The liver of woodchucks chronically infected with the woodchuck hepatitis virus contains foci of virus core antigen-negative hepatocytes with both altered and normal morphology

The livers of woodchucks chronically infected with woodchuck hepatitis virus (WHV) contain foci of morphologically altered hepatocytes (FAH) with "basophilic", "amphophilic" and "clear cell" phenotypes, which are possibly pre-neoplastic in nature. Interestingly, most fail to express detectable levels of WHV proteins and nucleic acids. We studied sections of WHV-infected liver tissue to determine if all foci of hepatocytes that failed to express detectable levels of WHV, as assessed by immunoperoxidase staining for WHV core antigen, could be classified morphologically as FAH. We found that at least half of the foci of WHV core antigen-negative hepatocytes did not show clear morphological differences in either H&E or PAS (periodic acid Schiff) stained sections from surrounding hepatocytes, and were therefore not designated as FAH. In the second approach, we assayed core antigen-negative foci for the presence of fetuin B, a serum protein produced by normal hepatocytes, but not by neoplastic hepatocytes in hepatocellular carcinomas. Basophilic and amphophilic FAH had reduced levels of fetuin B compared to hepatocytes present in the surrounding liver; fetuin B staining was detected in clear cell FAH but the level could not be accurately assessed because of the displacement of fetuin B to the cell periphery by accumulated glycogen. The foci of morphologically normal WHV core antigen-negative hepatocytes had similar levels of fetuin B to that of the surrounding hepatocytes. The co-existence of at least four types of WHV core antigen-negative foci, including those with no obvious morphologic changes, raises the possibility that the different foci arise from distinct primary events. We hypothesize that a common event is loss of the ability to express WHV, allowing these hepatocytes to escape immune mediated cell death and to undergo clonal expansion to form distinct foci.     

Induction and modulation of hepatic preneoplasia and neoplasia in the rat by dehydroepiandrosterone

Dehydroepiandrosterone (DHEA), the main adrenal steroid in humans and a precursor in androgen and estrogen biosynthesis, acts as a peroxisome proliferator and as a hepatocarcinogen in rats. Neoplasms emerge from a glycogenotic/amphophilic/basophilic preneoplastic cell lineage. A higher female tumor incidence suggests a relevant influence of sex hormones. DHEA enhances hepatocarcinogenesis induced by N-nitrosomorpholine (NNM), which is characterized by the glycogenotic/basophilic cell lineage. The tumor promoting effect is related to an additional amphophilic/basophilic preneoplastic lesion sequence and to faster proliferation of the basophilic preneoplastic lesions. Nevertheless, hepatocellular carcinomas provided under DHEA treatment seem to have a less malignant phenotype compared to tumors induced by NNM only. Further, DHEA treatment reduces growth and generation of glycogen storage foci (GSF) in initial NNM-treated rats. Thus, DHEA treatment results in both, a growth stimulation of the late basophilic lesion type with an additional amphophilic lesion sequence, and in a growth inhibition of early preneoplastic lesions, addressing especially GSF. DHEA also inhibits the growth of physiologically proliferating liver tissue. This might be explained by a DHEA related cellular metabolism, which results in significant energy consumption. Additionally, a DHEA-induced alteration of cytokine levels might contribute to this growth inhibition as well.     

Gamma-glutamyl Transferase as Oncofetal Marker of Experimental Hepatocarcinogenesis in the Rat

γ-Glutamyl transferase (γ-GTase) is abundant in the fetal and neonatal rat liver, but almost completely disappears in the normal adult rat liver. Aflatoxin B₁ (AFB₁) was used in several hepatocarcinogenic models in the rat. The hepatocarcinogenesis was divided in early, preneoplastic and neoplastic phases during which liver histological changes were studied in correlation with histochemical analysis of γ-GTase.
Five types of hepatocyte populations were encountered: original hepatocytes, small newly formed hepatocytes and transitional cells, early liver cell regenerative areas, late liver cell foci and nodules and evidently malignant liver cells. A very marked γ-GTase activity always resurged reversibly in every area of proliferating newly formed hepatocytes and transitional cells and in every early regenerative area, but it reappeared irreversibly in every late appearing focus and nodule and in every hepatocellular carcinoma. The whole remaining parenchyma (original hepatocytes) showed no γ-GTase activity, as in the normal adult rat liver. The resurgence of γ-GTase in these subsequent cell populations, including the malignant hepatocytes, emphasizes that the development of the latter implicates the reacquiring of some embryonal biochemical characteristics. Furthermore it illustrates the similar immature state of specific and focal preneoplastic and neoplastic liver cell lesions. γ-GTase histochemically proved to be the most sensitive marker of the hepatocarcinogenic process.     

'Undifferentiated progenitor cells' in focal nodular hyperplasia of the liver

Focal nodular hyperplasia is a tumour like lesion, characterized by a central fibrous scar with irradiating fibrous septa that surround hyperplastic nodules and contain multiple bile ductules. The origin of the bile ductular structures is not clear. Recently, we found evidence for the existence of human counterparts of rat oval cells (potential stem cells) that have the ability of differentiating towards both bile duct cells and hepatocytes. These cells were found in regenerating human liver as well as in chronic cholestatic conditions. Because cholestatic features are seen in focal nodular hyperplasia, we initiated an immunohistochemical study on 23 surgical specimens using antibodies specific for cytokeratins 7 and 19 (bile duct type cytokeratins), OV6 (rat oval cell marker), chromogranin-A (shown to be positive in reactive bile ductules and human oval-like cells) and neural cell adhesion molecule—NCAM (shown to be positive in reactive bile ductules) to investigate whether ‘undifferentiated progenitor cells’ are also present in focal nodular hyperplasia. Electronmicroscopy was applied in five cases. Bile ductules invariably showed immunoreactivity for CK7 and 19, OV6, chromogranin-A and NCAM. In addition, small individual cells with an oval nucleus and a small rim of cytoplasm, in the vicinity of the septa, were immunoreactive for chromogranin-A, CK7 and 19 and OV6. These cells were hardly recognizable on routine light microscopy. Clusters of periseptal hepatocytes, seemingly in continuity with bile ductular structures, had a transitional phenotype: they stained positive for chromogranin-A, CK7 and OV6 and sometimes formed liver cell rosettes. The number of OV6-positive hepatocytes was greater than the number of chromogranin-A and CK7 positive hepatocytes. This indicates that, in human liver, OV-6 is not purely a marker of progenitor cells. Ultrastructurally, small immature cells, highly resembling rat oval cells, were recognized in the vicinity of septa. In addition, transitional cells displaying characteristics both of hepatocytes and bile duct cells were also present. These results confirm the presence of ‘undifferentiated progenitor cells’ in focal nodular hyperplasia and suggest that the ductular reaction in these lesions results, at least partly, from activation of these cells.     

Transdifferentiation into biliary ductular cells of hepatocytes transplanted into the spleen

AIMS: Transplantation of rat hepatocytes into the syngeneic rat spleen results in the appearance of cytokeratin (CK)7 and CK19 positive biliary cells that form ductules. We examined whether hepatocytes are the origin of these biliary ductular cells. METHODS: We transplanted rat dipeptidyl peptidase IV (DPPIV) positive hepatocytes into the liver of retrorsine-treated and partially hepatectomised DPPIV negative rats, which resulted in proliferation of DPPIV positive hepatocytes in the liver. Two months later, hepatocytes were prepared from chimaeric livers of these rats and transplanted into the spleen of DPPIV negative rats. Four weeks later, the expression of DPPIV in CK7 positive ductules in the spleen was examined by immunofluorescent double-staining. RESULTS: In the spleen of DPPIV negative rats transplanted with hepatocytes prepared from the chimaeric livers, DPPIV was found to be expressed in some CK7 positive biliary ductules where only a fraction of cells expressed DPPIV, whereas in the spleen of DPPIV negative rats transplanted with hepatocytes from livers of DPPIV positive rats, DPPIV was expressed in all CK7 positive biliary ductules. CONCLUSION: The present study indicates that hepatocytes transplanted into the spleen could transdifferentiate into biliary cells that aggregate to form ductular structures.     

Spermatocytic seminoma in the rat

During the necropsy of a 2-yr-old untreated control rat from a carcinogenicity study, a tan brown nodular mass was observed within the tunica albuginea of one testis. Both testes were processed for light and transmission electron microscopy. Histologically, the testicular mass was composed of round to polyhedral cells with distinct cell boundaries, amphophilic cytoplasm, fine nuclear chromatin, and a delicate fibrovascular supporting stroma. Ultrastructurally, the tumor cells displayed features associated with spermatocytic differentiation. Based on the light microscopic and the ultrastructural features, the tumor was considered a spermatocytic seminoma of the testis, a rare tumor in the rat.     

Preparation of monoclonal antibody to hepatocellular membranes and its application to induction of liver cell membrane damage

Monoclonal antibody (MoAb) to rat liver plasma membranes was prepared by hybridization of mouse immune lymphocytes with mouse myeloma cells, and was identified by the immunodiffusion method in a fraction of IgM secreted from the hybridoma thus obtained. In indirect immunofluorescence tests, specific fluorescence was detected only on the surface of rat hepatocytes, but neither on the cells from other organs of the rat nor on the hepatocytes of other species of animals, suggesting that the antibody may be organ- and species-specific. When the primary culture rat hepatocytes, labelled with isotopic chromium (51Cr), were treated with the MoAb together with complement, a specific release of 51Cr from the cells was found shortly after treatment, accompanied with bubbling of the cell membranes, and a significant release of 51Cr was observed at an MoAb concentration of 15 micrograms/ml or more. Without complement, or with inactivated complement, these reactions were not observed. These facts suggest strongly that the cell surface of the hepatocytes was damaged by the MoAb in the presence of complement.     

Acellular liver matrix improves the survival and functions of isolated rat hepatocytes cultured in vitro

Alternative approaches to overcome the shortage of donors for liver transplantation may be the use of hepatocytes for bioartificial devices or transplantation. Therefore, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of hepatocytes represents a formidable challenge. Aim of this study was to obtain a liver homologous acellular matrix (HAM) able to support viability and metabolic functions of rat hepatocytes in primary culture. HAMs were prepared by sequential incubation of rat liver slices in deoxycholic acid and DNase solutions. Dispersed rat hepatocytes were obtained by collagenase digestion and mechanical disaggregation. Isolated hepatocytes were seeded on uncoated and collagen- or HAM-coated tissue culture plastic wells. Cultures were examined by scanning electron microscopy (SEM), and the viability of hepatocytes and their ability to produce albumin and urea were assessed. The viability of freshly dispersed hepatocytes was about 98%. Hepatocytes seeded on HAM exhibited a significantly higher viability and a markedly lower apoptotic rate than those grown on plastic or collagen. Accordingly, albumin and urea nitrogen productions were significantly higher in HAM-cultured hepatocytes. SEM showed that hepatocytes seeded on HAM displayed a clustered organization, and were well anchored to the matrix and morphologically stable. Taken together, these findings indicate that HAM strongly improves viability and functional activity of rat hepatocytes cultured in vitro.     

Spleno-enteritis caused by adenovirus in psittacine birds: A pathological study

Psittacine birds with haemorrhagic enteritis were submitted for pathological study. Histopathological examination revealed haemorrhagic enteritis and necrotic areas in the spleen, with amphophilic and basophilic intranuclear inclusion bodies in mononuclear cells of the spleen, intestinal cell infiltrate, endothelial cells of small vessels in spleen, intestine, lung and liver, and in a few hepatocytes. Thrombi were observed in intestinal capillaries. The clinical and pathological features of this outbreak resembled haemorrhagic enteritis of turkeys and immunohistochemical staining showed positive immunoreaction to group II avian adenovirus antigens in the intranuclear inclusion bodies. The possible relationship between the virus replication in endothelial cells and intestinal haemorrhages in avian adenovirus infection is discussed. This outbreak could represent a new pathological process in psittacine birds.     

Genotoxicity testing of potassium canrenoate in cultured rat and human cells

Potassium canrenoate (PC), a competitive aldosterone antagonistused as a diuretic and in the treatment of hypertension, wasexamined for its capacity to produce genotoxic effects in culturedrat and human cells. At subtoxic concentrations (10–90µM) PC was found to induce a dose-dependent degree ofDNA fragmentation, as detected by the Comet assay, and of DNArepair synthesis, as measured by quantitative autoradiography,in primary cultures of hepatocytes from rat and human donorsof both genders. In rat hepatocytes both DNA fragmentation andDNA repair were more marked after 3 h than after 20 h exposureand in cultures from females than from males. In human hepatocytesfrom one male and two female donors, PC caused a similar effectin terms of DNA fragmentation, whereas DNA repair was detectedin cultures from only two of the same three donors and was lessmarked than in rat hepatocytes. A modest but statistically significantincrease in micronucleated cells was present in primary culturesof replicating rat hepatocytes exposed to 10 or 30 µMPC for 48 h, the response being, in this case also, more evidentin females than in males. In contrast, PC did not induce micronucleusformation in human hepatocytes from two female donors. Any evidenceof DNA fragmentation and micronucleus formation was absent incultured human lymphocytes. Taken as a whole these findingssupport the hypothesis that hepatocytes activate PC to DNA-damagingreactive species. PC induced the observed genotoxic effectsat concentrations close to those produced in humans by the administrationof therapeutic doses, but these effects were as a whole moremarked in rat than in human hepatocytes. Since PC shares the17-hydroxy-3-oxopregna-4,6-diene structure with cyproteroneacetate, chlormadinone acetate and megestrol acetate, previouslyfound to be genotoxic to both rat and human hepatocytes, thepotential carcinogenic hazard of this type of steroids cannotbe neglected. ¹ To whom correspondence should be addressed. Tel: +39 010 3538800; Fax: +39 010 538232; Email: farmdimi{at}unige.it     

PREPARATION OF MONOCLONAL ANTIBODY TO HEPATOCELLULAR MEMBRANES AND ITS APPLICATION TO INDUCTION OF LIVER CELL MEMBRANE DAMAGE

Monoclonal antibody (MoAb) to rat liver plasma membranes was prepared by hybridization of mouse immune lymphocytes with mouse myeloma cells, and was identified by the immunodiffusion method in a fraction of IgM secreted from the hybridoma thus obtained. In indirect immunofluorescence tests, specific fluorescence was detected only on the surface of rat hepatocytes, but neither on the cells from other organs of the rat nor on the hepatocytes of other species of animals, suggesting that the antibody may be organ- and species-specific. When the primary culture rat hepatocytes, labelled with isotopic cromium (⁵¹Cr), were treated with the MoAb together with complement, a specific release of ⁵¹Cr from the cells was found shortly after treatment, accompanied with bubbling of the cell membranes, and a significant release of ⁵¹Cr was observed at an MoAb concentration of 15μg/ml or more. Without complement, or with inactivated complement, these reactions were not observed. These facts suggest strongly that the cell surface of the hepatocytes was damaged by the MoAb in the presence of complement. ACTA PATHOL. JPN. 35: 1375–1383, 1985.     

Co-culture of rat embryos and hepatocytes: in vitro detection of a proteratogen

The technique of whole embryo culture developed by New [Environ Health Perspect 18:105-110, 1976] provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabolic activation system. We have developed methods for the co-cultivation of rat embryos with primary hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activation for in vitro assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy were co-cultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co-cultivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophosphamide (CP) in the absence of hepatocytes. When embryos were co-cultivated with hepatocytes and exposed to CP, a dose-related embryotoxicity was observed, indicating metabolic activation of the proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an increase in CP-induced malformations and embryotoxicity compared to those of embryos exposed to CP in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by the addition of metyrapone. When similar numbers of hepatocytes were used for metabolic activation of CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other species suggests the utility of this in vitro system for the investigation of species differences in sensitivity to chemical teratogens.     

Comparative study of proliferation of suckling and adult rat hepatocytes in primary culture in response to growth-stimulating factors

The replicative responses of suckling and adult rat hepatocytes in primary culture to growth-stimulating factors were compared. By addition of L-proline alone, the [3H]-thymidine labeling of suckling rat hepatocytes was dramatically enhanced, but that of adult ones was not. Epidermal growth factor (EGF), insulin, triiodothyronine (T3) and glucagon also enhanced the labeling of suckling rat hepatocytes regardless of the presence or the absence of L-proline. On the other hand, in the absence of L-proline, only EGF enhanced the labeling of adult rat hepatocytes, and, in the presence of L-proline, insulin as well as EGF enhanced the labeling. In the presence of growth factors and L-proline, the number of suckling rat hepatocytes increased up to about 143%, whereas that of adult rat hepatocytes hardly increased. Thus, a remarkable difference in replicative responses to growth factors and L-proline was observed between suckling and adult rat hepatocytes in primary culture.     

Pleiotrophin/heparin-binding growth-associated molecule as a mitogen of rat hepatocytes and its role in regeneration and development of liver

Previously pleiotrophin (PTN) was identified among proteins secreted by Swiss 3T3 cells as a mitogen for cultured adult rat hepatocytes. The present study showed that the growth of rat hepatocytes was enhanced when cultured with rat hepatic stellate cells (HSCs). HSCs expressed PTN mRNA and secreted its protein in the co-cultures. Recombinant PTN enhanced the growth of hepatocytes in culture, suggesting that HSCs stimulate the growth of hepatocytes through the action of PTN. To know the biological role of PTN in the growth of hepatocytes in vivo, we examined the expression of PTN in four regeneration models of adult liver and embryonic liver of rat. The expression of PTN mRNA in the liver was markedly up-regulated by the treatment with D-galactosamine (GalN) or with acetylaminofluorene followed by partial hepatectomy. HSCs expressed PTN mRNA in response to GalN treatment and its protein was found on hepatocytes. The mRNA expression of N-syndecan, a PTN receptor, was up-regulated in GalN-treated hepatocytes. The mesenchymal cells in the septum transversum enclosing the embryonic liver, but not embryonic HSCs, expressed PTN mRNA. We suggest that PTN is secreted from activated adult HSCs and embryonic mesenchymal cells as a mitogen of parenchymal cells in adult and embryonic liver, respectively.     

Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (ie, mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocytes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the 11 chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2:3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent in inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocytes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.     

Unusual hepatocellular lesions in primary biliary cirrhosis resembling but unrelated to hepatocellular neoplasms

Summary Structural, cellular and nuclear abnormalities of hepatocytes are a histological hallmark of well-differentiated, small hepatocellular carcinoma (HCC) or its borderline lesion. This study revealed that several hepatocellular abnormalities found in these hepatocellular neoplasms were also found in non-cirrhotic stages of primary biliary cirrhosis (PBC) in which HCC is unlikely to develop. These changes are small cell changes, consisting of the appearance of small hepatocytes arranged in thin trabecular or compact patterns with increased cellularity and basophilic cytoplasm. This was found in 36%, 71% and 100% in specimens of stages 1, 2 and 3, respectively. Large cell changes occurred and consisted of large hepatocytes with large nuclei and prominent nucleoli, found in 27%, 47% and 22% of the stages, respectively. Finally, liver cell rosettes were seen, showing variable acinar formation and present in 0%, 41% and 33% of the stages, respectively. These lesions were identified microscopically as irregularly shaped areas or vague nodules of hepatocytes without a fibrous rim, in the hepatic lobules. They showed an expansive growth or shaggy border against the surrounding hepatic parenchyma. Follow-up studies, including autopsies, failed to show development of HCC or its borderline lesion in PBC cases. Pathologists must make a diagnosis of HCC and its borderline lesion bearing in mind the occurrence of such unusual hepatocellular lesions probably of a reactive nature.     

Alloantigen-Mediated Adherence of Sat Hepatocytes to Antibody-Coated Polystyrene Dishes

Rat alloantigens expressed on normal hepatocytes have been utilized in a cell “panning” procedure to mediate the haplotype-specific adherence of collagenase-isolated hepatocytes to antibody-coated polystyrene dishes. A polyvalent F344 anti-WF alloantiserum was prepared by immunizing F344 rats with WF tissue. The alloantibody IgG purified from the alloantiserum, when reacted with either WF or (WF x F344)F₁ hybrid rat hepatocytes, caused the adherence of these hepatocytes to bacteriological-grade polystyrene dishes coated non-specifically with a xenoantibody, rabbit anti-rat IgG. Similarly-treated hepatocytes of the F344 strain, which lack WF alloantigenic determinants, failed to be adsorbed above background level to the coated dishes.     

Alloantigen-Mediated Adherence of Sat Hepatocytes to Antibody-Coated Polystyrene Dishes

Rat alloantigens expressed on normal hepatocytes have been utilized in a cell “panning” procedure to mediate the haplotype-specific adherence of collagenase-isolated hepatocytes to antibody-coated polystyrene dishes. A polyvalent F344 anti-WF alloantiserum was prepared by immunizing F344 rats with WF tissue. The alloantibody IgG purified from the alloantiserum, when reacted with either WF or (WF x F344)F₁ hybrid rat hepatocytes, caused the adherence of these hepatocytes to bacteriological-grade polystyrene dishes coated non-specifically with a xenoantibody, rabbit anti-rat IgG. Similarly-treated hepatocytes of the F344 strain, which lack WF alloantigenic determinants, failed to be adsorbed above background level to the coated dishes.     

Use of rat hepatocytes immobilized in agarose gel threads for biosynthesis of metabolites of potential cytostatics.

The aim of this work was to evaluate possibility of use of the rat isolated hepatocytes immobilized in agarose gel and continuously perfused for production of needed metabolites of two potential cytostatics, benfluron (5-(2-dimethylamino-ethoxy)-7-oxo-7H-benzo[c]fluorene) and oracin (6-[2-(hydroxyethyl) amino-ethyl]-5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]isoquinoline). The rat isolated hepatocytes obtained by two-step collagenase perfusion method were immobilized in agarose threads and perfused in a small bioreactor under a recirculation regimes. Biosynthesis of 9-hydroxybenfluron and 3-hydroxyoracin in immobilized rat hepatocytes was studied. Yields of the metabolites of interest in hepatocytes in immobilized and perfused rat hepatocytes was compared to production of metabolites in hepatocyte suspension and in rats in vivo. 9-hydroxybenfluron was presented during perfusion of immobilized rat hepatocytes in a relatively high amounts but total recovery all forms of benfluron was very low due to especially high binding to components of the perfusion system. More effective method remains the production of 9-hydroxybenfluron in rats in vivo. A considerable biosynthesis of 3-hydroxyoracin by immobilized rat hepatocytes in the bioreactor was found. Concentration of the metabolite in the perfusate rose continuously during 6 hours of perfusion. 3-hydroxyoracin production was increased several times with use of immobilized hepatocytes from rats treated for three days with methylcholanthrene. The yield of 3-hydroxyoracin in rats in vivo was comparably high but an advantage of in vitro synthesis is a much shorter interval to obtain the same amount of the metabolite of interest. In spite of some limitations in compounds exerting high trapping in the perfusion system, the method of the immobilized and perfused hepatocytes can be very useful and effective for production of some drug metabolites in biochemistry and pharmacology.