DNA Synthesis In Rat Hepatocytes Research Articles

Effect of hypolipidemic peroxisome proliferators on unscheduled DNA synthesis in cultured hepatocytes and on mutagenesis in Salmonella

The peroxisome proliferators Wy-14,643, BR-931, nafenopin and ciprofibrate were tested in the primary hepatocyte culture-unscheduled DNA synthesis assay and in the Ames Salmonella microsome mutagenicity assay. The amount of unscheduled DNA synthesis (UDS) in hepatocytes was determined by quantifying the amount of [ 3H] thymidine incorporated into DNA in the presence of hydroxyurea after isolation of nuclei from hepatocytes treated with the test agent. Wy-14,643 and BR-931 induced unscheduled DNA synthesis in rat hepatocytes, whereas nafenopin and ciprofibrate had no effect. All of the peroxisome proliferators were negative in the Ames Salmonella assay.

Unscheduled DNA synthesis of rat hepatocytes in monolayer culture

Monolayer cultures of rat hepatocytes activated tris(2,3-dibromopropyl)phosphate (Tris-BP) more efficiently than 2-acetylaminofluorene (AAF), to genotoxic products which caused mutations in co-cultures of S. typhimurium. In contrast, AAF caused a greater genotoxic response in the hepatocytes than Tris-BP, as judged by the increase in DNA-repair synthesis measured by liquid scintillation counting of 3H-TdR incorporated into DNA isolated from the nuclei of the hepatocytes. Covalent binding of 0.05 mM 3H-Tris-BP to cellular proteins occurred at a similar rate as covalent binding of 0.25 mM 14C-AAF. Tris-BP was the more cytotoxic of the two compounds as determined by leakage of cellular lactate dehydrogenase into the culture medium. The observed differences in the cytotoxic and genotoxic responses between Tris-BP and AAF were probably caused by differences in the nature of their reactive metabolites with respect to stability, lipophilicity and/or their interactions with variuos cellular nucleophilic sites. The relative DNA-repair synthesis induced by an AAF exposure for 18 h decreased with time after plating of isolated hepatocytes. Tris-BP first caused an increase in the relative DNA-repair synthesis up to 27 h after plating, whereafter the response declined reaching control values using cultures 75 h after plating. In parallel with the decreased relative response in DNA-repair synthesis with time, the background radioactivity in isolated nuclei from untreated cells increased both when the hepatocytes were incubated in the presence or absence of hydroxyurea to inhibit replicative DNA synthesis. Increased DNA-repair synthesis was demonstrated as early as 3 h after commencing exposure to the test substances. While the induced DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF increased with increased exposure time beyond 6 h. To assess the role of different metabolic pathways in the genotoxic and cytotoxic responses of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by cell-culture medium containing 3H-TdR and hydroxyurea. The cytochrome P-450 inhibitor metyrapone decreased both the genotoxic and cytotoxic effects of Tris-BP, while α-naphthoflavone reduced the genotoxic effect of AAF. The addition of glutathione (GSH) or N-acetylcysteine decreased both the cytotoxic and genotoxic effects of Tris-BP, while cellular depletion of GSH by diethylmaleate increased these effects. Manipulations in the cellular levels of sulhydryl-containing substances in the hepatocytes by these agents had little effects on the DNA-repair synthesis caused by AAF. The results indicate that such a hepatocyte culture system may be very useful as a tool to study mechanisms involved in the formation of cytotoxic and/or genotoxic metabolites from various xenobiotics.

The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin

A low concentration (10 −11 mol/l) of epidermal growth factor (EGF) and/or an equimolar (10 −14 mol/l) mixture of glucagon and insulin stimulated DNA synthesis in hepatocytes in 4-day-old primary cultures of neonatal rat liver. EGF seems to have acted by inducing quiescent hepatocytes to begin cycling, while the glucagon-insulin combination seems to have acted mainly by shortening the cell cycle time. Incubation in low calcium medium blocked untreated hepatocytes in the G 1 phase of their cycle and prevented EGF and the glucagon-insulin mixture from stimulating DNA synthesis. Nevertheless, hepatocytes in calcium-deficient medium did respond to these agents, as they reached a late stage of prereplicative development before being blocked: in fact, they initiated DNA synthesis soon after the addition of calcium. EGF, but not the glucagon-insulin combination, also enabled the already cycling hepatocytes (but not the newly activated ones) to overcome the block imposed by the extracellular calcium deficiency after a delay of several hours.

Age-dependence of the stimulatory effect of triiodothyronine on the DNA synthesis of rat hepatocytes

The effect of a single subcutaneous injection of triiodothyronine (T 3) on DNA synthesis rate and liver growth was studied in young and old female Wistar rats. The hormonal treatment was effective in stimulating DNA synthesis, resulting in an increase of the specific liver volume as well as volume and numerical density of nuclei/100 g body wt. in young animals. In the old animals T 3 injection induced a reduced and delayed synthesis of DNA, which did not affect the morphometric parameters of liver/100 g body wt.

Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene.

The purpose of this study was to examine the induction of unscheduled DNA synthesis (UDS) by the potent hepatocarcinogen technical grade dinitrotoluene (tgDNT; 76% 2, 4-DNT, 19% 2, 6-DNT) using the in vivo-in vitro hepatocyte DNA repair assay. Male Fischer-344 rats were treated by gavage and hepatocytes were isolated by liver perfusion and cultured with [ 3 H]thymidine. UDS was measured by quantitative autoradiography as net grains/nucleus (NG); ≥5 NG was considered positive. Controls consistently had − 3 to − 6 NG. A dose-related increase in UDS was observed 12 h after treatment, with 200 mg/kg tgDNT producing 26 NG. A 50-fold increase in the number of cells in S-phase was observed at 48 h after treatment. This increase in S-phase cells could be suppressed in the presence of 10–20 mM hydroxyurea (HU), while the same levels of HU did not affect the level of UDS at 12 h after treatment. 2, 4-DNT produced only a weak response, in contrast to 2, 6-DNT which was a potent inducer of UDS. Treatment of female rats with tgDNT yielded only modest increases in UDS and DNA replication relative to males. These results are consistent with the carcinogenicity studies and indicate that tgDNT is a potent genotoxic agent, with 2, 6-DNT contributing the major portion of the effect.

The induction of ornithine decarboxylase and DNA synthesis in rat hepatocytes after a single administration of diethylnitrosamine.

In primary monolayer culture of adult rat liver cells, the presence of a mixture of hormones stimulates DNA synthesis and proliferation. Under conditions of suboptimal hormone stimulation diethylnitrosamine was found to be effective in stimulation of DNA synthesis. Diethylnitrosamine also influences ornithine decarboxylase activity. The effect of some carcinogens and their structural related non-carcinogenic analogs on ornithine decarboxylase activity in primary cultured rat hepatocytes was studied. The addition of the carcinogens diethylnitrosamine, benzo[a]pyrene, methylmethanesulphonate and ethylmethanesulphonate caused significant increases in ornithine decarboxylase activity. Under the same conditions the non-carcinogenic analogs diethylformamide and pyrene did not show this change in enzyme activity.