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.

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.

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.

The effect of ferric-nitrilotriacetic acid on the profile of polyunsaturated fatty acids in the kidney and liver of rats.

Intraperitoneal injection of the iron-complex, ferric-nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage associated with oxidative damage in vivo. Fe-NTA induced a time-dependent decrease of several polyunsaturated fatty acids (PUFA), together with increased conjugated diene values and decreased cellular levels of alpha-tocopherol and glutathione. At the time of maximum detectable oxidation (3 h), after the injection of a sublethal dose of Fe-NTA there were clear reductions in the peak values over the controls for several fatty acids notably, 20:5 (eicosapentaenoic acid) (36%), 22:6 (docosahexanoic acid) (30%), 20:3 n6 (eicosatrienoic acid) (30%) and 20:4 (arachidonic acid) (28%) in the kidney. Fewer fatty acids showed a reduction in their residual values in the liver. 20:5 was reduced by 45% and for the 18:3 n3 and 18:3 n6, reductions of 35%, respectively. The profile of PUFAs is sensitive to the oxidative damage due to Fe-NTA and this may find applications as oxidative biomarker model.

Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions.

Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm(2) of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm(2) in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP(+) foci/cm(2) in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.

Liver cell proliferation induced by nafenopin and cyproterone acetate is not associated with increases in activation of transcription factors NF-kappaB and AP-1 or with expression of tumor necrosis factor alpha.

Our previous studies have shown a different pattern of immediate early gene and growth factor gene expression between compensatory liver regeneration occurring after cell loss/death and direct hyperplasia induced by primary mitogens. In the present study, modifications in the activation of two transcription factors, NF-kappaB and AP-1; steady-state levels of tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA); and induction of the inducible nitric oxide synthase (iNOS) were examined in rat liver during different types of cell proliferation. Compensatory regeneration was induced in male Wistar rats by partial hepatectomy of two thirds (PH) or a necrogenic dose of CCl4 (2 mL/kg), whereas direct hyperplasia was induced by a single administration of the primary mitogens lead nitrate (LN, 100 micromol/kg), cyproterone acetate (CPA, 60 mg/kg), or nafenopin (NAF, 200 mg/kg). Liver regeneration after treatment with CCl4 was associated with an increase in steady-state levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, and induction of iNOS. A strong and prolonged activation of NF-kappaB but not of AP-1 was observed in LN-induced hyperplasia. LN also induced an increase in hepatic levels of TNF-alpha and iNOS mRNA. On the other hand, direct hyperplasia induced by two other primary mitogens, NAF and CPA, occurred in the complete absence of modifications in the hepatic levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, or induction of iNOS, although the number of hepatocytes entering S phase 18 to 24 hours after NAF was similar to that seen after PH. These results add further support to the hypothesis that cell proliferation occurring in the absence of cell loss/death may be triggered by unknown signaling pathways different from those responsible for the transition of hepatocytes from G0 to G1 after PH or cell necrosis.

Hepatocyte proliferation induced by a single dose of a peroxisome proliferator.

In compensatory hyperplasia after partial hepatectomy or liver cell injury, hepatocyte proliferation is triggered by coordinated actions of growth factor such as hepatocyte growth factor and transforming growth factor-alpha and -beta. Initiation of hepatocyte DNA synthesis is preceded by the activation of the set of early growth response genes mediated by enhanced nuclear factor-kappa B binding to DNA. Using an experimental model to induce hepatocyte DNA synthesis in vivo by a single dose of a peroxisome proliferator, which does not induce liver cell necrosis (direct hyperplasia), we investigated whether peroxisome proliferator-induced hepatocyte proliferation involved an induction of known growth factors, an activation of early growth response genes, and nuclear factor-kappa B. A single intragastric administration of 250 mg/kg BR931 (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide) to male wistar rats induced a wave of hepatocyte DNA synthesis starting after 12 hours and peaking at approximately 24 to 36 hours. The response was dose dependent. The treatment also induced the expression of the mRNA for the peroxisomal bifunctional enzyme, one of the peroxisome-related fatty acid beta-oxidation enzymes. Pretreatment of rats with dexamethasone (2 mg/kg) inhibited both hepatocyte DNA synthesis and the induction of the peroxisomal bifunctional enzyme gene. Northern blot analyses of liver RNA during a period preceding the onset of DNA synthesis revealed no induction of hepatocyte growth factor, transforming growth factor-alpha, or tumor necrosis factor-alpha mRNAs. No induction of early growth response genes, liver regeneration factor-1, or c-myc was detected. Furthermore, gel mobility shift assays showed no enhanced nuclear factor-kappa B binding to its DNA consensus sequence after BR931 treatment, whereas control studies demonstrated a distinct increase in binding after partial hepatectomy or lead nitrate treatment. The results suggest that peroxisome-proliferator-induced hepatocyte proliferation may be triggered by signal transduction pathways different from those after partial hepatectomy and that the binding of peroxisome proliferators to their nuclear receptors may play a role in stimulation of DNA synthesis and peroxisome proliferation.