Stephen C. Strom

Cryopreservation of isolated rat hepatocytes

SummaryIsolated parenchymal hepatocytes from adult rats were frozen in media containing 10% glycerol, 10% dimethylsulfoxide (DMSO), or 20% DMSO. Three microsome-associated functions were compared in nonfrozen cells and cells frozen in each of the above cryoprotectant solutions. Freezing in DMSO maintains cytochromes P-450 and b5 and NADPH-cytochrome C reductase at levels nearer to control values than does freezing in glycerol. Cells frozen and subsequently thawed and cultured for 24 h lose a greater amount of cytochrome P-450 than do nonfrozen cultured cells. The levels of cytochrome b5 and reductase in frozen-thawed cells remain close to control values. Cell viability (trypan blue dye exclusion and percentage of attached cells) after freezing is maintained better using DMSO as a cryoprotectant. Dimethylsulfoxide protects the hepatocytes from freeze-induced damage to the extent that many viable cells attach to collagen-coated petri dishes, survive for at least 24 h, and still maintain significant levels of enzymes of importance to drug and carcinogen metabolism.

Assessment of unscheduled and replicative DNA synthesis in hepatocytes treated in vivo and in vitro with unleaded gasoline or 2,2,4-trimethylpentane

In a recent chronic inhalation exposure study, unleaded gasoline (UG) produced kidney tumors in male rats and liver tumors in female mice, but did not increase the incidence of liver tumors in male mice or rats of either sex. To examine the possible basis for this pattern of hepatocarcinogenesis, unscheduled DNA synthesis (UDS) as an indicator of genotoxic activity and replicative DNA synthesis (RDS) as an indicator of cell proliferation were measured in rat and mouse hepatocytes following in vivo and in vitro exposures to UG and 2,2,4-trimethylpentane (TMP), a nephrotoxic component of UG. Primary hepatocyte cultures, prepared from cells isolated from Fischer-344 rats, B6C3F1 mice, or human surgical material, were incubated with [3H]thymidine and the test agent. UDS was measured by quantitative autoradiography as net nuclear grains (NG). By similar methods, UDS and RDS (S-phase cells) were measured in hepatocytes isolated from rats and mice treated by gavage with TMP (500 mg/kg) or UG (100 to 5,000 mg/kg). A dose-related increase in UDS activity was observed in rat hepatocytes treated in vitro with 0.05 to 0.10% (v/v) UG. These doses were, however, toxic in both mouse and human hepatocyte cultures. Weak UDS activity was observed in hepatocytes isolated from male and female mice treated 12 hr previously with UG. No UDS was induced in rat hepatocytes treated in vivo or in vitro with TMP. Twenty- and fourfold increases in the percentage of cells in S-phase were observed 24 hr after treatment with TMP in male and female mice, respectively, as compared to a fivefold increase in male rats. UG increased the percentage of S-phase cells in male mice by ninefold but failed to induce RDS in females. Thus, there appears to be genotoxic compounds in UG that can be detected in cultured hepatocytes and in the livers of exposed mice. The lack of UDS activity in rat liver was consistent with the reported lack of liver tumors in chronically exposed rats. However, neither the UDS nor the RDS responses in mice exposed by gavage correlated to the sex-specific pattern of liver tumors observed in the 2-year bioassay.

Effect of phenobarbital and 3-methylcholanthrene on aldehyde dehydrogenase activity in cultures of HepG2 cells and normal human hepatocytes

Aldehyde dehydrogenase (ALDH) activity was measured in primary cultures of normal human hepatocytes and of the human hepatoma cell line HepG2 after application of phenobarbital (PB) or 3-methylcholanthrene (MC) for 5 days. Treatment with PB alone resulted in a significant increase in both protein and DNA content at concentrations of 2 and 3 mM. Treatment with MC at a concentration as low as 5 microM led to a significant loss of cells when it lasted more than 5 days. Concentrations of 3-5 mM of PB in the media of HepG2 cell cultures caused a 2-fold enhancement of the activity of ALDH, as measured with NAD and propionaldehyde (P/NAD) or benzaldehyde (B/NAD). On the other hand, MC-treated cultures (5 microM) showed a 20-fold increase in enzyme activity measured with NADP and benzaldehyde (B/NADP), and a 2-fold increase in B/NAD activity. Combined treatment with both PB and MC led to an effect of dynamic synergism as far as B/NAD and B/NADP activities are concerned, suggesting a metabolite of MC as the mediator for the increase of ALDH activity. Normal human hepatocytes in primary cultures responded to PB (3 mM) in a similar way as HepG2 cells as far as DNA and protein content and ALDH activity are concerned. It is concluded, that HepG2 hepatoma cells behave similar to the normal hepatocytes in terms of ALDH regulation and can be used for studies on the activity of ALDH as modified by added xenobiotics.

Induction of DNA repair in human and rat hepatocytes by 1,6-dinitropyrene

1,6-Dinitropyrene (DNP) was found to be an extremely potent genotoxicant in metabolically competent primary cultures of human and rat hepatocytes. Dose-dependent increases in DNA repair as measured by unscheduled DNA synthesis (UDS) were observed in the range from 0.05 to 5 microM 1,6-DNP for both species, indicating that the rat-hepatocyte assay is an appropriate model for assessing genotoxic potential in human hepatocytes for this class of compound. Unlike some nitroaromatic compounds, 1,6-DNP did not require gut flora for metabolic activation. No DNA repair was observed in hepatocytes isolated from rats treated with 50 mg/kg 1,6-DNP in corn oil by gavage 2, 12 or 24 h previously. The reason for the lack of a response in vivo is not known, but may relate to detoxification or distribution of the compound in the animal.

Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes

SummaryIsolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10−5M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10−5M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide.

Enhancement of aldehyde dehydrogenase activity in human and rat hepatocyte cultures by 3-methylcholanthrene

Aldehyde dehydrogenase was measured in primary cultures of hepatocytes obtained with a two-step collagenase perfusion either from human hepatic tissue or from livers of Fisher rats. Basal enzyme activity declines gradually as a function of time in culture, but remains at all times higher when measured with propionaldehyde and NAD (P/NAD) than with benzaldehyde and NADP (B/NADP). Treatment of the cultures with 2 μM of 3-methylcholanthrene for four days significantly increased the B-NADP activity of human and rat hepatocytes (tenfold and eightfold respectively). In human hepatocytes 3-methylcholanthrene increases also the P/NAD activity, but to a lesser extent (twofold), compared to the B/NADP activity. Due to the significant enhancement of B/NADP activity in cultures of human and rat hepatocytes after application of 3-methylcholanthrene, the initial difference in the basal activity levels between the P/NAD and B/NADP forms diminishes or, in the case of human hepatocytes, is even inverted. These results show for the first time that aldehyde dehydrogenase activity is increased in cultured human hepatocytes. This biochemical property is preserved in human and rat hepatocyte cultures, despite the rather quick loss of the basal aldehyde dehydrogenase activity.

Radiation protection of rat parenchymal hepatocytes with S-2-(3-aminopropylamino)ethylphosphorothioic acid.

The ability to protect parenchymal hepatocytes from ionizing radiation damage with S-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR-2721) was investigated. The clonogenic survival of irradiated hepatocytes was determined with an in vivo transplantation assay system. Injection of WR-2721 (400 mg/kg) immediately after irradiation was without protective effect. In contrast, an intraperitoneal (IP) injection of WR-2721 30 min prior to 60Co irradiation resulted in a significant protection of parenchymal hepatocytes. The mean lethal dose (Do) and extrapolation number (n) of the survival curve for unprotected hepatocytes as 2.9 Gy and 1.8, respectively. The Do and n values for hepatocytes protected with WR-2721 were 5.7 Gy and 1.8, respectively. Thus, WR-2721 at a concentration of 400 mg/kg acted as a dose modifying agent with a dose modifying factor of approximately 2.0. Decreasing the concentration of WR-2721 to 200 mg/kg did not significantly reduce the radioprotective effectiveness of the drug. However, at concentrations less than 200 mg/kg, the radioprotective effect decreased in a dose-response manner with a concentration of 150 mg/kg providing 50% of the maximum effect observed.

Characterization of liver epithelial cells transfected withmyc and/orras oncogenes

While many liver tumors contain activatedmyc andras oncogenes, the mechanisms by which these genes contribute to cellular transformation is poorly understood. Activated versions of the cellular oncogenes,c-myc and/orc-H-ras were transfected into normal rat liver epithelial cells to identify cellular pathways that are altered in the cells containing the oncogenes. The results of these and other investigations indicate that the biological properties associated with the transfection ofc-myc include immortalization, reduced contact inhibition of growth, activation of phospholipase A2-mediated pathways, increased sensitivity to transformation with aras gene, and greatly increased sensitivity to growth factors. The biological properties associated with the transfection of theras gene include morphological transformation, anchorage-independent growth, tumorigenicity, increased phosphotidylinositol metabolism, the induction of growth-factor processing and secretion, which leads to (exogenous) growth factor-independent tumor growth, and a marked resistance to normal inhibitors of growth such as TGF-β. It is proposed that the complementary actions of themyc andras genes in cellular transformation may be related to theras-induced secretion of autocrine growth factors by cells sensitized to their effects by themyc gene. The increased stimulus for growth coupled to aras-induced insensitivity to growth inhibitors may lead to clonal expansion of these cells and tumor development.

Induction of cytochrome P1450 RNA and benzo[a]pyrene metabolism in primary human hepatocyte cultures with benzanthranene

Exposure of cells to microsomal enzyme inducers can modify the potency of many carcinogens. We have examined the steady-state level of RNA from the P1450 gene and the metabolism of benzo[a]pyrene (BP) in primary cultures of human hepatocytes exposed for up to 4 days to 12.5 μm benzanthracene (BA), and in uninduced control cultures. While the steady-state levels of RNA from the P1450 gene were nondetectable in uninduced (DMSO only) human hepatocytes, 12.5 μm BA-induced AHH activity, BP metabolism, and/or P1450-specific RNA in hepatocytes from seven human cases were investigated. RNA levels specific for the P1450 gene appeared maximal at 24 hr following exposure to BA, whereas, the protein, as determined by AHH enzyme activity from BA-induced hepatocytes, continued to increase up to the last time point examined, 72 hr. BA induction for 96 hr increased metabolism of BP (initial concentration of BP, 10 μm) over a time course of 3, 6, 12, and 24 hr of incubation with BP compared with that of controls. The major metabolites of BP produced by human hepatocytes in culture were the unidentified polar BP metabolite(s), possibly polyhydroxylated. BA induction caused approximately a twofold increase in these metabolites. BA-induced cultures showed an increase in glutathione conjugation compared to that in controls. The percentage of glucuronide and sulfate conjugates remains similar in all cultures. Total binding of tritium label BP to DNA was 1.3-fold to fivefold greater in induced cultures, and related more to total metabolism than to production of a specific metabolite. Exposure of human hepatocytes in vitro to BA leads to a large increase in the steady-state level of the RNA specific for the P1450 gene and an increase metabolism of BP.