DNA Synthesis In Liver Research Articles

The whole transcriptome effects of the PPAR\u03b1 agonist fenofibrate on livers of hepatocyte humanized mice

BackgroundThe role of PPARα in gene regulation in mouse liver is well characterized. However, less is known about the role of PPARα in human liver. The aim of the present study was to better characterize the impact of PPARα activation on gene regulation in human liver. To that end, chimeric mice containing hepatocyte humanized livers were given an oral dose of 300 mg/kg fenofibrate daily for 4 days. Livers were collected and analyzed by hematoxilin and eosin staining, qPCR, and transcriptomics. Transcriptomics data were compared with existing datasets on PPARα activation in normal mouse liver, human primary hepatocytes, and human precision cut liver slices.ResultsOf the different human liver models, the gene expression profile of hepatocyte humanized livers most closely resembled actual human liver. In the hepatocyte humanized mouse livers, the human hepatocytes exhibited excessive lipid accumulation. Fenofibrate increased the size of the mouse but not human hepatocytes, and tended to reduce steatosis in the human hepatocytes. Quantitative PCR indicated that induction of PPARα targets by fenofibrate was less pronounced in the human hepatocytes than in the residual mouse hepatocytes. Transcriptomics analysis indicated that, after filtering, a total of 282 genes was significantly different between fenofibrate- and control-treated mice (P < 0.01). 123 genes were significantly lower and 159 genes significantly higher in the fenofibrate-treated mice, including many established PPARα targets such as FABP1, HADHB, HADHA, VNN1, PLIN2, ACADVL and HMGCS2. According to gene set enrichment analysis, fenofibrate upregulated interferon/cytokine signaling-related pathways in hepatocyte humanized liver, but downregulated these pathways in normal mouse liver. Also, fenofibrate downregulated pathways related to DNA synthesis in hepatocyte humanized liver but not in normal mouse liver.ConclusionThe results support the major role of PPARα in regulating hepatic lipid metabolism, and underscore the more modest effect of PPARα activation on gene regulation in human liver compared to mouse liver. The data suggest that PPARα may have a suppressive effect on DNA synthesis in human liver, and a stimulatory effect on interferon/cytokine signalling.

Modulation of Rat Liver Regeneration after Partial Hepatectomy by Dietary Cholesterol.

The aim of study was to evaluate impact of long-term dietary cholesterol overload on the cholesterol homeostasis and liver regeneration. Serum lipid parameters, 14C-cholesterol incorporation, liver DNA synthesis and protein expression was determined in partially hepatectomized (PH) rats fed with a standard (SLD) or hypercholesterolemic (CHOL) diet. 29-day intake of CHOL diet before PH produced increase in serum total cholesterol, LDL lipoprotein, and triglyceride concentration. PH provoked decrease in serum total cholesterol and triglyceride concentration in both groups. PH was associated with increase in serum ALT activity more pronounced in CHOL animals. Hepatic DNA synthesis was increased after PH in both groups, but lower in CHOL. Hypercholesterolemic diet reduced the absorption of radiolabelled cholesterol in intestine and then activity in blood and liver. The 14C-cholesterol hepatic activities tend to increase after PH in both groups. CHOL diet produced up-regulation of Acyl-CoA:cholesterol acyltransferase-2 protein expression. PH was associated with increase of LDL receptor and Acyl-CoA:cholesterol acyltransferase-2 protein expression in both dietary groups. Liver regeneration after PH is negatively influenced by CHOL diet. The increased uptake of cholesterol in the liver after PH associated with up-regulation of LDL receptor protein expression suggests preferential use of extrahepatic cholesterol by the liver.

Long-lived Snell dwarf mice display increased proteostatic mechanisms that are not dependent on decreased mTORC1 activity.

Maintaining proteostasis is thought to be a key factor in slowed aging. In several growth-restricted models of long-life, we have shown evidence of increased proteostatic mechanisms, suggesting that proteostasis may be a shared characteristic of slowed aging. The Snell dwarf mouse is generated through the mutation of the Pit-1 locus causing reductions in multiple hormonal growth factors and mTORC1 signaling. Snell dwarfs are one of the longest lived rodent models of slowed aging. We hypothesized that proteostatic mechanisms would be increased in Snell compared to control (Con) as in other models of slowed aging. Using D2O, we simultaneously assessed protein synthesis in multiple subcellular fractions along with DNA synthesis in skeletal muscle, heart, and liver over 2 weeks in both sexes. We also assessed mTORC1-substrate phosphorylation. Skeletal muscle protein synthesis was decreased in all protein fractions of Snell compared to Con, varied by fraction in heart, and was not different between groups in liver. DNA synthesis was lower in Snell skeletal muscle and heart but not in liver when compared to Con. The new protein to new DNA synthesis ratio was increased threefold in Snell skeletal muscle and heart compared to Con. Snell mTORC1-substrate phosphorylation was decreased only in heart and liver. No effect of sex was seen in this study. Together with our previous investigations in long-lived models, we provide evidence further supporting proteostasis as a shared characteristic of slowed aging and show that increased proteostatic mechanisms may not necessarily require a decrease in mTORC1.

Tyrosine/Cysteine Cluster Sensitizing Human γD-Crystallin to Ultraviolet Radiation-Induced Photoaggregation in Vitro

Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. HγD-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs. We investigated the role of the tyrosines of HγD-Crys by replacing pairs with alanines and monitoring photoaggregation using light scattering and SDS-PAGE. Mutating both tyrosines in the Y16/Y28 pair to alanine slowed the formation of light-scattering aggregates. Further mutant studies implicated Y16 as important for photoaggregation. Mass spectrometry revealed that C18, in contact with Y16, is heavily oxidized during UVR exposure. Analysis of multiple mutant proteins by mass spectrometry suggested that Y16 and C18 likely participate in the same photochemical process. The data suggest an initial photoaggregation pathway for HγD-Crys in which excited-state Y16 interacts with C18, initiating radical polymerization.

Potential mechanisms of hepatitis B virus induced liver injury.

Chronic active hepatitis (CAH) is acknowledged as an imperative risk factor for the development of liver injury and hepatocellular carcinoma. The histological end points of CAH are chronic inflammation, fibrosis and cirrhosis which are coupled with increased DNA synthesis in cirrhotic vs healthy normal livers. The potential mechanism involved in CAH includes a combination of processes leading to liver cell necrosis, inflammation and cytokine production and liver scaring (fibrosis). The severity of liver damage is regulated by Hepatitis B virus genotypes and viral components. The viral and cellular factors that contribute to liver injury are discussed in this article. Liver injury caused by the viral infection affects many cellular processes such as cell signaling, apoptosis, transcription, DNA repair which in turn induce radical effects on cell survival, growth, transformation and maintenance. The consequence of such perturbations is resulted in the alteration of bile secretion, gluconeogenesis, glycolysis, detoxification and metabolism of carbohydrates, proteins, fat and balance of nutrients. The identification and elucidation of the molecular pathways perturbed by the viral proteins are important in order to design effective strategy to minimize and/or restore the hepatocytes injury.

The effects of sorafenib on liver regeneration in a model of partial hepatectomy

BackgroundSorafenib is currently approved for advanced hepatocellular carcinoma (HCC) and is presently being studied as an adjuvant treatment for HCC following resection. The effects of sorafenib on liver regeneration have not been clearly defined. Our objective was to identify the effects of sorafenib on liver regeneration in a murine partial hepatectomy (PH) model. Materials and methodsWe performed PH in C57Bl/6 mice treated with a range of sorafenib doses with assessments at several time points. Liver sinusoidal endothelial cells (LSEC) and hepatocyte DNA synthesis and proliferation were assessed with 5-bromo-2′-deoxyuridine (BrdU) and Ki67 by flow cytometry and immunohistochemistry. ResultsTreatment with sorafenib did not result in any deaths following PH. When we measured BrdU uptake to assess DNA synthesis, there was a statistically significant increase at 48 h post-PH for nonfibrotic LSEC following treatment with 60 mg/kg of sorafenib. However, BrdU and Ki67 staining among LSEC and hepatocytes was not significantly affected by sorafenib at any of the other doses or time points. BrdU and Ki67 flow cytometry data correlated with immunohistochemistry findings and postoperative liver weights. ConclusionIn a murine PH model, sorafenib did not alter the repair response of normal or fibrotic livers following PH as measured by changes in liver weight, DNA synthesis, and cellular proliferation. These findings suggest sorafenib administered following hepatic resection does not impair liver regeneration.

Methyleugenol Genotoxicity in the Fischer 344 Rat Using the Comet Assay and Pathway-Focused Gene Expression Profiling

Methyleugenol (MEG), a constituent of human food, induces malignant tumors in multiple tissues of rats and mice. Although MEG forms DNA adducts and induces unscheduled DNA synthesis in rat liver, it is negative in many in vitro genetic toxicity assays. In the present study, we evaluated MEG-induced DNA damage in the rat using (1) the alkaline Comet assay, (2) the oxidative Comet assay, and (3) expression profiling of genes associated with DNA damage pathways. Male F344 rats received single oral doses of 400 or 1000 mg/kg body weight (bw) MEG and DNA damage was assessed by the Comet assay in liver, bladder, bone marrow, kidney, and lung 3 h and 24 h later. MEG failed to produce any increase in DNA damage. In addition, rats were given a single oral dose of 2000 mg/kg bw MEG, and Comet assays were performed with liver, bone marrow, and bladder 1, 3, 6, and 8 h later. With one exception (bone marrow at 8 h), no DNA damage was detected. Enzyme-modified Comet assays were conducted in parallel with standard Comet assays in liver. Whereas no MEG-induced DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in increases in DNA damage at the 6- and 8-h sampling times. Gene expression analysis on the livers from MEG-exposed rats showed significant reduction in genes associated with DNA repair. The results indicate that MEG induces DNA damage in rat liver and that oxidative DNA damages may be partly responsible for the genotoxicity of MEG in rodents.

Effects of peroxisome proliferators on zonated DNA synthesis in rat and mouse liver

Effects of peroxisome proliferators on zonated DNA synthesis in rat and mouse liver

T Cell-Derived Lymphotoxin Regulates Liver Regeneration

The ability of the liver to regenerate hepatic mass is essential to withstanding liver injury. The process of liver regeneration is tightly regulated by distinct signaling cascades involving components of the innate immune system, cytokines, and growth factors. However, the role of the adaptive immune system in regulation of liver regeneration is not well-defined. The role of adaptive immune system in liver regeneration was investigated in lymphocyte-deficient mice and in conditional lymphotoxin-deficient mice. A model of liver regeneration after 70% partial hepatectomy was used, followed by examination of liver pathology, survival, DNA synthesis, and cytokine expression. We found that mice deficient in T cells show a reduced capacity for liver regeneration following partial hepatectomy. Furthermore, surface lymphotoxin, provided by T cells, is critical for liver regeneration. Mice specifically deficient in T-cell lymphotoxin had increased liver damage and a reduced capacity to initiate DNA synthesis after partial hepatectomy. Transfer of splenocytes from wild-type but not lymphotoxin-deficient mice improved liver regeneration in T cell-deficient mice. We found that an agonistic antibody against the lymphotoxin beta receptor was able to facilitate liver regeneration by reducing liver injury, increasing interleukin-6 production, hepatocyte DNA synthesis, and survival of lymphocyte-deficient (Rag) mice after partial hepatectomy. The adaptive immune system directly regulates liver regeneration via a T cell-derived lymphotoxin axis, and pharmacological stimulation of lymphotoxin beta receptor might represent a novel therapeutic approach to improve liver regeneration.

Species-Specific Kinetics and Zonation of Hepatic DNA Synthesis Induced by Ligands of PPARα

Peroxisome proliferator-activated receptor alpha (PPARalpha) ligands evoke a profound mitogenic response in rodent liver, and the aim of this study was to characterize the kinetics of induction of DNA synthesis. The CAR ligand, 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene, caused induction of hepatocyte DNA synthesis within 48 h in 129S4/SvJae mice, but the potent PPARalpha ligand, ciprofibrate, induced hepatocyte DNA synthesis only after 3 or 4 days dosing; higher or lower doses did not hasten the DNA synthesis response. This contrasted with the rapid induction (24 h) reported by Styles et al., 1988, Carcinogenesis 9, 1647-1655. C57BL/6 and DBA/2J mice showed significant induction of DNA synthesis after 4, but not 2, days ciprofibrate treatment. Alderley Park and 129S4/SvJae mice dosed with methylclofenapate induced hepatocyte DNA synthesis at 4, but not 2, days after dosing and proved that inconsistency with prior work was not due to a difference in mouse strain or PPARalpha ligand. Ciprofibrate-induced liver DNA synthesis and growth was absent in PPARalpha-null mice and are PPARalpha dependent. In the Fisher344 rat, hepatocyte DNA synthesis was induced at 24 h after dosing, with a second peak at 48 h. Lobular localization of hepatocyte DNA synthesis showed preferential periportal induction of DNA synthesis in rat but panlobular zonation of hepatocyte DNA synthesis in mouse. These results characterize a markedly later hepatic induction of panlobular DNA synthesis by PPARalpha ligands in mouse, compared to rapid induction of periportal DNA synthesis in rat.

The Influence of Repeated Blood Withdrawals before Surgery on Clinical Outcome

The aim of this study was to find the influence of blood withdrawals and diet iron on elective surgery. Male Wistar rats (n=24) were divided: 1. group (SLD) ate standard laboratory diet (SLD), 2. group (FE) an iron enriched diet (FE) with one blood withdrawal after 9 weeks. 3. group (SLD-w) SLD and 4. group (FE-w) ate the FE diet; with 9 withdrawals once a week. The rats were sacrificed 18 hour after partial hepatectomy (PH) in the 10th week. Liver DNA synthesis (3H-thymidin - kBq/mg DNA) was performed. Serum hepcidin (pg/ml), iron concentration, respiratory burst of polymorfonucleares (RB, spontaneous; stimulated, %), count of blood cells were determined. FE-w had a higher (2.36+/-0.36) liver DNA synthesis after PH vs. SLD (1.21+/-0.49). Higher hemoglobin in erythrocytes (pg) was in FE-w and SLD-w vs. FE and SLD. PMN count in SLD-w, FE-w increased vs. SLD, FE. Hepcidin after PH decreased in SLD (78.0), FE (68.0), FE-w (97.0), but increased in SLD-w (217). Serum iron increased in SLD-w. RB after PH increased in FE-w (4.5; 47.6) vs. SLD (1.15; 29.1), FE (3.20;17.8), SLD-w (3.30;13.7). The iron diet with stimulation of haematopoesis by withdrawals improves an organism's condition expressed as better response to elective surgery and better PMN functions.

An autoradiographic study of cellular proliferaton, DNA synthesis and cell cycle variability in the rat liver caused by phenobarbital-induced oxidative stress: the protective role of melatonin.

The protective effect of melatonin against phenobarbital-induced oxidative stress in the rat liver was measured based on lipid peroxidation levels (malondialedyde and 4-hydroxyalkenals). Cellular proliferation, DNA synthesis and cell cycle duration were quantitated by the incorporation of 3H-thymidine, detected by autoradiography, into newly synthesized DNA. Two experiments were carried out in this study, each on four equal-sized groups of male rats (control, melatonin [10 mg/kg], phenobabital [20 mg/kg] and phenobarbital plus melatonin). Experiment I was designed to study the proliferative activity and rate of DNA synthesis, and measure the levels of lipid peroxidation, while experiment II was for cell cycle time determination. Relative to the controls, the phenobarbital-treated rats showed a significant increase (P < 0.01) in the lipid peroxidation levels (30.7%), labelling index (69.4%) and rate of DNA synthesis (37.8%), and a decrease in the cell cycle time. Administering melatonin to the phenobarbital-treated rats significantly reduced (P < 0.01) the lipid peroxidation levels (23.5%), labelling index (38.2%) and rate of DNA synthesis (29.0%), and increased the cell cycle time. These results seem to indicate that the stimulatory effect of phenobarbital on the oxidized lipids, proliferative activity, kinetics of DNA synthesis and cell cycle time alteration in the liver may be one of the mechanisms by which the non-genotoxic mitogen induces its carcinogenic action. Furthermore, melatonin displayed powerful protection against the toxic effect of phenobarbital.

Portal Blood Flow Regulates Volume Recovery of the Rat Liver after Partial Hepatectomy: Molecular Evaluation

Background/Aim: Liver regeneration is a finely tuned process that is closely regulated by multiple cell cycle steps. Although the portal blood flow affects liver regeneration, the molecular mechanism by which the blood flow regulates gene expression and liver function is largely unknown. The aim of this study was to investigate the molecular effect of portal blood flow on hepatocyte proliferation and gene regulation during liver regeneration. Materials and Methods: We developed a simple surgical rat model to investigate the relation between portal blood flow and liver regeneration by partially ligating the portal trunk with 8-0 Proline sutures under microscopy to reduce the blood flow by 40%. We investigated recovery of liver volume, DNA synthesis, and gene expression associated with cell cycle regulators, comparing partially hepatectomized (PH) rats without (PH group; n = 30) and with partial portal ligation (PHPL group; n = 30) for 7 days after the operation. Results: The hepatic tissue blood flow and the recovery ratio between liver weight and body weight in the PHPL group were significantly lower than in the PH group after hepatectomy. The peak 5-bromo-2′-deoxyuridine labeling index in the PHPL group was delayed and weak compared with the PH group. The expression of CT-1 and cyclin D, E, and B mRNAs indicated that the liver regeneration in the PHPL group was delayed and weak. In addition, there was reciprocal expression of C/EBPα and C/EBPβ mRNAs, an observation supported by their nuclear protein levels. Furthermore, the cytochrome P-450 protein level in the PHPL group was higher than that in the PH group 1 day after hepatectomy. Conclusion: The portal blood flow regulates the activity of liver regeneration and the gene expression associated with cell cycle regulators, while the functions are maintained.

Styrene monomer does not induce unscheduled DNA synthesis in the mouse liver following inhalation exposure

Styrene monomer is a commercially important chemical used extensively in the production of plastics. It has been shown to induce lung tumours in the mouse via the inhalation route. Styrene monomer has shown a low reactivity with DNA and also a lack of genotoxic response in the mouse lung in vivo. Together with the fact that the mouse lung tumours were late occurring and mostly benign, which suggest a promotional effect rather than initiation, these factors have led to a suggestion that the tumours may not be of genotoxic origin. The studies examining the genotoxicity of styrene monomer in vivo have to date been predominantly cytogenetic assessments, although low levels of DNA adducts have been reported in the mouse liver and lung. In order to extend this evaluation, a mouse liver unscheduled DNA synthesis study has been performed to assess the ability of styrene monomer to induce DNA damage/repair. The negative response observed in this assay is consistent with the theory that tumours observed in mouse oncogenicity studies are non-genotoxic in origin.

IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF)

Several lines of evidence suggest a role of insulin-like growth factor I (IGF-I) in the regulation of apoptosis. Up to now its impact on many specific cells is unknown. We therefore studied the effect of IGF-I on two similar mesenchymal matrix-producing cell types of the liver, the hepatic stellate cells (HSC) and the myofibroblasts (rMF). The present study aimed to reveal the influence of IGF-I on cell cycle and apoptosis of HSC and rMF and to elucidate responsible signaling. While IGF-I significantly increased DNA synthesis in HSC, cell number decreased and apoptosis increased. In rMF IGF-I also increased DNA synthesis, which is, however, followed by proliferation. Blocking extracellular signal regulating kinase (ERK) revealed that in HSC, bcl-2 upregulation and bax downregulation are effected downstream of ERK, whereas downregulation of NFκB and consecutive of bcl-xL is mediated upstream. In the rMF upregulation of both, the antiapoptotic bcl-2 and bcl-xL is mediated upstream of ERK. The expression of the proapoptotic bax is not regulated by IGF-I in rMF. The studies demonstrate a completely different effect and signaling of IGF-I in two morphologically and functionally similar matrix-producing cells of the liver.

Induction of hepatocyte proliferation by retinoic acid.

Retinoids have been shown to exert an anticarcinogenic effect through suppression of the cell cycle, induction of apoptosis and/or differentiation. In rat liver, in particular, retinoic acid has been shown to inhibit regeneration after partial hepatectomy, most probably through repression of the expression of c-fos and c-jun. Surprisingly enough, in spite of the proposed therapeutic effects of all-trans retinoic acid (tRA) no data are available on its effect on normal adult liver. Here, we show that tRA administration in the diet (150 mg/kg) increased DNA synthesis in mouse liver, at 1 and 2 weeks, with a return to control values at 4 weeks (labelling index was 16.5, 8.3 and 3.3%, respectively, versus control values of 1.4, 1.3 and 2.5%). Increase in mitotic index paralleled that of bromodeoxyuridine incorporation. Kinetic studies showed that entry into S phase began between 24 and 48 h, with a peak between 96 and 120 h. Histological observation of the liver and biochemical evaluation of the levels of serum glutamate-pyruvate transaminases did not reveal any evidence of cell death demonstrating that increased DNA synthesis was not due to tRA-induced liver damage and regeneration, but rather the consequence of a direct mitogenic effect. In addition, analysis of total hepatic DNA content after a 7-day treatment showed a significant increase in tRA-fed mice compared with controls (21.11 mg/100 g body wt in tRA-fed mice versus 15.67 mg/100 g body wt of controls). Hepatocyte proliferation in tRA-fed mice was associated with increased hepatic levels of cyclin D1, E and A, and enhanced expression of the member of pRb family, p107. In conclusion, the results showed that tRA induces hepatocyte proliferation in the absence of cell death, similarly to other ligands of steroid/thyroid hormone nuclear receptor superfamily. The mitogenic effect of tRA cautions about its possible use for antitumoral purposes in liver carcinogenesis.

Use of radiolabelled iododeoxyuridine as adjuvant treatment for experimental tumours of the liver.

The aim of the study was to determine whether hepatic regeneration stimulates growth of tumour residing within the liver, and whether a difference in the rate of DNA synthesis in liver and tumour may be used to target cancer using the radiolabelled thymidine analogue 5-iodo-2'-deoxyuridine (IUdR). Partial hepatectomy was performed on Buffalo rats bearing solitary nodules of syngeneic Morris hepatoma. Liver and tumour DNA synthesis was measured by incorporation of radioactive IUdR. [(125)I]IUdR was tested as an adjuvant therapy after hepatectomy in Buffalo rats bearing diffuse microscopic Morris hepatomas to simulate the clinical situation. Liver regeneration enhanced liver and tumour DNA synthesis as measured by incorporation of radioactive IUdR. Liver DNA synthesis returned to baseline by 7 days, whereas tumour DNA synthesis remained above baseline level. Hepatectomy enhanced the growth of microscopic liver tumours. [(125)I]IUdR (250 micro Ci or 1 mCi/kg) administered 4 days after hepatectomy significantly reduced tumour growth without signs of systemic toxicity or liver dysfunction. The local environment of the regenerating liver stimulates tumour growth. The thymidine analogue [(125)I]IUdR may be used preferentially to target tumour DNA synthesis in the regenerating liver, and may prove useful as an adjuvant therapy for hepatic tumours after surgical resection.

In vivo genotoxicity studies with 3-monochloropropan-1,2-diol.

3-Monochloropropan-1,2-diol (3-MCPD) is a contaminant of polyamine flocculants used in the production of drinking water, but more significantly for human exposure it can arise also in certain foodstuffs containing acid-hydrolysed vegetable protein. It is carcinogenic in the rat, producing tumours in males in the testes, mammary gland and the preputial gland and also kidney tumours in both sexes. It has given positive results in in vitro mutagenicity studies, but there have been no satisfactorily conducted in vivo studies in somatic cells published in the peer reviewed literature. As a result, and because of the absence of appropriate in vivo evidence, several international regulatory agencies had previously judged it prudent to assume that 3-MCPD possessed mutagenic activity in vivo and considered 3-MCPD to be a genotoxic carcinogen. We present in this paper results from two in vivo mutagenicity studies with 3-MCPD, namely a bone marrow micronucleus test in the rat and unscheduled DNA synthesis in the rat liver, both conducted in accordance with relevant OECD protocols. These studies show that 3-MCPD does not possess genotoxic activity in vivo in the tissues examined. On the basis of these findings, and along with evidence that tumours may be induced by mechanisms involving either hormonal disturbances or sustained cytotoxicity, we believe that 3-MCPD may now be considered to be carcinogenic to rodents via a non-genotoxic mechanism.

Retrorsine: a kinetic study of its influence on rat liver regeneration in the portal branch ligation model

Background: Retrorsine, a naturally occurring pyrrolizidine alkaloid, impairs liver regeneration after partial hepatectomy by mechanisms that are still unclear. Aim: The aim of the study was to clarify the influence of retrorsine on cell cycle progression in the regenerating liver lobes of rats after portal branch ligation (PBL). Methods: Liver weight, protein and DNA contents, DNA synthesis (5′-bromodeoxyuridine (BrdU) incorporation) and cellular levels of Cyclin E, CDK-2, CDK-4 and proliferating cell nuclear antigen (PCNA) were assessed before and 24, 48, 72 and 168 h after PBL. Results: Before surgery, higher levels of cyclin E, CDK-2, CDK-4 and PCNA as well as BrdU incorporation were found in the liver of retrorsine-treated rats than in untreated rats. Liver weight gain, protein and DNA synthesis as well as induction of cell cycle related proteins were all strongly impaired by retrorsine in the regenerating lobes after PBL. Conclusions: In conclusion, retrorsine impairs liver regeneration in the PBL model not only by an S or G2/M phase block, but also by a block located before the G1/S transition of the cell cycle.

The mitogenic activity of the liver growth factor is mediated by tumor necrosis factor alpha in rat liver

Background/Aims: Liver growth factor (LGF) is a hepatic mitogen, however, the hepatic stimulation pathway remains to be characterized. The aim of this study was to determine whether tumor necrosis factor alpha (TNF-α) stimulation constitutes a step in the mitogenic pathway of LGF. Methods: Rats were injected with 4.5 μg LGF/rat, and LGF activity was measured both by liver DNA synthesis stimulation and ‘proliferating cell nuclear antigen (PCNA)-positive’ hepatocytes in rats injected with LGF or +anti-TNF-α. TNF-α expression was evaluated by reverse-transcription polymerase chain reaction. TNF-α-producing cells were immunodetected. Human endothelial cells (HUVEC) were stimulated by LGF. TNF-α was detected in the supernatant, and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial adhesion molecule-1 (VCAM-1) by flow cytometry analysis. Results: LGF-injected rats showed higher intrahepatic TNF-α expression. DNA synthesis and PCNA-positive hepatocytes induced by LGF were inhibited by anti-TNF-α, PCNA-positive hepatocytes being especially abundant around the central vein when LGF was injected alone, but TNF-α exhibited increased signal intensity in endothelial cells of the portal vein. LGF stimulated TNF-α secretion in HUVEC, but did not stimulate ICAM-1 or VCAM-1 up-regulation. Conclusions: The mitogenic cascade initiated by LGF in rat liver in vivo depends, at least in part, on TNF-α stimulation. Portal vein endothelial cells seem to be a source of TNF-α.