Effects of insulin, glucagon and triiodothyronine on DNA synthesis in rat hepatocyte primary cultures induced by liver tumour promoters and EGF

Parzefall, Wolfram; Erber, Eva; Kainzbauer, Eveline; Schulte‐Hermann, Rolf

doi:10.1016/0887-2333(95)00107-7

Cited by 8 publications

(9 citation statements)

References 62 publications

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“…In previous studies with rat hepatocytes in culture, we and others have shown that the cells under established experimental conditions can be stimulated to enter DNA synthesis and mitosis by liver mitogens of growth-factor type, such as EGF or rat serum (Parzefall et al 1982) and also by xenobiotics with tumor-promoting activity, such as hexachlorocyclohexane, nafenopin, and cyproterone acetate (Parzefall et al , 1996. Hepatocyte growth responses can be inhibited by TGF-b1 in culture (Nakamura et al 1985;Carr et al 1986;Russell 1988), and apoptosis can be induced (Oberhammer et al 1991).…”

Section: Discussionmentioning

confidence: 97%

“…Since we have previously shown (Parzefall et al 1996) that thymidine-labeling indices from autoradiographies correlate well with short-term (2 h) thymidine incorporation, expressed as specific activity (cpm/lg DNA), we have used the latter for routine analysis of hepatocyte DNA synthesis in the present series of experiments. Harvesting of cultures and determination of thymidine incorporation and DNA content was performed as described (Oberhammer et al 1991) and expressed as DNA specific activity.…”

Section: Determination Of Dna Synthesismentioning

confidence: 99%

“…Viability of the cell isolates was 80±7% by trypan-blue exclusion test, and the cell yield was on average 32±17 million cells per mouse liver. Rat perfusions were performed essentially as described earlier (Parzefall et al 1996).…”

Section: Media and Buffersmentioning

confidence: 99%

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Response of isolated hepatocytes from carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals inducing replication or apoptosis

Parzefall

Kainzbauer

Qin

et al. 2002

Archives of Toxicology

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The mouse strain C3H shows high incidence of liver tumors in carcinogenicity testing, while the strain C57BL exhibits low incidence. The F1 generation hybrids, B6C3F1, which are widely used in long-term carcinogenesis bioassays, are of intermediate sensitivity. We asked whether this strain difference could be due to different susceptibility of the parenchymal cells to signals inducing replication or apoptosis. Hepatocytes were isolated and cultured according to standard protocols. We tested (1) for the induction of DNA synthesis by epidermal growth factor (EGF), (2) for its inhibition by TGF-beta1, and (3) for the induction of apoptosis by TGF-beta1. Basal rates of DNA synthesis in untreated hepatocytes cultured from C3H and B6C3F1 mice were 6.5 and 3.5 times higher, respectively, than in hepatocytes from C57BL on day 3. Moreover, addition of EGF (10 ng/ml) increased DNA synthesis on day 3 in hepatocytes from C3H (4.2-fold) and B6C3F1 (2.7-fold) more strongly than in hepatocytes from C57BL. Treatment with TGF-beta1 inhibited basal and EGF-stimulated DNA synthesis dose-dependently. Inhibition was maximal at 1 ng TGF-beta1/ml in cultures from C57BL mice, and at 0.3 ng/ml in hepatocytes from C3H mice. In untreated hepatocytes from both strains virtually no apoptotic figures (condensed or fragmented nuclei, Hoechst 33285 staining) were found. After treatment with TGF-beta1 the incidence of apoptotic nuclei in hepatocytes from C57BL was higher than in cells from C3H mice (1.7% vs 3% on day 3). Thus it appears that hepatocytes from C57BL mice possess a lower growth potential, as indicated by a low basal rate of DNA synthesis and low inducibility by EGF, but a higher sensitivity to induction of apoptosis by TGF-beta1 than hepatocytes of the C3H strain. These findings may be helpful to explain the different susceptibility to induction of hepatocarcinogenesis in C3H and C57BL mice.

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Section: Discussionmentioning

confidence: 97%

Section: Determination Of Dna Synthesismentioning

confidence: 99%

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Response of isolated hepatocytes from carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals inducing replication or apoptosis

Parzefall

Kainzbauer

Qin

et al. 2002

Archives of Toxicology

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“…15,26,[31][32][33] We have already demonstrated that metofluthrin can increase replicative DNA synthesis in cultured rat hepatocytes in vitro 21 as well as in vivo. For example, a number of compounds that can activate either the CAR or the PPARα in rodent liver can induce replicative DNA synthesis in cultured rat and mouse hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

“…Studies with other CAR activators and PPARα activators, including some hypolipidaemic drugs, have shown that these compounds stimulate replicative DNA synthesis in cultured rodent hepatocytes but not in cultured human hepatocytes, 13,15,26,32,33,37 and they also do not stimulate replicative DNA synthesis in chimeric mice with human hepatocytes. Studies with other CAR activators and PPARα activators, including some hypolipidaemic drugs, have shown that these compounds stimulate replicative DNA synthesis in cultured rodent hepatocytes but not in cultured human hepatocytes, 13,15,26,32,33,37 and they also do not stimulate replicative DNA synthesis in chimeric mice with human hepatocytes.…”

Section: Toxicology Research Papermentioning

confidence: 99%

Lack of effect of metofluthrin and sodium phenobarbital on replicative DNA synthesis and Ki-67 mRNA expression in cultured human hepatocytes

et al. 2015

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High doses of metofluthrin have been shown to produce hepatocellular tumours in rats. Previous in vivo and in vitro mode of action (MOA) studies have demonstrated that metofluthrin induces hepatic microsomal cytochrome P450 (CYP) 2B enzymes and hepatocellular replicative DNA synthesis in rats, suggesting that the MOA for liver tumour formation is similar to that of other rodent non-genotoxic hepatocarcinogens that are constitutive androstane receptor (CAR) activators. To evaluate the potential human carcinogenic risk of metofluthrin, in this study the effect of metofluthrin (7.7-770 µM) on replicative DNA synthesis (as determined by BrdU labeling) was examined in cultured human hepatocytes from four different donors. The effect of sodium phenobarbital (NaPB), a well-known rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. In addition, the effect of metofluthrin on the expression of Ki-67, CYP2B6 and CAR mRNAs in cultured human and rat hepatocytes was examined. Treatment with 10 and 100 ng mL −1 hepatocyte growth factor (HGF) produced a concentration-dependent increase in BrdU labeling in human hepatocyte preparations. However, no increase in BrdU labeling was observed after treatment with metofluthrin or NaPB. Treatment with HGF significantly increased Ki-67 mRNA expression in both human and rat hepatocytes. However, while metofluthrin increased Ki-67 mRNA expression in rat hepatocytes, treatment with metofluthrin and NaPB had no effect on Ki-67 mRNA expression in human hepatocytes. Treatment with NaPB produced an increase in CYP2B6 mRNA levels in human hepatocytes, with metofluthrin also producing a small effect. Neither metofluthrin nor NaPB significantly changed CAR mRNA expression levels in both cultured rat and human hepatocytes. Thus, while metofluthrin and NaPB could activate CAR in cultured human hepatocytes, neither compound increased BrdU labeling and Ki-67 mRNA expression. As human hepatocytes are refractory to the mitogenic effects of metofluthrin, in contrast to rat hepatocytes, the data suggest that the MOA for metofluthrin-induced rat liver tumour formation is not relevant for humans and hence that metofluthrin does not pose a carcinogenic hazard for humans. Fig. 2 Effect of NaPB and metofluthrin (MFT) on cell viability (MTT assay) in cultured human hepatocytes. Results are presented as mean ± SD of 12 replicate wells. No values were statistically significantly different (all p > 0.05) from the control (DMSO only treated).Paper Toxicology Research 906 | Toxicol. Res., 2015, 4, 901-913 This journal is

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Stimulation of DNA synthesis by tumor promoters in primary rat hepatocytes is not mediated by arachidonic acid metabolites

Lee

Edwards

2001

Journal Cellular Physiology

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Studies in vivo using inhibitors of eicosanoid synthesis suggested that prostaglandins may play a role in mediating tumor promotion in liver by agents such as phenobarbital (PB). However, it is not clear whether any stimulation of arachidonic acid metabolism/prostaglandin formation results directly from the action of tumor promoters on hepatocytes or indirectly from effects of promoters on Kupffer cells or other non-hepatocytes. Our laboratory has been utilizing relatively pure populations of rat hepatocytes under the defined conditions of primary cultures, to investigate growth-stimulatory actions of tumor promoters, an important element in the promotion stage of carcinogenesis. It has been shown that most if not all liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with factors such as EGF, insulin, and glucocorticoid. In the present study, we sought evidence for a role of prostaglandins (PGs) in the direct growth-stimulatory actions of tumor promoters on hepatocytes. PGE(2), PGF(2 alpha), and PGD(2) cause concentration-dependent stimulation of hepatocyte DNA synthesis, while arachidonic acid was without any effect. PGE(2) and PGF(2 alpha) required the presence of dexamethasone to exert significant effects. These PGs did not further augment the stimulatory effect of EGF. In contrast, PGD(2) stimulated DNA synthesis in the presence or absence of insulin, dexamethasone, or EGF. The effect of tumor promoters on arachidonic acid metabolism, as measured by [(3)H]arachidonic acid release and PGE(2) production, was determined. The phorbol ester TPA significantly increased [(3)H]arachidonic acid release as well as PGE(2) formation in hepatocytes in line with known effects in other cell types. However, liver tumor promoters phenobarbital (PB), alpha-hexachlorocycohexane (HCH), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pregnenolone-16 alpha-carbonitrile (PCN) were without effects. Finally, inhibitors of arachidonic acid metabolism were tested for effects on the ability of TPA or liver tumor promoters to stimulate DNA synthesis by direct action on cultured hepatocytes. In all cases, lack of selective inhibition was observed. Taken together, the results show that while prostaglandins may directly stimulate DNA synthesis in hepatocytes, they are unlikely to mediate the direct growth-stimulatory actions of liver tumor promoters.

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Effects of insulin, glucagon and triiodothyronine on DNA synthesis in rat hepatocyte primary cultures induced by liver tumour promoters and EGF

Cited by 8 publications

References 62 publications

Response of isolated hepatocytes from carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals inducing replication or apoptosis

Response of isolated hepatocytes from carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals inducing replication or apoptosis

Lack of effect of metofluthrin and sodium phenobarbital on replicative DNA synthesis and Ki-67 mRNA expression in cultured human hepatocytes

Stimulation of DNA synthesis by tumor promoters in primary rat hepatocytes is not mediated by arachidonic acid metabolites

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