Cultures Of Fetal Hepatocytes Research Articles

Regulation of IGF-I and -II by insulin in primary cultures of fetal rat hepatocytes.

During perinatal development, insulin and nutrients, rather than GH, regulate the IGF system. A selective primary culture of fetal rat hepatocytes has been established in our laboratory to elucidate the molecular mechanism of action of the above regulatory factors on IGF-I and -II gene expression during the late fetal period of the rat. In this model we have previously reported a regulatory role for glucose on IGF-I and -II synthesis and secretion. In the same experimental model, we now report that doses of insulin (0.1-5 microM) within the physiological range in rat fetuses during the last stages of gestation evoke an increase of IGF-I and -II mRNA abundance. Insulin regulated in a parallel manner IGF peptide secretion, and an excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to the hormone. Finally, the insulin-induced rise in IGF-I and -II mRNA was not mediated by stimulation of gene transcription but by increased transcript stability. The results support the hypothesis that insulin plays a major role in IGF regulation at immature stages of development.

Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency.

Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in utero. IGFBP-1 is primarily made in the liver, and it mostly inhibits IGF actions at the cellular level. IGFBP-1 is elevated in the fetal circulation of human and animal pregnancies complicated by IUGR caused by placental insufficiency and in utero hypoxia and is believed to restrict fetal growth by sequestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effects of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes were isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry, using specific antibodies and indirect immunofluorescence. Cultures consisted primarily (>90%) of cells positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascular cells and less than 8% macrophages. Identification of isolated hepatocytes was further confirmed by morphology. Hepatocytes were cultured in defined medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under hypoxic conditions (2% O(2)), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O(2) did not demonstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-fold under hypoxic (2% O(2)), compared with normoxic, conditions. Western ligand blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared with normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypoxic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.

Glucocorticoid-induced DNA demethylation and gene memory during development.

Glucocorticoid hormones were found to regulate DNA demethylation within a key enhancer of the rat liver-specific tyrosine aminotransferase (Tat) gene. Genomic footprinting analysis shows that the glucocorticoid receptor uses local DNA demethylation as one of several steps to recruit transcription factors in hepatoma cells. Demethylation occurs within 2-3 days following rapid (< 1 h) chromatin remodeling and recruitment of a first transcription factor, HNF-3. Upon demethylation, two additional transcription factors are recruited when chromatin is remodeled. In contrast to chromatin remodeling, the demethylation is stable following hormone withdrawal. As a stronger subsequent glucocorticoid response is observed, demethylation appears to provide memory of the first stimulation. During development, this demethylation occurs before birth, at a stage where the Tat gene is not yet inducible, and it could thus prepare the enhancer for subsequent stimulation by hypoglycemia at birth. In vitro cultures of fetal hepatocytes recapitulate the regulation analyzed in hepatoma cells. There fore, demethylation appears to contribute to the fine-tuning of the enhancer and to the memorization of a regulatory event during development.

Expression of cyclooxygenase-2 promotes the release of matrix metalloproteinase-2 and -9 in fetal rat hepatocytes

Treatment of primary cultures of fetal hepatocytes with proinflammatory cytokines, lipopolysaccharide (LPS), and hepatocyte growth factor promoted the expression of cyclooxygenase-2 (COX-2) and the synthesis of high amounts of prostaglandins (PGs). Under these conditions, the active forms of the matrix metalloproteinases-2 and -9 (MMPs) were released to the extracellular medium. This process was inhibited when the synthesis of PGs was suppressed pharmacologically with COX-2 inhibitors. Addition to the cell cultures of PGE2 promoted the release of MMPs through a mechanism that involved the expression of COX-2 and the synthesis of additional PGs. Kinetic analysis of the secretion of MMPs in response to LPS and PGE2 showed a similar time course, with a lag period of 6 hours, which suggests that PGE2 does not act directly on the mechanism of MMP processing and release. Inhibitors of protein kinase A, p38 MAP kinase, phosphatidylinositol-3-kinase, and nuclear factor κB (NF-κB) activation impaired the release of MMPs in response to PGE2 challenge, indicating the involvement of multiple steps in the process. The ability of fetal hepatocytes to release MMPs in response to growth factors and inflammatory stimuli constitutes a model for the study of the extracellular matrix remodeling that accompanies most liver diseases. (HEPATOLOGY 2001;33:860-867.)

Growth Regulation via p38 Mitogen-activated Protein Kinase in Developing Liver

During normal development in the rat, hepatocytes undergo marked changes in the rate of proliferation. We have previously observed transient G(1) growth arrest at term, re-activation of proliferation immediately after birth, and a gradual transition to the quiescent adult hepatocyte phenotype after postnatal day 4. We hypothesized that these changes in proliferation are due in part to growth inhibitory effects mediated by the p38 mitogen-activated protein kinase pathway. p38 kinase activity measurements showed an inverse relationship with hepatocyte proliferation during the perinatal and postnatal transitions, whereas p38 content remained constant. Anisomycin activated the p38 pathway in fetal hepatocyte cultures while inducing growth inhibition that was sensitive to the p38 inhibitor, SB203580. Activation of p38 in these cultures, via transient transfection with a constitutively active form of its upstream kinase MKK6, also inhibited DNA synthesis as well as reducing cyclin D1 content. Transfection with inactive MKK6 did neither. Furthermore, MKK6-induced growth arrest was sensitive to SB203580. Finally, administration of SB203580 to near-term fetal rats in utero abrogated the transient hepatocyte growth arrest that occurs at term. These findings indicate a role for the p38 mitogen-activated protein kinase pathway in the physiological regulation of hepatocyte proliferation during normal development in the rat.

Effects of four extracellular matrices associated with growth factors on clonal culture and proliferation of murine fetal hepatocytes

Effects of four extracellular matrices associated with growth factors on clonal culture and proliferation of murine fetal hepatocytes

Serine and glycine transport in fetal ovine hepatocytes

The role of hepatic serine and glycine transport in the regulation of the biosynthesis of serine by the fetal liver has not been studied. The goal of this study was to characterize serine and glycine transport and utilization at physiologic concentrations in primary cultures of fetal ovine hepatocytes. Primary culture of hepatocytes from mid gestation (≈90 days) and term (≈135 days) fetal sheep were studied after overnight serum free culture. At both gestational ages, the initial rate for sodium dependent 300 μM serine transport (1697±131 pmoles/min/mg protein at mid, 1765±544 at term) was fourfold greater than sodium dependent 300 μM glycine transport (309±54 at mid, 579±252 at term). At physiologic concentrations (300 μM), 69±7% of serine and 49±8% of glycine transport was sodium dependent. At term, sodium dependent serine transport has a V max of 1751±348 pmoles/min/mg protein and a K m of 159±111 μM. Sodium independent serine transport has a V max of 904±185 and a K m of 416±188 μM. Sodium dependent glycine transport has a V max of 410±69 and a K m of 2290±895 μM while sodium independent glycine transport exhibits non-saturable kinetics. Glycine (300 μM) sodium dependent transport was not inhibited by methyl-AIB while sodium dependent 300 μM serine transport was inhibited (31%). n-Ethylmaleimide inhibited sodium dependent serine and glycine transport by 36±9% and 37±2% respectively in term hepatocytes. Cysteine inhibited sodium dependent serine transport by 37%. Sodium independent glycine transport at 300 μM was higher in low glucose (1.1 mM) medium (881±76 pmoles/min/mg protein) compared to high glucose (5.5 mM) medium (510±60 P=0.004). There were no significant differences in serine or glycine incorporation into RNA, DNA, glycogen or lipid and protein. The predominance of serine transport over glycine at physiologic concentrations suggests that inward cellular amino acid transport of serine and glycine is not likely to be a regulatory mechanism that would favor serine biosynthesis in fetal ovine hepatocytes.

Regulation of Insulin-like Growth Factor-I and -II by Glucose in Primary Cultures of Fetal Rat Hepatocytes

A selective primary culture of fetal rat hepatocytes was established in our laboratory in order to elucidate the molecular mechanisms of action of different factors and conditions on insulin-like growth factor (IGF)-I and -II gene expression during the perinatal period of the rat. In this model we report that, in a serum-free condition and the presence of non-stimulatory doses of insulin, 5-20 mM glucose evoked an increase of IGF-I and -II mRNA abundance. Glucose regulated in a parallel manner IGF peptide secretion, and an excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to the carbohydrate. The experiment with 2-deoxyglucose suggests that glucose 6-phosphate, but not its further metabolism, is necessary for the induction of IGF transcript abundance in cultured fetal hepatocytes. Finally, the glucose-induced rise in IGF-II mRNA, the main IGF in fetal stages, was mediated by stimulation of gene transcription and increased transcript stability. The results support the idea that IGFs belong to a family of genes that are positively regulated by glucose.

The relationship between differentiation and proliferation in late gestation fetal rat hepatocytes.

Hepatocyte proliferation and differentiation occur simultaneously during late mammalian gestation. We hypothesized that regulation of hepatocyte growth and differentiation would be coordinated in late gestation fetal hepatocyte cultures such that proliferation would be most active in a population of less well-differentiated cells. Cultured fetal hepatocytes (embryonic d 19 and 21; E19 and E21) were studied using double staining immunofluorescent microscopy. Differentiation was assessed as staining for alpha-fetoprotein (AFP), three markers of enzymic differentiation (glucokinase [GK], phosphoenolpyruvate carboxykinase [PEPCK], and carbamoyl phosphate synthase [CPS]), and a hepatocyte cell-cell adhesion molecule (C-CAM). Proliferation was assessed using immunocytochemical detection of proliferating cell nuclear antigen (PCNA) or 5-bromo-2'-deoxy-uridine (BrdU) incorporation into DNA. Fetal hepatocyte cultures consisted of a heterogeneous population of cells, slightly more than half of which were proliferative under defined, growth factor-free conditions. These cultures were heterogeneous for AFP expression. There was no correlation between the expression of AFP and PCNA or AFP and S-phase entry (BrdU staining) during the first 48 h in culture. Similar results were obtained in staining for the enzymic differentiation markers and C-CAM. In addition, the differentiation status of cultured fetal hepatocytes was unrelated to a presumed indicator of mature growth regulation, mitogenic responsiveness to transforming growth factor alpha (TGFalpha), and hepatocyte growth factor (HGF). Finally, absence of any correlation between proliferation and differentiated phenotype was supported by in vivo studies using staining for PCNA, AFP, CPS, and PEPCK in liver sections. These results indicate that the developmental program governing differentiation of late gestation fetal rat hepatocytes is independent from mechanisms controlling proliferation.

Fatty acid desaturation: effect of alphafetoprotein on alpha-linolenic acid conversion by fetal rat hepatocytes

Freshly isolated fetal hepatocytes transformed 4.3, 8.5 and 19.2 pmol/min/106cells of stearic, linoleic and α-linolenic acids, respectively, complexed to albumin or α-fetoprotein (AFP), to more unsaturated derivatives. Thus, fetal hepatocytes displayed high elongase and ≜9, ≜6, ≜5-desaturase activities, as well as an ability to synthesize hexaene derivatives. Desaturase activities decreased when the time of culture of fetal hepatocytes (previous to incubation with the substrate) was prolonged, being practically undetectable after 24 h of culture. However, the rate of fatty acid uptake remained nearly constant. When AFP was used as the carrier the amount of hexaene fatty acid derivatives of α-linolenic acid recovered in cells was reduced up to 50% by albumin. This effect was associated with an increase of radioactivity found in the culture medium of hepatocytes incubated with AFP compared to albumin. Both observations taken together could be explained by an efflux of hexaene derivatives from cells caused by AFP.

Growth induction of rat primary hepatocytes using antisense oligonucleotides

We examined growth control of adult and fetal hepatocytes by regulating the expression of cell-cycle-related proteins using antisense S-oligonucleotides to tumor suppressors retinoblastoma (RB) protein and p53, and cyclin-dependent kinase (CDK) inhibitors p21 and p27. The protein expression in both adult and fetal hepatocytes was significantly suppressed with the addition of corresponding antisense oligonucleotides at a concentration of 2.5 μM. For the evaluation of growth, 3H-thymidine incorporation and DNA content were measured and the results demonstrated that all the antisense oligonucleotides had growth-promoting effects and the promoting potential was equivalent or slightly greater than that with the addition of hepatocyte growth factor (HGF) (10 ng/ml). The growth-promoting effect of the antisense oligonucleotides was enhanced by HGF in both adult and fetal hepatocyte cultures, and the effects on hepatocyte growth were also observed in a suspension culture.

Growth and differentiation of cultured fetal hepatocytes isolated various developmental stages.

We examined the relationship between cell proliferation and differentiation of cultured rat fetal and newborn hepatocytes isolated from various developmental stages. The albumin production rate increased along with cell growth under in vitro culture and became maximal two days after the growth cessation. AFP was secreted by both fetal and newborn hepatocytes with growth ability. Furthermore, the responses to HGF addition in fetal hepatocyte cultures were observed in terms of growth stimulation and down-regulated of the Met receptor. We also studied the changes in RB and liver enriched transcription factors (C/EBPs) for investigating the mechanism underlying proliferation and differentiation of fetal hepatocytes. Western blot analysis of hepatocytes taken from various gestation stages of rat liver showed that the expression of RB and C/EBP beta increased as gestation stage proceeded. When RB antisense S-oligonucleotide was added to the culture medium, proliferation and AFP expression increased, while C/EBP alpha and albumin expressions decreased. These results indicated that the tumor suppressor gene product RB had a profound role not only in cell proliferation but also hepatocyte differentiation.

SERINE AND GLYCINE TRANSPORT IN FETAL OVINE HEPATOCYTES

43 Serine (ser) and glycine (gly) are poorly transported across the placenta from the mother to the fetus. Although we have shown that the fetal liver is an important primary site of fetal ser synthesis for the unique fetal metabolism of ser and gly, the regulation of this unique fetal ser and gly metabolism is uncertain. The goal of this study was to characterize ser and gly transport in primary cultures of fetal ovine hepatocytes. Transport was determined after primary culture of hepatocytes from term (≈135 days) fetal lambs using sodium free or sodium containing KRP and the appropriate U-14C labeled amino acid after overnight culture. (Table) At 300µM concentrations, sodium dependent transport was inhibited by NEM (ser 36±9% and gly 37±2%) with similar inhibition by MeAIB. There was competitive inhibition of total gly transport by ser but not of ser transport by gly. Sodium free gly transport was higher in low glucose (1.1 mM) media (881±76) compared to high glucose (10 mM) media (510±60 p=0.004). Gly transport was inhibited by sarcosine. These data suggest that ser is primarily transported via system L and ASC in term fetal ovine hepatocytes while gly is transported primarily by system L with less of a contribution from ASC and gly. There is a contribution from system A to both ser and gly transport in term fetal ovine hepatocytes. The predominance of ser transport over gly at physiologic concentrations suggests that inward cellular amino acid transport of ser and gly is not likely to be a regulatory mechanism that would favor ser biosynthesis in fetal ovine hepatocytes except in conditions that would favor sodium free transport.TABLE

Response of Cultured Fetal and Adult Rat Hepatocytes to Growth Factors and Cyclosporine

Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in experimental animal models with genetic disorders of liver metabolism and liver failure. Fetal hepatocytes have several characteristics that make them potentially suitable as donor cells. In contrast to adult hepatocytes, fetal hepatocytes are thought to be highly proliferative, which may facilitate engraftment, expansion of transplanted cell population, and gene transfer requiring active DNA synthesis. The present study was undertaken to evaluate the proliferative capacity of fetal and adult rat hepatocytes under standardized culture conditions. Fetal (20 days of gestation) and adult hepatocytes were cultured in serum-free media at low densities and treated with growth factors. Proliferation was assessed by [ 3H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about ×100 higher and after 10 days in culture ×20 higher in fetal compared to adult hepatocytes. When epidermal growth factor (EGF) was added, maximal DNA synthesis in fetal hepatocytes was seen at 48 h, whereas in adult hepatocytes at 72 h. For adult hepatocytes, the average increase compared to untreated cells was ×13.8 with EGF, ×18.5 with transforming growth factor alpha (TGF-α), and ×7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-α or HGF. EGF-, TGF-α- and HGF-dependent DNA synthesis was inhibited by transfroming growth factor beta-1 (TGF-β1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-α- and HGF-dependent DNA synthesis in fetal hepatocyte cultures decreased by 36–46%, whereas in adult hepatocytes by 19–27%. These results show that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independently of growth factors and are relatively resistant to the inhibitory effect of TGF-β1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.

Initial expression of phosphoenolpyruvate carboxykinase gene in fetal hepatocytes: role of transcription factors

Background/Aims: Hepatic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) gene is absent in fetal liver. However, this gene can be initially expressed in 20-day-old fetal hepatocyte primary cultures under specific hormonal stimulation. The role of transcriptional factors involved is also studied. Methods: Primary 20-day-old fetal hepatocytes have been cultured and Northern-blot and nuclear run-on transcription assays have been performed. Results: Fetal hepatocytes in culture initially expressed PEPCK gene by dibutyryl cAMP, in the presence of dexamethasone. Dibutyryl cAMP increased by 8-fold the rate of transcription of PEPCK gene at 30 min, and produced a 50-fold increase in its mRNA content at 3 h. This induction of PEPCK expression by cAMP occurred in the presence of sustained levels of CCAAT/enhancer binding protein (C/EBP)α-δ mRNAs, and was accompanied by an increase in the rate of transcription and mRNA content of C/EBPβ gene, and a decrease in the expression of c-myc, in the absence of c-fos expression. In addition, insulin or phorbol esters decreased by 50% the PEPCK rate of transcription and its mRNA accumulation induced by dibutyryl cAMP. This inhibitory effect of insulin or phorbol esters on PEPCK gene expression was accompanied by an increase in the rate of transcription and mRNA content of nuclear factors such as c-fos and c-myc, the expression of C/EBPs remaining essentially unmodified.

Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes

In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5′ long terminal repeat controlling transcription of the β-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of β-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to β-galactosidase activity and cell survival. For maximum transfection of cells, 10 μg/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling β-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.

Bacterial lipopolysaccharide antagonizes transforming growth factor beta 1-induced apoptosis in primary cultures of hepatocytes

Incubation of primary cultures of fetal hepatocytes with lipopolysaccharide (LPS) elicited the expression of nitric oxide (NO) synthetase as well as antagonized the apoptotic cell death evoked by treating the cells with transforming growth factor beta 1 (TGF-beta 1). In addition to LPS, exposure of the cells to chemical NO donors also protected against apoptotic cell death when assayed at concentrations in the low micromolar range. Treatment of hepatocytes with large concentrations of NO donors promoted both apoptotic and necrotic cell death. These results suggest that NO synthesis by hepatocytes might be involved in the protection against apoptotic death. (Hepatology 1996 May;23(5):1200-7)

Bacterial lipopolysaccharide antagonizes transforming growth factor beta 1-induced apoptosis in primary cultures of hepatocytes.

Incubation of primary cultures of fetal hepatocytes with lipopolysaccharide (LPS) elicited the expression of nitric oxide (NO) synthetase as well as antagonized the apoptotic cell death evoked by treating the cells with transforming growth factor beta 1 (TGF-beta 1). In addition to LPS, exposure of the cells to chemical NO donors also protected against apoptotic cell death when assayed at concentrations in the low micromolar range. Treatment of hepatocytes with large concentrations of NO donors promoted both apoptotic and necrotic cell death. These results suggest that NO synthesis by hepatocytes might be involved in the protection against apoptotic death.

DNA adducts and P450 induction in human, rat and avian liver cells after exposure to polychlorobiphenyls

Polychlorinated biphenyls (PCBs) are industrial chemicals which have been detected in fish, birds and humans. They are known to exert marked effects on the liver. The induce hepatocellular carcinoma in rats and birds, and are suspected of being carcinogenic to humans. To better understand the genotoxic effects of PCBs, we used 32P-postlabelling to investigate DNA adduct formation, after exposure to PCBs (Aroclor 1254 and 3,′,4,4′-tetrachlorobiphenyl), in primary cultures of fetal hepatocytes from two animal species and in a human cell line (Hep G2). We also studied the induction of 7-ethoxyresorufin- O-deethylase (EROD) in these PCB-treated cells. The three cell types used are are known to express different cytochrome P450 families. The aim was to see whether a correlation could be established between EROD activity (a CYP1A1-related activity) and DNA adduct formation. DNA adducts were found in all three models after exposure to 50 μM 3,3′,4,4′-tetrachlorobiphenyl. The number of adducts was higher in quial hepatocytes (37 adducts per 10 9 nucleotides) than in rat hepatocytes or Hep G2 cells (20 adducts per 10 9 nucleotides in both cases). The major adduct was the same in all three cell types, but some adducts were found in only one or two species. These inter-species differences probably reflect metabolic differences leading to different ultimate carcinogens. Exposure to Aroclor 1254 failed to produce significant levels of DNA adduct, suggesting that pre-treated cells are required to magnify Aroclor 1254 metabolism. No correlation was found between adduct formation and the level of EROD induction.

Glucose regulation of glucose transporters in cultured adult and fetal hepatocytes

GLUT2 is the major glucose transporter of adult hepatocytes. In vivo, membrane GLUT1 is localized to a ring of perivenous cells and increases slightly after fasting or insulin deprivation. GLUT1 also increases in vitro after prolonged culture of isolated adult hepatocytes. We have previously shown that GLUT1 mRNA, protein, and activity are present in the rat fetal hepatocyte, and that both GLUT1 and GLUT2 are important for the pattern of glucose transport in the fetal hepatocyte. We tested the hypothesis that the postnatal increase in circulating glucose is one of the regulators of the changed pattern of GLUT1 and GLUT2 in the hepatocyte after the fetal to neonatal transition. Fetal and adult rat hepatocytes were cultured for 45 hours in supplemented Dulbecco's modified Eagle's medium at glucose concentrations of 1, 8.3, or 30 mmol/L. Culture at 8.3 and 30 mmol/L glucose diminished GLUT1 mRNA as compared with culture in 1 mmol/L glucose in both fetal and adult hepatocytes ( P < .05), but GLUT1 mRNA levels were lower in adult versus fetal hepatocyte cultures at 8.3 and 30 mmol/L ( P < .05). Similarly, GLUT1 protein levels were significantly diminished in hepatocytes cultured at higher medium glucose ( P < .05 for fetal cells at 30 v 1 mmol/L; P < .05 for adult cells at 8.3 and 30 v 1 mmol/L). GLUT2 mRNA abundance was enhanced by medium glucose in adult hepatocytes ( P < .05 at 8.3 and 30 v 1 mmol/L) and was unchanged by medium glucose in fetal hepatocytes. In contrast, GLUT2 protein level was unchanged by medium glucose in adult hepatocytes, and was diminished at 30 mmol/L as compared with 1 mmol/L glucose in fetal hepatocytes ( P < .05). In confirmation of these findings, uptake of 2-deoxyglucose (2-DOG) by fetal hepatocytes was significantly diminished after culture in 8.3 or 30 mmol/L glucose versus 1 mmol/L glucose ( P < .05 and <.01, respectively). These studies confirm that the fetal hepatocyte glucose transporter pattern could be maintained in part by low fetal portal glucose levels. However, the resistance of the fetal hepatocyte glucose transporter pattern as compared with that of the adult hepatocyte to the effects of hyperglycemia suggests additional undefined control mechanisms.