Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures.

Experiments were designed to determine the role of cell proliferation in the initiation of liver carcinogenesis induced by chemicals. To investigate this, two methylating carcinogens, N-methyl-N-nitrosourea and 1,2-dimethylhydrazine, were used as the initiating carcinogens. The initiated hepatocytes were monitored by selectively stimulating them to grow into focal islands of presumptive preneoplastic hepatocytes. The experimental approach in brief consisted of the following. Rats received a nonnecrogenic dose of the carcinogen; at a time period when the carcinogen could no longer be detected in the system, they were subjected to either partial or sham hepatectomy. The initiated cell thus formed were selectively stimulated to grow into foci of preneoplastic hepatocytes using three different selection regimens: (a) feeding a diet containing 0.02% 2-acetylaminofluorene plus one administration of carbon tetrachloride (2 ml/kg body weight) intragastrically; (b) feeding a diet containing 0.05% phenobarbital; and (c) feeding a choline-deficient diet. The foci were quantitated by staining them for the presence of gamma-glutamyltransferase. The results obtained indicate that irrespective of the type of selection procedure used foci of preneoplastic hepatocytes were seen only in rats that received the carcinogen coupled with a cell-proliferative stimulus such as partial hepatectomy. Very few or no foci were seen in rats that received the carcinogen plus sham hepatectomy. These results suggest that cell proliferation plays an important role in the initiation of liver carcinogenesis by chemicals.

Cell proliferation induced by triiodothyronine in rat liver is associated with nodule regression and reduction of hepatocellular carcinomas.

Previous studies have demonstrated that short-term treatment with peroxisome proliferators decreased the size and number of gamma-glutamyl transpeptidase or placental glutathione S-transferase (GSTP)-positive hepatic hyperplastic lesions. In this study, we have examined the effect of the hormone triiodothyronine (T3), which, similarly to peroxisome proliferators, is a strong liver mitogen and a ligand of nuclear receptors, on the growth of GSTP-positive nodules generated by the resistant hepatocyte model and on the development of hepatocellular carcinoma. Hepatic hyperplastic nodules were induced in male Fischer rats by a single dose (150 mg/kg) of diethylnitrosamine, followed by a 2-week exposure of the animals to 2-acetylaminofluorene and partial hepatectomy. Nine weeks after diethylnitrosamine administration, rats were switched to a diet containing 4 mg/kg T3 for 1 week (experiment 1) and sacrificed during T3 feeding or were exposed to seven cycles of T3-supplemented diet (1 week/month per 7 months), and sacrificed 6 months after the last cycle (experiment 2). Results showed that T3 treatment for 1 week caused a 70% reduction in the number of GSTP-positive nodules (14/cm2 in T3-fed rats versus 44/cm2 of control animals), as well as GSTP-positive area (12% versus 43% of controls). Reduction in the number of GSTP-positive nodules observed 1 week after T3 feeding was associated with a strong increase in the labeling index of enzyme-altered nodules compared with that of controls (labeling index was 64 and 31%, respectively). No significant differences in the apoptotic index were observed between the two groups. Results from experiment 2 did reveal that although rats treated with diethylnitrosamine + 2-acetylaminofluorene developed 100% hepatocellular carcinoma and 33% of them showed lung metastasis, only 50% of rats exposed to repeated cycles of triiodothyronine developed hepatocellular carcinoma with no lung metastasis. This study indicates that cell proliferation per se might not necessarily represent a promoting condition for putative preneoplastic lesions and demonstrates an anticarcinogenic effect of T3.

Inability of mitogen-induced liver hyperplasia to support the induction of enzyme-altered islands induced by liver carcinogens.

Experiments were designed to determine whether liver cell proliferation induced by direct mitogens is as effective as compensatory cell proliferation consequent to previous cell loss, in supporting the growth of enzyme-altered islands in the liver induced by chemical carcinogens. Male Wistar rats were given injections of a single nonnecrogenic dose of N-methyl-N-nitrosourea or benzo(a)pyrene during the S phase following the administration of four different liver mitogens, namely, lead nitrate, ethylene dibromide, nafenopin, and cyproterone acetate, or during compensatory cell proliferation following partial hepatectomy or a necrogenic dose of CCl4. The carcinogen-altered hepatocytes were monitored as gamma-glutamyltransferase- or placental glutathione S-transferase-positive foci using a 2-wk promoting regimen consisting of 0.03% 2-acetylaminofluorene coupled with a necrogenic dose of CCl4. The results indicate that, unlike compensatory cell proliferation induced by partial hepatectomy or CCl4, the mitogen-induced cell proliferation did not result in a significant number of enzyme-altered islands, despite the fact that the extent of cell proliferation at the time of carcinogen administration, as monitored by the examination of labeled cells, is similar with both types of proliferative stimuli.

Induction of cellular DNA synthesis in the pancreas and kidneys of rats by peroxisome proliferators, 9-cis retinoic acid, and 3,3',5-triiodo-L-thyronine.

We recently suggested that peroxisome proliferators (PPs), 3,3',5-triiodo-L-thyronine (T3), and 9-cis retinoic acid (9-cis RA) induce hepatocyte proliferation in rats through the activation of their nuclear receptors, PP-activated receptors, T3 receptors, and retinoid X receptors. To test whether nuclear hormone receptor-mediated cell proliferation can be observed in organs other than liver, we examined the effects of these agents on the pancreas and kidneys of male Wistar rats using BrdUrd immunohistochemistry. A single s.c. injection of T3 (2 mg/kg) and single intragastric administration of 9-cis RA (40 mg/kg) or 4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide (200 mg/kg) induced a wave of DNA synthesis in the pancreatic acinar cells and in the proximal tubular epithelial cells of the kidneys, peaking after 24 h. No stimulation of DNA synthesis was observed in ductal or islet cells of the pancreas and in glomeruli of the kidneys. All-trans-retinoic acid, a ligand for retinoic acid receptor, at a dose (200 mg/kg) that induced hepatocyte proliferation, had no effects on cell proliferation of the pancreas and the kidneys. The results suggest that T3, 9-cis RA, and PP activate genes that regulate cell proliferation in target cells through receptor-mediated pathways and initiate cellular DNA synthesis.

In vivo hepatocyte proliferation is inducible through a TNF and IL-6-independent pathway.

Recent studies in mice harboring a targeted disruption of genes encoding TNF receptor 1 (TNFR-1) or Interleukin 6 (IL-6) suggested a critical role for TNF and IL-6 in initiation of liver regeneration after 2/3 partial hepatectomy. However, hepatocyte proliferation can also occur following treatment with agents that do not induce tissue loss (primary mitogens). To determine whether the above cytokines could also be involved in mitogen-induced liver cell proliferation, we studied the hepatocyte proliferative response after treatment with primary mitogens in mice knock-out for TNFR-1 or IL-6. Our results showed no difference in the proliferative response of the liver between the wild type and the knock-out mice following treatment with the mitogens 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), or the peroxisome proliferator, ciprofibrate, suggesting that TNF or IL-6 may not play a major role in this type of proliferation. Gel shift assay indicated that TCPOBOP-induced hepatocyte proliferation is not associated with activation of STAT3 transcription factor, a major target of IL-6 and other growth factors/cytokines. Our results thus indicate that hepatocyte proliferation can be induced by at least two different pathways; compensatory regeneration being TNF and IL-6-dependent, and mitogen-induced direct hyperplasia which does not require TNF or IL-6.

Increased expression of c-fos, c-jun and LRF-1 is not required for in vivo priming of hepatocytes by the mitogen TCPOBOP.

The notion that an increased expression of immediate early genes such as c-fos and c-jun is an absolute requirement for the G0-G1 transition of the hepatocytes has recently been challenged by the finding that rat liver cell proliferation induced by primary mitogens may occur in the absence of such changes (Columbano and Shinozuka, 1996). To further investigate the relationship between immediate early genes and hepatocyte proliferation, we have compared the hepatic levels of c-fos, c-jun and LRF-1 transcripts during mouse liver cell proliferation in two conditions: (i) direct hyperplasia induced by the non-genotoxic hepatocarcinogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, and (ii) compensatory regeneration caused by a necrogenic dose of carbon tetrachloride. The results show striking differences in the activation of early genes. In spite of a rapid stimulation of S phase by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (approximately 8% of hepatocytes were BrdU-positive as early as 24 h after mitogen treatment versus 1% of labelled hepatocytes after 2/3 partial hepatectomy), no changes in the expression of c-fos, c-jun and LRF-1 could be observed. Moreover, no change in steady state mRNA hepatic levels of IGFBP-1 (a gene highly expressed in rat liver following partial hepatectomy), and only a slight increase in c-myc and PRL-1, was found after mitogen administration. On the contrary, a rapid, massive and transient increase in the hepatic mRNA levels of all these genes was observed during carbon tetrachloride induced regeneration. The results indicate that increased expression of immediate early genes may be dependent upon the nature of the proliferative stimulus, and it may not be a prerequisite in certain in vivo conditions such as proliferation induced in the absence of liver tissue damage.

Cell proliferation, cell death and hepatocarcinogenesis.

The carcinogenic process in the liver is a multistep process, characterised by an altered ratio between cell proliferation and cell death. In the last few years, we have undertaken studies aimed at determining the possible differences exhibited by two different types of cell proliferation, namely compensatory regeneration and direct hyperplasia at a molecular and cellular level. These two types of proliferative stimuli appear to play different roles in liver carcinogenesis. The scope of this article is to summarise the present knowledge about the differences in the expression of genes involved in the entry of liver cells into cell cycle, between liver regeneration following cell loss and/or cell death and direct hyperplasia induced by primary mitogens.

Induction of pancreatic acinar cell proliferation by thyroid hormone.

Thyroid hormone is known to elicit diverse cellular and metabolic effects in various organs, including mitogenesis in the rat liver. In the present study, experiments were carried out to determine whether thyroid hormone is able to stimulate cell proliferation in another quiescent organ such as the pancreas. 3,5,3'-L-tri-iodothyronine (T3) added to the diet at a concentration of 4 mg/kg caused a striking increase in nuclear bromodeoxyuridine (BrdU) incorporation of rat acinar cells 7 days after treatment (the labeling index was 46.7% in T3-treated rats vs 7.1% in controls). BrdU incorporation was limited to the acinar cells, with duct cells and islet cells being essentially negative. The increase in DNA synthesis was accompanied by the presence of several mitotic figures. Histological examination of the pancreas did not exhibit any sign of T3-induced toxicity. Determination of the apoptotic index, measurement of the serum levels of alpha-amylase and lipase, and glycemia determination did not show any increase over control values, suggesting that the enhanced proliferation of acinar cells was a direct effect induced by T3 and not a regenerative response consequent to acinar or beta-cell injury. Additional experiments showed that DNA synthesis was induced as early as 2 days after T3 treatment (the labeling index was 9.4 vs 1.9% in controls) and was associated with increased protein levels of cyclin D1, cyclin A and proliferating cell nuclear antigen, with no substantial differences in the expression of the cyclin-dependent kinase inhibitor p27. The mitogenic effect of T3 on the pancreas was not limited to the rat, since extensive acinar cell proliferation was also observed in the pancreas of mice treated with T3 for 1 week (the labeling index was 28% in T3-treated mice vs 1.8% in controls). Treatment with three other ligands of nuclear receptors, ciprofibrate, all-trans retinoic acid and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, induced little or no pancreatic cell proliferation. These results demonstrated that T3 is a powerful inducer of cell proliferation in the pancreas and suggested that pancreatic acinar cell proliferation by selected agents may have potential for therapeutic use.

Involvement of DNA polymerase beta in proliferation of rat liver induced by lead nitrate or partial hepatectomy.

We have studied the expression pattern of DNA polymerase beta in two different models of in vivo cell proliferation. Both mRNA levels and enzyme activity of DNA polymerase beta markedly increased before and/or during DNA synthesis in proliferating hepatocytes in mitogen-treated and partially hepatectomized rats. The time-courses of the expression of the gene coding for DNA polymerase beta were significantly different in the two cell systems. A 5-fold increase in DNA polymerase beta mRNA was observed 8 h after lead nitrate administration, i.e. well before the onset of DNA synthesis. In the regenerative liver cells a 3-fold increase in the amount of mRNA was observed 24-48 h after partial hepatectomy, the event being coincident with extensive DNA synthesis. In both systems, the increase of mRNA levels was always paralleled by an increase in enzyme activity, suggesting that DNA polymerase beta activity may be regulated at a pre-translational level.

Antiapoptotic compound to enhance hypothermic liver preservation.

BACKGROUND: Apoptosis (programmed cell death) occurs as a consequence of global organ ischemia during isolation and storage prior to transplantation. If apoptosis is inhibited during ischemia, organ preservation should be improved, and the length of time for permissible storage may be increased. The objective of this study was to test the effect of a newly developed antiapoptotic compound, LXR-015, during extended hypothermic liver preservation. METHODS: Three groups of 12 rats each were studied. In the normal group, liver function was studied immediately after harvesting. In the study group, harvested livers were flushed with Euro-Collins solution (30 ml/kg body weight) containing LXR-015 at a concentration equivalent to 9 mg/kg animal body weight (300 microg/ml). The livers were then stored at 4 degrees C for 24 hr before liver function was studied. In the control group, harvested livers were flushed with Euro-Collins solution without LXR-015 and then stored at 4 degrees C for 24 hr before liver function was studied. RESULTS: Portal venous flow was higher (P<0.05) in the normal and study groups compared with the control group. Portal venous resistance was lower (P<0.05) in the normal and study groups compared with the control group. Liver tissue oxygen consumption in the study group was significantly higher than in both the normal and control groups (P<0.05). Liver enzyme production (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase) was higher in the control group than in either the study or normal group (P<0.05). Bile production in both the normal and study groups was higher than in the control group (P<0.05). The liver tissue wet to dry weight ratio in both the normal and study groups was lower than in the control group (P<0.05). Histopathology studies revealed fewer apoptotic bodies (P<0.05) in both the normal (1.70+/-0.15 per high-power field) and study groups (2.08+/-0.10 per high-power field) than in the control group (7.92+/-.33 per high-power field). CONCLUSIONS: Adding an antiapoptotic compound, LXR-015, to Euro-Collins solution significantly improves hypothermic preservation of the rat liver compared with Euro-Collins solution alone.

Liver cell proliferation induced by the mitogen ethylene dibromide, unlike compensatory cell proliferation, does not achieve initiation of rat liver carcinogenesis by diethylnitrosamine.

The purpose of this investigation was to determine whether mitogen-induced cell proliferation is as effective as compensatory cell proliferation in achieving initiation of carcinogenesis in rat liver. Male Wistar rats were injected with a single non-necrogenic dose of the hepatocarcinogen diethylnitrosamine (DENA) during the peak of DNA synthesis following the administration of the hepatic mitogen ethylene dibromide (EDB) or a necrogenic dose of CCl4. After subjecting the animals to a promoting procedure, the rats were sacrificed and the initiated hepatocytes were monitored as gamma-glutamyltranspeptidase (gamma-GT) positive foci. The results indicate that while DENA administration during compensatory cell proliferation results in the formation of GT positive foci, no enzyme-altered foci were produced when the carcinogen was given during liver hyperplasia induced by EDB, despite the fact that at the time of carcinogen administration, the extent of cell proliferation, as monitored by thymidine incorporation into DNA, was the same in both the groups.

Dietary orotic acid, a new selective growth stimulus for carcinogen altered hepatocytes in rat.

It was observed that orotic acid (OA), a precursor for pyrimidine nucleotide biosynthesis, when supplied exogenously at 1% level in the diet selectively stimulated the growth of hepatocytes modified by 1,2-dimethylhydrazine (1,2-DMH) to form gamma-glutamyltransferase (gamma-GT) (EC 2.3.2.2) positive islands. Increasing the duration of OA diet from 5 to 10 weeks resulted in an increase in the number of foci from 6 to 14/cm2. Rats that received the carcinogen and basal diet, however, developed only 1-2 foci/cm2. This unique effect of OA can be further accentuated by supplying a liver cell proliferative stimulus, such as a single necrogenic dose of CCl4.

Induction of rat liver glutathione transferase subunit 7 by lead nitrate.

The effect of a single dose of lead nitrate (10 microM/100 g body wt), a hepatic mitogen, on rat liver glutathione transferase (GST) subunit expression was investigated. Using SDS-polyacrylamide gel electrophoresis and Western blot technique evidence for the induction of GST 7-7 is shown. This occurrence is identical to that observed in preneoplastic nodules generated in rat liver by different models of chemical carcinogenesis, suggesting that lead nitrate may be a very simple model for investigation of the mechanism of glutathione transferase 7-7 gene expression in chemical hepatocarcinogenesis.

Studies on the kinetics of expression of cell cycle dependent proto-oncogenes during mitogen-induced liver cell proliferation.

The present study was undertaken to determine the kinetics of DNA synthesis and expression of cell cycle dependent proto-oncogenes in response to two types of cell proliferative stimuli in male Wistar rat liver. The peak of DNA synthesis was approximately 24 h after a compensatory cell proliferative stimulus induced by 2/3 partial hepatectomy and approximately 36 h following a mitogenic stimulus obtained with a single dose of lead nitrate (10 micromol/100 g body wt, through femoral vein). Even though both proliferative stimuli induced the expression of c-fos, c-myc and c-Ha-ras, the extent of the increase in c-fos expression was 4- to 5-fold less in mitogen-induced cell proliferation. In addition, while the expression of c-myc, following partial hepatectomy returned to basal level by 4 h, the induced expression of c-myc persisted for up to 40 h during the lead nitrate-induced liver cell proliferation.

Stimulation of DNA synthesis by rat plasma following in vivo treatment with three liver mitogens.

The present study was undertaken to determine the effect of two different types of liver cell proliferative stimuli, namely compensatory regeneration and direct hyperplasia on DNA synthesis of normal and preneoplastic isolated hepatocytes. Platelet-poor plasma (PPP) isolated from male Wistar rats treated with three different hepato-mitogens, lead nitrate (LN), cyproterone acetate (CPA) and ethylene dibromide (EDB), or subjected to surgical partial hepatectomy (PH), was tested for its ability to stimulate DNA synthesis in normal and preneoplastic hepatocytes in primary cultures. Induction of DNA synthesis was detected as early as 30 min after CPA, EDB and PH administration and persisted up to 5 days after the LN administration. In addition, hepatocytes isolated from preneoplastic liver nodules were also able to respond in culture to the DNA synthesis stimulus induced by these factors.

Ciprofibrate and triiodothyronine do not suppress in vivo induction of placental glutathione S-transferase expression in rat hepatocytes.

Studies on hepatocyte primary cultures have suggested that loss of expression of the placental form of glutathione S-transferase in peroxisome proliferator (PP)-induced hepatocarcinogenesis is due to inhibition of glutathione S-transferase P (GSTP) transcription by the PPs. In the present study, we have analyzed the effect of a PP, ciprofibrate, and of another ligand of nuclear receptors, 3,3', 5-triiodo-L-thyronine (T3), on GSTP mRNA and protein levels in an in vivo model where GSTP expression was induced in Wistar rats by pre-treatment with a single dose of lead nitrate. Results indicate that administration of ciprofibrate or T3, immediately after lead nitrate treatment, did not exert any inhibitory effect on GSTP mRNA and protein levels, as revealed by both Western and immunohistochemical analysis. The results indicate that PPs do not inhibit hepatocyte GSTP expression induced in vivo by lead nitrate and suggest that inhibition of GSTP expression by PPs may not necessarily be the cause for the rapid disappearance of GSTP-positive preneoplastic lesions observed after a short term exposure to these agents.

Compensatory regeneration, mitogen-induced liver growth, and multistage chemical carcinogenesis.

Liver cell proliferation has often been implicated to play a major role during different steps of the carcinogenic process. Most of the experimental studies indicating a close association between cell proliferation and liver cancer development have made use of a compensatory type of proliferative stimulus. However, liver growth may also be caused by direct hyperplasia after administration of primary mitogens. Our recent studies examined the possible differences between these two types of cell proliferation. Our studies indicate that a) increased expression of proto-oncogenes such as c-fos, c-jun, and c-myc is not necessary for entry into the cell cycle during mitogen-induced liver growth; b) mitogen-induced liver growth does not support initiation of chemical hepatocarcinogenesis; c) repeated proliferative stimuli induced by primary mitogens do not stimulate the growth of initiated cells to a focal and/or nodular stage; and d) mitogen-induced liver growth, unlike compensatory regeneration, is followed by a particular mode of cell death, namely, apoptosis. This type of cell death may be responsible for the elimination of carcinogen-initiated cells.

Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis.

During multistage liver carcinogenesis, there is a sequential decrease in gap junctional intercellular communication (GJIC), associated with reduced expression of a major liver gap-junction protein (connexin 32). There are also several lines of evidence indicating that the induction of cell proliferation plays an important role during liver carcinogenesis. The relationship between GJIC and cell proliferation and their roles in liver carcinogenesis are not yet known. Results from various experiments suggest that there is a close relationship between the inhibition of GJIC and stimulation of liver cell proliferation. However, our results also suggest that different stimuli may affect cell proliferation and GJIC differentially by different mechanisms.

Genotoxic and non-genotoxic activities of 2,4- and 2,6-diaminotoluene, as evaluated in Fischer-344 rat liver.

Among aminoaromatics, 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) represent a conflicting couple of isomers; despite showing the same structural alert to DNA reactivity (and thus potential genotoxicity), they are different in terms of carcinogenicity. Of the two, 2,4-DAT alone is a potent rodent carcinogen, the liver being its major target. According to the literature, assays using various short-term genotoxicity tests have not discriminated satisfactorily between the carcinogenic and non-carcinogenic isomer, both chemicals producing overall positive results. To investigate their mechanism of action, we assayed both 2,4-DAT and 2,6-DAT in F-344 rat liver for their ability to induce DNA adducts, as detected by the 32P-postlabelling technique, and to enhance the induction of preneoplastic foci, as detected by GGT-staining in diethylnitrosamine (DENA)-initiated hepatocytes. Our expectation was that, using the correct target/metabolism, a classic genotoxicity assay and an assay detecting non-genotoxic activities could, together, reflect the different carcinogenic behaviour of the two isomers. The results indicate that, at the single equimolar dose of 250 mg/kg i.p., 2,4-DAT was able to induce approximately 6500 times more DNA adducts than 2,6-DAT; the estimated RAL values for the two isomers were 18.6 x 10(-6) and 0.29 x 10(-8), respectively. Moreover, of the two, only 2,4-DAT was able to significantly enhance the growth of DENA-initiated hepatocytes. Indeed, liver sections from rats treated with 2,4-DAT (30 daily doses of 25 mg/kg, i.g.) exhibited an average total number and area of foci of 10.53/cm2 and 1.22 mm2/cm2 vs. 4.46/cm2 and 0.33 mm2/cm2, for their respective controls. By contrast, no effect on the growth of GGT-positive foci was observed when liver sections from rats treated with 2,6-DAT (30 daily doses of 50 mg/kg, i.g.) were scored (5.54 foci per cm2 and total area of 0.42 mm2/cm2). The results indicate that in spite of the structural alert common to the two isomers, 2,4-DAT and 2,6-DAT, only the former appears to significantly affect the carcinogenic process in the liver.

9-cis retinoic acid is a direct hepatocyte mitogen in rats.

We recently suggested that peroxisome proliferators (PP)-induced hepatocyte DNA synthesis may be mediated by a specific peroxisome proliferator activated receptor (PPAR). Heterodimers of the PPAR with the retinoid nuclear receptor, RXR, activate transcription after binding to DR1 response elements of the target genes. DR1 elements are also activated by RXR homodimers formed in the presence of 9-cis retinoic acid (9 cis RA) suggesting that PP and 9 cis RA might regulate an overlapping set of target genes. The present study was therefore designed to test whether 9-cis RA stimulates hepatocyte DNA synthesis. Male Wistar rats were given a single intragastric dose of 9-cis RA (10-100 mg/Kg) or all trans retinoic acid (RA)(200 mg/Kg and 100 mg/Kg), and levels of hepatocyte DNA synthesis after 24 hours were determined by BrdU immunohistochemistry. Effects of 9-cis RA and RA(10(-9)-10(-5)M) on hepatocyte DNA synthesis in primary culture were also examined. Over 10 fold increases in the levels of BrdU incorporation were noted 24 hours after a single dose of 9 cis RA at a dose of 60 and 100 mg/Kg. RA at a dose of 200 mg/Kg induced a 5-6 fold increases in BrdU labeling, while a dose of 100 mg/Kg had no significant effects. Since the RA effect only occurs at higher doses, it may be only after conversion to 9-cis RA. In primary culture of hepatocytes, neither 9-cis RA nor RA with or without EGF had stimulatory effects on hepatocyte DNA synthesis. This is the first report to demonstrate a potent stimulatory effect of 9-cis RA on DNA synthesis of rat hepatocytes in vivo. It is suggested that 9-cis RA exerts this effect through receptor mediated mechanisms similar to PP, both activating genes that regulate hepatocyte proliferation.