 was completely lost. Ethanol and clofibrate had the same effect as in pure culture. These results show, by modifying culture medium conditions and cell-cell interactions, that it is possible to maintain reasonable xenobiotic-metabolizing-enzyme expression; however, these conditions have to be improved in order to preserve better P-450 expression. The mechanism of these effects and the inducibility of these systems remain to be elucidated by a study at molecular level.     

Primary cultures of hepatocytes on human fibroblasts.

Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.     

Primary cultures of hepatocytes on human fibroblasts

Summary Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.     

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.     

Culture of primary rat hepatocytes within a flat hollow fibre cassette for potential use as a component of a bioartificial liver support system

Primary rat hepatocytes were cultured in a flat, hollow-fibre cassette, `The Tecnomouse', which provided direct oxygenation and a homogeneous environment for cells within the cassette. Most hollow fibre systems utilise media oxygenators to provide O₂ to cells; in the Tecnomouse cassette, cells are provided with direct oxygenation via gas channels in the silicone membrane surrounding the hollow fibres. Hepatocyte functionality was monitored by following urea production, albumin production and cytochrome P-450 enzyme activities. The system could maintain cells in a viable state and the presence of specific hepatocyte functions including albumin production and cytochrome P-450 activity. Electron microscopy showed aggregated spherical hepatocytes and apparent high extent of necrosis.     

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.     

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.     

Inhibition of protein synthesis: a basis for tunicamycin-induced decrease in rat liver cytochrome P-450

Tunicamycin caused a dose and time dependent decrease in cytochrome P-450 in rat liver. A dose of 50 micrograms/kg caused a decrease of about 50% in 72 hours. A similar decrease in the activities of rat liver microsomal aniline hydroxylase, aminopyrine N-demethylase and ethoxycoumarin O-deethylase were also seen after the tunicamycin treatment. Tunicamycin also suppressed food and water intake but the decrease in cytochrome P-450 was not related to these effects. NADPH cytochrome c reductase was not markedly decreased by tunicamycin. A decrease in cytochrome P-450 was also observed in cultured rat hepatocytes treated with tunicamycin. It decreased incorporation of [35S]-methionine into total proteins as well as into various cytochrome P-450 isozymes of rat hepatocytes. This indicates that a decrease in protein synthesis may be responsible for the tunicamycin-induced decrease in cytochrome P-450 and drug metabolism.     

Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes

Specific polyclonal antibodies were used to investigate the distribution of two cytochrome P-450 isozymes (5 and 8), NADPH cytochrome c reductase, and epoxide hydrolase in adult human hepatocytes cultured alone or co-cultured with rat liver epithelial cells. The enzymes were localized by the indirect immunoperoxidase technique following fixation with a paraformaldehyde-glutaraldehyde mixture and membrane permeabilization with saponin. The pattern of distribution of the four enzymes after 24 hr in culture was similar to that found in vivo. Virtually all the hepatocytes exhibited nearly homogeneous positive staining for cytochrome P-450-8, whereas only 60-80% were positive for cytochrome P-450-5. Nearly homogeneous staining was also observed in all hepatocytes for NADPH cytochrome c reductase and epoxide hydrolase. During the first 12 days in pure culture, the intensity of staining, as well as the number of positively stained cells, decreased slightly except for epoxide hydrolase, which did not show any obvious change. In contrast, even after 15 days in co-culture the extent of staining for all the enzymes decreased less than in pure culture. These results indicate that adult human hepatocytes continue to express specific drug-metabolizing enzymes for several days in culture and provide further evidence that those cells are more stable than rodent hepatocytes in primary culture.     

Maintenance of microsomal cytochromes b5 and P-450 in primary cultures of parenchymal liver cells on collagen membranes

In previous reports from various laboratories, the levels of the microsomal cytochromes b₅ and P-450 in hepatocytes in primary culture have been found to be very low and difficult to measure. The studies reported in this paper demonstrate that cytochromes b₅ and P-450 in hepatocytes cultured on floating collagen membranes for periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of liver $\text{in}$$\text{vivo}$. Addition of high levels of hydrocortisone (10^?4M) to the culture medium for periods up to 10 days resulted in further increases in the levels of these cytochromes. Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained.     

Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome P-450 in rat liver cell culture. Potent maintenance by metyrapone.

The loss of cytochrome P-450 in cultured rat hepatocytes can be prevented by substituted pyridines, especially isonicotinamide, 3-hydroxypyridine and metyrapone. The effect of these compounds is independent of protein synthesis, suggesting that they maintain pre-existing cytochrome P-450. The efficiency of pyridines at maintaining cytochrome P-450 in hepatocyte culture is highly correlated with their ability to bind to this cytochrome, suggesting that ligand formation with cytochrome P-450 prevents its accelerated turnover in liver cell culture.     

Induction of cytochrome P-448 isozyme(s) in primary cultured rat hepatocytes by drugs which induce different isozymes in vivo

Effect of 2-methoxy-4-aminoazobenzene (2-MeO-AAB) and 3-methylcholanthrene (MC) on the induction of microsomal cytochrome P-448 isozymes in primary cultured rat hepatocytes was examined by means of immunochemical methods such as protein A-enzyme-linked immonosorbent assay and immuno-blots using anti-rat cytochrome P-448 monoclonal antibodies and by means of bacterial mutation tests. Although 2-MeO-AAB selectively induced cytochrome P-448H and MC induced both cytochrome P-448H and a low spin form of cytochrome P-448 (P-448L) in the liver of rats, addition of these chemicals to primary cultured rat hepatocytes resulted in selective induction of cytochrome P-448L, as determined by the immunological methods. This was substantiated by the bacterial mutation test using Salmonella typhimurium TA 98 bacteria and two aromatic amine substrates with different specificities to the cytochrome P-448 isozymes. These results suggest that the responses of rat hepatocytes to cytochrome P-450 inducers are different in in vivo and in vitro.     

Glucocorticoid-dependent induction of HMG-CoA reductase and malic enzyme gene expression by polychlorinated biphenyls in rat hepatocytes

Administration of xenobiotics to rats results in hypercholesterolemia and in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and malic enzyme. To investigate the mechanism of the induction of the enzymes by xenobiotics, the effects of xenobiotics on gene expressions for HMG-CoA reductase, malic enzyme, and cytochrome P-450 in rat liver and in cultured hepatocyte were investigated. The treatment of rats with polychlorinated biphenyls (PCB) as a xenobiotic induced mRNAs for HMG-CoA reductase and malic enzyme as well as CYP2B1/2 (cytochrome P-450b/e). Other xenobiotics, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and chloretone, also increased HMG-CoA reductase mRNA. In an investigation of diurnal rhythm of mRNA for HMG-CoA reductase, the induction by PCB was observed in a dark period. Induced expressions of HMG-CoA reductase gene and malic enzyme gene by PCB were observed in primary cultured rat hepatocytes and showed that the action of PCB on the gene expression relating to lipid metabolism was directed on hepatocytes. The induction was observed only in hepatocytes cultured on Engelbreth-Holm-Swarm sarcoma basement membrane gel (EHS-gel), not on type I collagen, which is usually used for monolayer culture of hepatocytes. The induction of CYP2B1/2 gene expression also was observed only in the cells cultured on EHS-gel. The induction of HMG-CoA reductase and malic enzyme by PCB required dexamethasone. However, the addition of dexamethasone per se to medium containing insulin did not show an inductive effect on levels of mRNA for HMG-CoA reductase and malic enzyme. From the data of diurnal variation and hepatocyte culture experiment, HMG-CoA reductase and malic enzyme are considered to be induced by PCB through the so-called "permissive effect" of glucocorticoid.     

Ligands maintain cytochrome P-450 in liver cell culture by affecting its synthesis and degradation

Rat hepatocytes cultured for 24 h lose 68% of their cytochrome P-450. It is shown that this loss is due to the failure of cultured hepatocytes to synthesize cytochrome P-450 as well as enhanced degradation. Compounds that form ligands with cytochrome P-450, eg metyrapone, prevent the loss of cytochrome P-450. Ligands are generally considered to protect proteins from degradation but the present work suggests that the effect of metyrapone on cytochrome P-450 synthesis is of equal importance to its effect on degradation in preventing the loss of cytochrome P-450 in hepatocyte culture.