Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

The effects of several extracellular matrix components (EMCs)--fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen--on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [3H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [3H]thymidine uptake exhibited in the cells cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density.     

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 hepatocyte primary culture/DNA repair test using hepatocytes from several species

The hepatocyte primary culture/ DNA repair test, originally validated with rat hepatocytes, has been extended to use hepatocytes from other species including mouse, hamster, guinea pig, rabbit, monkey and human. Both qualitative and quantitative differences have been observed when chemicals are examined in the hepatocyte primary culture/DNA repair test using hepatocytes from more than one species. Examples are discussed that illustrate that the genotoxicity of a chemical can be a species-specific response and that multi-species testing permits a more complete assessment of genotoxicity.Abbreviations HPC hepatocyte primary culture - MOCA 4,4-methylene-bis-2-chloroaniline     

Mechanism of stimulation of DNA synthesis induced by epinephrine in primary culture of adult rat hepatocytes

Addition of epinephrine to primary cultured adult rat hepatocytes stimulated their DNA synthesis dose-dependently, especially in presence of insulin and epidermal growth factor. This effect of epinephrine was strongly inhibited by an alpha 1-antagonist, prazosin, but not by a beta-antagonist, propranolol, and was also slightly inhibited by an alpha 2-antagonist, yohinbin. These results indicate that the stimulation of DNA synthesis of hepatocytes by epinephrine is mediated predominantly by an alpha 1-action. 12-o-Tetradecanoylphorbol-13-acetate (TPA) or Ca2+-ionophore A-23187 stimulated DNA synthesis of Swiss 3T3 cells, but did not induce DNA synthesis of hepatocytes either singly or in combination. The fact that pretreatment of hepatocytes with TPA caused down-regulation of the stimulatory effect of epinephrine on DNA synthesis of hepatocytes within 15 min suggested that the effect of epinephrine on hepatocytes is mediated by its alpha 1 receptor and that TPA activated protein kinase c in the hepatocytes. Addition of dibutyryl cGMP did not induce DNA synthesis of hepatocytes. Therefore, the alpha 1-action of epinephrine that induce stimulation of DNA synthesis of primary cultured adult rat hepatocytes was apparently not mediated by either activation of phospholipid-dependent protein kinase or Ca2+ mobilization. Possible alternative mechanism was discussed.     

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.     

Characterization of gene-specific DNA repair by primary cultures of rat hepatocytes

At present, almost all the information on gene-specific DNA repair in mammals comes from studies with transformed cell lines and proliferating primary cells obtained from rodents and humans. In the present study, we measured the repair of specific DNA regions in primary cultures of nondividing rat hepatocytes (parenchymal cells). DNA damage was induced by irradiating the primary cultures of hepatocytes with ultraviolet (UV) light, and the presence of cyclobutane pyrimidine dimers (CPDs) was measured by using T4 endonuclease V in the following: a 21-kb BamHI fragment containing the albumin gene, a 14-kb BamHI fragment containing the H-ras gene, and the genome overall. The frequency of CPDs in the two BamHI fragments and the genome overall were similar and ranged from 0.5 to 1.3 CPDs per 10 kb for UV doses of 5–30 J/m². However, the removal of CPDs from the DNA fragment containing the albumin gene was significantly higher than from that of the genome overall and the DNA fragment containing the H-ras gene. Within 24 hr, approximately 67% of the CPDs was removed from the DNA fragment containing the albumin gene versus less than 40% for the genome overall and the DNA fragment containing the H-ras gene. The lower repair observed for the 14-kb fragment containing the H-ras gene is probably indicative of repair of the nontranscribed region of this fragment because the H-ras gene makes up only 2.4 kb of the 14-kb fragment. Primary cultures of hepatocytes removed CPDs from the transcribed strand of albumin fragment more efficiently than from the nontranscribed strand; however, no differences were observed in the repair of the two strands of the fragment containing the H-ras gene. These results demonstrate that primary cultures of nondividing rat hepatocytes show differential repair of UV-induced DNA damage that is comparable to what has been reported for transformed, proliferating mammalian cell lines. J. Cell. Physiol. 176:314–322, 1998. © 1998 Wiley-Liss, Inc.     

The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes

Summary Bicarbonate in the culture medium is essential for DNA synthesis of primary cultured rat hepatocytes stimulated by epidermal growth factor (EGF). When primary cultured hepatocytes in supplemented Leibovitz L15 medium were placed in a 100% air incubator, no increase in DNA synthesis was observed even after stimulation by EGF. However, when these cells were cultured with NaHCO₃ and EGF and placed in a 5% CO₂:95% air incubator, a stimulus of DNA synthesis more than 10-fold greater than in cultures in air only was seen, and many mitotic figures could be identified. Furthermore, NaHCO₃ added to supplemented DMEM/F12 medium enhanced the DNA synthesis of primary cultured rat hepatocytes in this medium. The ideal pH of the medium for DNA synthesis of cultured hepatocytes was in the range of 7.6 to 8.0. A dose response of NaHCO₃ in several media showed that DNA synthesis of the cells increased as the concentration of NaHCO₃ increased and that 25 to 30 mM NaHCO₃ in the medium was optimal for the replication of DNA by primary cultured rat hepatocytes. The investigations described in this study were supported in part by grants CA-07175, CA-22484, and CA-45700 from the National Cancer Institute, Bethesda, MD.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits DNA synthesis in rat primary hepatocytes

Treatment of Sprague-Dawley (SD) rats with a dosing regimen of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) maintaining a steady-state liver concentration of 150 ng/g results in enhanced hepatocyte proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule (Fox et al. (1993) Cancer Res., 53, 2265–2271). Here, we report an initial characterization of the actions of TCDD on hepatocyte proliferation by monitoring DNA synthesis in primary hepatocytes isolated from SD rats. TCDD caused a dose-dependent inhibition (EC₅₀ = 10 pM) of DNA synthesis in primary hepatocytes isolated from either male or female SD rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor, hepatocyte growth factor, and transforming growth factor ). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with transforming growth factor-β1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is non-responsive to estradiol, independent of TGF-β, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.     

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.     

Rapid and random turnover of mitochondrial DNA in rat hepatocytes of primary culture

It is known that mitochondrial DNA (mtDNA) replication is independent of the cell cycle. Even in post-mitotic cells in which nuclear DNA replication has ceased, mtDNA is believed to still be replicating. Here, we investigated the turnover rate of mtDNA in primary rat hepatocytes, which are quiescent cells. Southwestern blot analysis using 5-bromo-2'-deoxyuridine (BrdU) was employed to estimate the activity of full-length mtDNA replication and to determine efficient doses of replication inhibitors. Southern blot analysis showed that a two-day treatment with 20mM 2',3'-dideoxycytidine and 0.2mug/ml ethidium bromide caused a 37% reduction in the amount of mtDNA, indicating that the hepatocytes had a considerably high rate of turnover of mtDNA. Further, pulse-chase analysis using Southwestern analysis showed that the amount of newly synthesized mtDNA labeled with BrdU declined to 60% of the basal level within two days. Because the rate of reduction of the new mtDNA was very similar to the overall turnover rate described above, it appears that degrading mtDNA molecules were randomly chosen. Thus, we demonstrated that there is highly active and random turnover of mtDNA in hepatocytes.     

A method for the comparative study of replicative DNA synthesis in GGT-positive and GGT-negative hepatocytes in primary culture isolated from carcinogen-treated rats

Summary The presence of gamma-glutamyl transpeptidase (GGT) in focal nodules of hepatocytes is a commonly used marker for the identification of preneoplastic cell populations. Female Fischer 344 rats were initiated with a single intragastric administration of 200 mg diethylnitrosamine/kg, altered cells were selected after 0.02% 2-acetylaminofluorene was given in the diet; this was followed by a partial hepatectomy and promotion with dietary sodium phenobarbital for 4 wk. A mixed-cell population of GGT-positive and GGT-negative hepatocytes was obtained after collagenase perfusion and Percoll purification. An enriched population of GGT-positive hepatocytes was obtained by a modified “panning” technique. With quantitative scintillation spectrometry and autoradiography of [³H]thymidine incorporation, replicative DNA synthesis of GGT-positive and GGT-negative rat hepatocytes was observed in both the mixed-cell population and the enriched GGT-positive and GGT-negative cell populations. Under the culture conditions used, GGT-positive cells showed a higher level of replicative DNA synthesis than did GGT-negative cells; this indicates that such altered hepatocytes in the stage of promotion possess an inherently greater capacity for all replication, as previously suggested from studies in vivo.     

Stimulation of DNA synthesis and mitotic activity of chick embryo hepatocytes in primary culture

Summary Monolayer cultures were prepared from hepatocytes of 15 d chick embryos and maintained at high cell density in a chemically defined medium. In the absence of growth stimulatory conditions DNA synthesis was observed only during the first 10 to 16 h of culture. Thus, after a 12 h exposure to [³H]thymidine ([³H]dThd, 4 to 16 h) 9.1±1% ( ,n=4) of the hepatocyte nuclei were labeled. Labeled mitotic nuclei, up to late telophase, were regularly observed in these cultures. Beyond 16 h less than 2% labeled nuclei were found (12 h of [³H]dThd), which indicates that the hepatocytes entered proliferative quiescence. DNA synthesis of “resting” hepatocytes was stimulated by insulin and, only slightly, by hydrocortisone, glucagon, or fetal bovine serum. Triiodothyronine (T₃), or the nucleoside inosine (i) did not stimulate. Combination of insulin (I) with hydrocortisone (H), T₃ (T), or glucagon (G) resulted in a more than additive effect. Nearly maximal stimulation occurred with the combinations IHT and ITG. Labeling increased at 10 ng/ml of each component and was maximal at 1 to 10 μg/ml. A lag period of 8 to 10 h after hormone administration (IHiTG, 10 μg/ml) was observed before nuclear labeling increased. Within the subsequent 10 h a considerable proportion of the hepatocytes (up to 30% or more) entered DNA synthesis. Mitotic activity (with nuclei in prophase up to late telophase) also was stimulated. An increase of both total DNA and protein content was measured in several experiments. Hormonal stimulation of hepatocyte DNA synthesis and mitotic activity was associated with decreased β-naphthoflavone-mediated induction of cytochrome P450. A causal relationship between these two phenomena remains to be established. It is suggested that chick embryo hepatocyte cultures are a useful tool for studies on hepatocyte proliferation and differentiation. The present study is based on original observations by Dr. F. R. Althaus (presently at the Institute of Pharmacology and Biochemistry, University of Zürich, Switzerland). This contribution of his and his incisive criticism are acknowledged. The study was supported by Grant 3.893.81 from the Swiss National Research Foundation.     

Inhibition of DNA synthesis of adult rat hepatocytes in primary culture by dibutyrylcytidine 3', 5'-cyclic monophosphate

The effect of dibutyrylcytidine 3',5'-cyclic monophosphate (Bt2cCMP) on DNA synthesis of adult rat hepatocytes in primary culture was examined. Bt2cCMP caused dose-dependent inhibition of the DNA syntheses stimulated by various growth factors including human hepatocyte growth factor (hHGF). Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) inhibited the DNA synthesis more effectively than Bt2cCMP, but dibutyrylguanosine 3',5'-cyclic monophosphate (Bt2cGMP) and n-butyrate had a slight or null inhibitory effect. When added at the onset of DNA synthesis, Bt2cAMP was much less effective, but Bt2cCMP was still effective. Thus Bt2cCMP is able to inhibit growth factor-stimulated hepatocyte proliferation.     

The occurrence of thermotolerance in non-proliferating rat hepatocytes in primary culture

The occurrence of thermotolerance, induced by an initial heat treatment at 42 degrees C for 30 min, was studied in adult non-proliferating rat hepatocytes in primary culture. Heat treatment at 42 degrees C for 30 min did not affect cell morphology, cell attachment, Na+, K+ pump activity, K+ content and lactate dehydrogenase accumulation into the medium. In contrast, after exposure to 44 degrees C for 30 min a dramatic change in all these parameters was observed. However, of the cells, which remained attached to the substratum 24 h after treatment, Na+, K+ pump activity and K+ content appeared to be normal compared with untreated cells. Cells, pre-treated at 42 degrees C for 30 min, followed by incubation at 37 degrees C for 16 h, were found to be completely thermal resistant against heat treatment at 44 degrees C, as judged by cell morphology, detachment from the substratum, lactate dehydrogenase accumulation, Na+, K+ pump activity and K+ content. These results show that induction and development of thermotolerance can be studied in non-proliferating cells in primary culture.