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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.

The ultrastructure of primary hepatocytes cultured for 2 to 17 days on floating collagen membrane was evaluated. A pellet of the hepatic cell suspension used to inoculate the collagen membranes contained some single cells and many aggregates of two, three, or four cells. Desmosomes were split during the perfusion, but tight junctions and gap junctions remained intact. By 2 days in culture, the hepatocytes had formed a monolayer of cells of polygonal shape with newly synthesized desmosomes between cells. Since the flexible floating collagen membrane decreases in size as the monolayer forms, the hepatocytes do not flatten out, as is characteristic of cells cultured on a rigid substrate. Hepatocytes in culture for 10 days or less exhibited large lamellar arrays of rough endoplasmic reticulum, well developed Golgi complexes, and structures resembling bile canaliculi, which possess tight junctions and desmosomes separating them from the intercellular space. Microfilaments oriented parallel to the plasma membranes of adjoining cells and in an intermeshed network at the edge of the monolayer and beneath the plasma membrane bordering the medium increased in size and number in older cultures. After 17 days in culture, the cells maintained tight junctions, desmosomes, Golgi complexes, and rough endoplasmic reticulum in small lamellar stacks and in small vesicles. Since hepatocytes on the floating collagen membrane retain most of the subcellular structural elements characteristic of normally functioning hepatocytes for 2.5 weeks, this system may be valuable for future experiments involving drug metabolism and carcinogenesis in vitro.

The role of adrenergic agents in DNA synthesis was investigated in two models of stimulated hepatocyte growth: in vitro primary serum-free cultures of adult parenchymal hepatocytes, and in vivo liver regeneration after two-thirds partial hepatectomy. In both systems the alpha 1-adrenergic receptor appeared to be involved in mediating stimulatory effects. In primary hepatocyte cultures norepinephrine acted via this receptor to enhance the DNA synthesis stimulated by epidermal growth factor (EGF), and heterologously downregulated EGF receptors. In liver regeneration the administration of an alpha 1 blocking agent interfered with the first wave of regenerative DNA synthesis, and this effect was preceded by an elevation in EGF receptor number. Measurements of plasma catcholamines demonstrated that elevated levels of norepinephrine and epinephrine were in circulation within 2 h after partial hepatectomy. Surgical hepatic sympathectomy also interfered with early liver regeneration, suggesting that locally delivered adrenergic agents are important to initiation of DNA synthesis. These data suggest that stimulation at the alpha 1-adrenergic receptor is among the early signals for liver regeneration and that heterologous regulation of EGF receptors, similar to that observed in vitro, may be a part of the regenerative response.

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

Primary cultures of adult rat hepatocytes were tested for their suitability to assess sister chromatid exchange (SCE)-inducing DNA damage produced by both directly and indirectly acting mutagens/carcinogens. Compared to other genotoxicity assay systems which utilize the metabolizing activity of liver microsomes, this system is at least 1-2 orders of magnitude more sensitive. The approximate drug concentrations leading to a doubling of control SCE levels were 2.5 X 10(-4) M for cyclophosphamide, 4.5 X 10(-5) M for dimethylnitrosamine, 2.5 X 10(-6) M for N-methyl-N-nitro-N-nitrosoguanidine, 2 X 10(-10) M for aflatoxin B1 (AFB1) and 30 mJ for u.v. The most potent inducer of SCE proved to be AFB1, leading to a significantly elevated level of exchanges at a concentration of 10(-12) M. The increased background SCE levels observed (0.75 SCE/chromosome) appears to reflect the sensitivity of hepatocytes to SCE-inducing DNA damage resulting from the dietary intake of mutagenic/carcinogenic compounds. In view of the high sensitivity and versatility of this genotoxicity assay system, it will be of use for the detection of the low levels of mutagenic/carcinogenic compounds found in the environment.

Nicotinamide administered in the drinking water of male Fischer 344 rats increased the number of renal tubular cell tumors of rats treated with an i.p. injection of diethylnitrosamine (DEN) (25-mg/kg body weight). The incidence of kidneys with tumors in rats treated with DEN alone was 5%. In rats which received DEN and then were promoted with either 30 or 6.7 mM nicotinamide in their drinking water, the incidence of kidneys with tumors rose to 59 and 28%, respectively. Rats which were on 30 mM nicotinamide but did not receive DEN had no kidney tumors present. These results show that nicotinamide promoted DEN-induced renal tubular cell tumorigenesis.

Repeated periods of DNA synthesis activity (each period consisting of two to three cycles) separated by intervals of quiescence in primary rat hepatocytes can be stimulated by sequential addition and removal of 2% dimethyl sulfoxide (DMSO) in the presence of epidermal growth factor (EGF). Hepatocytes can be kept in nonproliferating cultures for 7 days in media supplemented with 2% DMSO and EGF. If DMSO is removed while EGF is maintained, rat and human hepatocytes enter a 3 to 4 day period of DNA synthesis that declines rapidly by days 4 and 5. If DMSO is reintroduced into cultures at that point, kept on for 3 more days and removed again, hepatocytes reenter into proliferation with another self-limited response of 3 to 4 days. Similar phenomena can seen with hepatocytes maintained in the presence of 3 mM phenobarbital. These protocols demonstrate that loss of responsiveness to mitogens in primary hepatocyte cultures is not an irreversible process. They also raise the possibility that signals for termination of DNA synthesis in hepatocytes emanate from hepatocytes themselves. These studies also suggest for the first time the possibility of designing in vitro systems that will allow clonal expansion of differential hepatocytes.

Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats (Michalopoulos, G., Cianciulli, H. D., Novotny, A. R., Kligerman, A. D., Strom, S. C., and Jirtle, R. L. Cancer Res., 42: 4673-4682, 1982). The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of [3H]thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity. The ability of hepatocytes to activate 2-acetylaminofluorene to reactive intermediates capable of binding to DNA and inhibiting new DNA synthesis decreased as a function of time in culture. gamma-Glutamyl-transferase-positive hepatocytes from diethylnitrosamine-treated rats were observed to be relatively insensitive to carcinogen inhibition of DNA synthesis.