Bovine lactoferrin potently inhibits liver mitochondrial 8-OHdG levels and retrieves hepatic OGG1 activities in Long-Evans Cinnamon rats

BACKGROUND/AIMS: To assess the effect of lactoferrin on oxidative liver damage and its mechanism, we used Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant-like hepatitis and lethal hepatic failure. METHODS: Four-week-old female LEC rats were divided into the untreated and treated groups. The latter was fed bovine lactoferrin at 2% mixed with conventional diet. RESULTS: The cumulative survival rates were 75.0% vs. 100% at 14 weeks, 37.5% vs. 91.7% at 15 weeks, and 12.5% vs. 91.7% at 16 weeks, respectively, for untreated and treated rats (P=0.0008). The 8-OHdG levels in liver mitochondrial DNA and malondialdehyde in plasma and liver tissues were significantly lower in treated than untreated rats (P<0.001, =0.017 and 0.034, respectively). Mitochondrial DNA mutations were more common in untreated rats. OGG1 mRNA and protein expression levels were significantly lower in untreated than treated rats (P=0.003 and 0.007, respectively). Hypermethylation of the second CpG island located upstream of OGG1 gene was observed in untreated rats. CONCLUSIONS: Our findings indicated that lactoferrin inhibits oxidative liver damage in LEC rats. Lactoferrin could be potentially useful for the treatment of oxidative stress-induced liver diseases.     

Ethanol-Mediated Fetal Dysmorphology and its Relationship to the Ontogeny of Maternal Liver Metallothionein

Background: Fetal zinc (Zn) deficiency arising from ethanol‐induction of the Zn‐binding protein metallothionein (MT) in the mother’s liver has been proposed as a mechanism of teratogenicity. Here, we determine the ontogeny of MT and Zn homeostasis in rats and mice and then examine the effect of acute ethanol exposure in early embryonic development on this relationship. The protective effect of Zn against ethanol‐mediated fetal dysmorphology is also examined. Methods: Study 1: Maternal liver MT and Zn homeostasis was determined in Sprague–Dawley rats and C57BL/6J mice throughout gestation. Study 2: Rats were administered ethanol (25% in saline, intraperitoneal 0.015 ml/g) or vehicle alone on gestational day (GD) 9. Maternal liver MT and Zn, and plasma Zn was determined over the ensuing 24 hours. Study 3: Pregnant rats were treated with ethanol and Zn (s.c. 2.5 μg Zn/g) on GD9 and fetal dysmorphology was assessed on GD 19. Results: Study 1: Maternal liver MT began to rise around GD 9 peaking on GD 15 before falling to nonpregnant levels around term. The pregnancy‐related increase in MT was associated with a fall in plasma Zn which was significantly lower on GD 15 thereafter returning to nonpregnant levels by parturition. Study 2: Ethanol administered to pregnant rats on GD 9 resulted in a 10‐fold induction of MT in the maternal liver and was associated with a 33% rise in liver Zn and a 30% fall in plasma Zn, 16 hours after treatment. Study 3: Ethanol treatment on GD 9 resulted in a significant increase in craniofacial malformations which were prevented by concurrent Zn treatment. Conclusions: The findings indicate that maternal liver MT levels are lowest in early gestation (before GD 10) making this a sensitive period where ethanol‐induction of MT can affect fetal Zn homeostasis and cause fetal dysmorphology. The study further provides evidence of a protective role for Zn against ethanol‐mediated teratogenicity.     

Dietary copper triggers onset of fulminant hepatitis in the Long-Evans cinnamon rat model

AIM:To investigate the impact of dietary copper given at different time points on the onset of fulminant hepatitis. METHODS:The Long-Evans cinnamon (LEC) rat model of Wilson's disease (WD) was used to study the impact of high dietary copper (hCu) on the induction of fulminant hepatitis at early or late time points of life. High Cu diet was started in rat pups or in adults (month 5) for three months. Animals that received reduced dietary copper (rCu) throughout their lifetime served as a control. Hepatitis-associated serum markers (alanine aminotransferase, aspartate transaminase, bilirubin) were analyzed in animal groups receiving hCu or rCu. Liver copper content and liver histology were revealed at sacrifice. A set of 5 marker genes previously found to be affected in injured liver and which are related to angiogenesis (Vegfa), fat metabolism (Srebf1), ex-tracellular matrix (Timp1), oxidative stress (Hmox1), and the cell cycle (Cdkn1a) were analyzed by real-time polymerase chain reaction. RESULTS:Regardless of the time point when hCu was started, LEC rats (35/36) developed fulminant hepatitis and died. Animals receiving rCu (36/36) remained healthy, did not develop hepatitis, and survived long term without symptoms of overt disease, although liver copper accumulated in adult animals (477 ± 75 μg/g). With regard to start of hCu, onset of fulminant hepatitis was significantly (P 0.001) earlier in adults (35 ± 9 d) that showed pre-accumulation of liver copper as compared to the pup group (77 ± 15 d). Hepatitis-associated serum markers, liver copper and liver histology, as well as gene expression, were affected in LEC rats receiving hCu. However, except for early and rapid onset of hepatitis, biochemical and molecular markers were similar at the early and late time points of disease. CONCLUSION:Rapid onset of fulminant hepatitis in asymptomatic LEC rats with elevated liver copper suggests that there is a critical threshold of liver copper which is important to trigger the course of WD.     

Changes in free radical-metabolizing enzymes and lipid peroxides in the liver of Long-Evans with cinnamon-like coat color rats

We report changes in free radical-metabolizing enzymes and the increased generation of lipid peroxides associated with extreme metal accumulation in the liver of the Long-Evans with cinnamon-like coat color (LEC) rat, a new mutant strain displaying hereditary hepatitis and subsequent hepatocellular carcinoma. The activity of free radical-metabolizing enzymes and lipid peroxides, and the concentration of metal in the liver were determined sequentially after birth. Mn-superoxide dismutase activity significantly increased immediately after the onset of hepatitis in LEC rats, whereas no remarkable change was observed in control rats. Cu, Zn-superoxide dismutase activity in LEC rats was similar to that in control rats. Glutathione reductase activity increased, while glutathione peroxidase activity was lower in LEC rats than in control rats throughout the observation periods. Lipid peroxides, estimated by thiobarbituric acid reaction, also increased 4-to 5-fold immediately after the onset of hepatitis in LEC rats. Copper concentration was 30-to 50-fold higher in the liver of LEC rats than in control rats, and the iron content also increased significantly before and after the onset of hepatitis. These findings suggested that an oxidant injury generated by toxic metals could be one of the factors responsible for hepatocellular damage in this unique hereditary hepatitis.     

Inhibition of hereditary hepatitis and liver tumor development in Long-Evans cinnamon rats by the copper-chelating agent trientine dihydrochloride

Trientine dihydrochloride (trientine) is an alternative medicinal copper chelating agent for patients with Wilson's disease of penicillamine intolerance. We examined the effects of trientine on the spontaneous development of hepatitis and hepatic tumors, by its short- term and long-term administration to Long-Evans cinnamon (LEC) rats with an accumulation of copper in the liver, as animal models of Wilson's disease. Male rats were given trientine in their drinking water at 1500 ppm for 18 weeks, from 6 weeks to 24 weeks of age in short-term experiment, and 1500 ppm for 27 weeks then 750 ppm for 52 weeks, from 8 to 87 weeks of age in the long-term experiment. Development of hepatitis was observed in the control LEC rats at 18 weeks of age. They had high levels of plasma transaminases (glutamic oxaloacetic transaminase [GOT], glutamic pyruvic transaminase [GPT]), and on pathological examination, hepatocyte destruction was observed. Histological findings revealed that short-term administration of trientine inhibited the development of hepatitis remarkably. The plasma GOT and GPT levels of treated animals were only slightly higher than those of normal LEA (Long-Evans with agouti coat color) rats, a sibling line of LEC rats. Copper levels in the liver were decreased by a maximum of 50 percent. In the long-term administration of trientine, the incidence of hepatic cell carcinoma (HCC) in the treated rats was 67 percent that of the untreated LEC rats, and the number of HCCs per rat in the treated group was 0.7 +/- 0.5, being significantly lower as compared with 4.7 +/- 3.5 in the untreated rats. Additionally, the development of cholangiofibrosis in LEC rats was completely prevented by long-term administration of the agent. The copper level in the liver of treated rats was reduced by 33 percent at 87 weeks of age. Development of HCC in LEC rats might be partly, but not totally, because of copper accumulation. No effects on the levels of copper, iron, or zinc in the liver of LEA rats was detected, and no adverse effects were detected in either LEC or LEA rats after both short- and long-term administration of trientine in drinking water. (Hepatology 1996 Apr;23(4):764-70)     

Relationship between copper, zinc and metallothionein in hepatocellular carcinoma and its surrounding liver parenchyma

BACKGROUND/AIM: Accumulation of copper (Cu) in hepatocellular carcinoma (HCC), especially in small tumors, is greater than that in the surrounding liver parenchyma. Metallothionein (MT) is considered to be present as Cu-MT, Zn,Cu-MT or Zn-MT. The aim of this study was to determine the presence and localization of Cu-MT and Zn-MT in HCC and surrounding liver parenchyma. METHODS: In 16 HCC patients, surgically resected specimens including HCC and surrounding liver parenchyma were evaluated. RESULTS: The level of Cu present in small HCC (<4 cm in diameter) was significantly greater than that in the surrounding liver parenchyma (p<0.05). However, the level of Cu in large HCC (>4 cm in diameter) was similar to that in the surrounding liver parenchyma. Analysis by Sephadex G-75 gel filtration revealed that the peak fraction due to Cu was identical to that due to MT in 14 (87.5%) of 16 HCC, the peak fraction due to Cu and Zn was identical to that due to MT in 2 (12.5%) HCC, and the peak fraction due to Zn was identical to that of MT in none of 16 HCC. CONCLUSIONS: Accumulation of Cu in small HCC, in which Cu was present as Cu-MT or Zn, Cu-MT, was greater than that in the surrounding liver parenchyma. Cu accumulation and the presence of MT in the liver may be related to carcinogenesis of HCC, because of the similarity of these findings in the experimental data of Long-Evans rats with a cinnamon-like coat color who develop HCC spontaneously.     

Tetrathiomolybdate in the treatment of acute hepatitis in an animal model for Wilson disease

BACKGROUND/AIMS: Tetrathiomolybdate (TTM) is a potent copper-chelating agent that has been shown to be effective in Wilson disease patients with neurological symptoms. Here, we investigate the potential use of TTM in treating the acute hepatic copper toxicosis in Long-Evans Cinnamon (LEC) rats, an authentic model for Wilson disease. METHODS: After the onset of acute hepatitis, LEC rats were treated once with 10 mg TTM/kg. After 1 and 4 days, parameters of liver toxicity and the subcellular distribution and binding of copper and iron were studied. RESULTS: In 11 out of 12 rats TTM rapidly improved acute hepatitis. Hepatic copper decreased through removal from cytosolic metallothionein and lysosomal metallothionein polymers. The remaining lysosomal copper forms a metallothionein-copper-TTM complex. In an almost moribund rat, however, TTM caused severe hepatotoxicity with fatal outcome. CONCLUSIONS: TTM is effective in treating acute hepatitis in LEC rats when applied before the animals become moribund. TTM appears to act by removing the presumable reactive copper associated to lysosomal metallothionein polymers. The remaining lysosomal copper seems to be inactivated by forming a complex with TTM. Moreover, TTM removes copper from cytosolic copper-containing metallothionein. As a consequence, metallothionein is degraded and the uptake of copper-metallothionein into the lysosomes and the formation of the metallothionein polymer associated copper is reduced.     

Ethanol decreases zinc transfer to the fetus in normal but not metallothionein-null mice

BACKGROUND: Ethanol causes significant teratogenicity in normal (MT+/+) but not metallothionein-null (MT-/-) fetuses. Impaired maternal fetal zinc (Zn) transfer is indicated, because ethanol significantly reduces plasma Zn concentrations in MT+/+ dams while increasing concentrations in MT-/- dams. In this study we examined maternal-fetal Zn homeostasis in response to ethanol in MT+/+ and MT-/- mice and the origins of the increase in plasma Zn in MT-/- mice. METHODS AND RESULTS: Mice were treated with saline or ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) on day 12 of gestation. An additional subcutaneous injection of 65Zn tracer was administered after the second ethanol injection before mice were killed 3 hr later. Maternal liver MT levels were not different between ethanol and saline MT+/+ mice. Both liver Zn and 65Zn levels were higher in MT+/+ mice. Plasma Zn concentrations were higher in MT-/- mice, with MT-/- ethanol-treated mice having levels greater than those of MT-/- saline-treated controls. MT+/+ ethanol-treated fetuses exhibited lower 65Zn transfer and whole Zn concentrations compared with MT+/+ and MT-/- saline and MT-/- ethanol fetuses. So we could examine changes in plasma Zn after ethanol treatment, MT+/+ and MT-/- mice were injected with 65Zn 3 days before they received ethanol treatment. Muscle and skin showed a decrease in 65Zn retention in both genotypes over 3 hr. There was a trend toward greater 65Zn release from skin and muscle at an earlier time in MT-/- mice: 24% vs. 2% decrease (MT-/- vs. MT+/+) for muscle and 28% vs. 15% decrease (MT-/- vs. MT+/+) for skin at 2 hr. CONCLUSIONS: The results show (a) that ethanol interferes with the transfer of Zn to the fetus, and that this is MT dependent, and (b) that the increase in plasma Zn seen in MT-/- mice after ethanol administration is a result of Zn release from the skin and muscle, in the absence of hepatic Zn sequestration.     

Short-term zinc supplementation attenuates Helicobacter felis-induced gastritis in the mouse

BACKGROUND: Mucosal damage by H. pylori infection is mainly caused by neutrophils producing large quantities of reactive oxygen species (ROS). Metallothionein (MT) an intracellular, low-molecular, cysteine-rich protein, which is inducible by dietary zinc (Zn), has been implicated in sequestering ROS. This study examines the effects of Zn supplementation on Helicobacter colonisation and associated gastritis and the relationship with gastric MT levels. METHODS: C57Bl/6 mice were inoculated with either 10(8) H. pylori or H. felis and were infected for 4 weeks or 6 and 12 weeks, respectively. Mice infected with H. pylori (4 weeks) or H. felis (6 weeks) were treated with either Zn acetate (ZnA; 1 mg/ml), or Zn sulphate (ZnSO4; 5 mg/ml) for 2 weeks with 0.1 ml oro-gastric gavage twice daily. H. pylori load and H. felis colonisation density were determined by culture and microscopy, respectively. MT levels and H. felis-induced gastritis were also determined. RESULTS: Zn treatment showed no significant difference in Helicobacter load and gastric MT, however, ZnSO4 treatment showed a significant (p<0.05) increased in gastric MT in H. felis infected mice. Both Zn-treated groups showed a significant (p<0.05) difference in gastritis score in the antrum of the stomach within the basal and submucosal compartments compared to H. felis-infected controls. CONCLUSIONS: We found that H. felis-induced gastritis can be attenuated by short-term treatment of Zn. This observation suggests that Zn alone may be effective for the suppression of gastric mucosal inflammation induced by Helicobacter.     

Lentiviral gene transfer ameliorates disease progression in Long-Evans cinnamon rats: an animal model for Wilson disease

OBJECTIVE: Wilson disease is a copper storage disorder caused by mutations in the ATP7B gene leading to liver cirrhosis. It has previously been shown that lentiviral vectors can govern an efficient delivery and stable expression of a transgene. The aim of this pilot study was to prove the principle of a lentiviral gene transfer in the Long-Evans cinnamon (LEC) rat, an animal model of Wilson disease. MATERIAL AND METHODS: LEC rats were treated either by systemic application of lentiviral vectors or by intrasplenic transplantation of LEC-rat hepatocytes lentivirally transduced with ATP7B. The ATP7B gene expression was analyzed by RT-PCR and immunofluorescence analysis. The therapeutic effect was assessed by analysis of liver histology, serum ceruloplasmin oxidase activity, and liver copper content. RESULTS: Hepatic expression of the transgene was detected at different time-points post-treatment and lasted for up to 24 weeks (end of experiment). Liver copper levels were lowered in all treatment groups compared to untreated LEC rats. Twenty-four weeks after treatment, the area of the examined liver-tissue sections occupied by fibrosis was 48.3-57.9% in untreated LEC rats and 10.7-19.8% in rats treated with cell therapy. In systemically treated rats, only small fibrous septa could be observed. CONCLUSIONS: These data prove for the first time that lentiviral ATP7B gene transfer is feasible in Wilson disease. In our pilot study the systemic approach was more promising in ameliorating disease progression than the transplantation of lentivirally transduced hepatocytes.     

Early cell transplantation in LEC rats modeling Wilson's disease eliminates hepatic copper with reversal of liver disease

BACKGROUND & AIMS: The Long-Evans Cinnamon (LEC) rat is an excellent model of Wilson's disease with impaired copper excretion, hypoceruloplasminemia, and copper toxicosis. We hypothesized that early hepatocyte transplantation would improve copper excretion and liver disease in Wilson's disease. METHODS: Normal syngeneic Long-Evans Agouti rat hepatocytes were transplanted intrasplenically into 2-week-old LEC rats. Untreated LEC pups were controls. Liver repopulation was shown by changes in serum ceruloplasmin, hepatic atp7b messenger RNA, and bile and liver copper levels. Histologic analysis of the liver was performed. RESULTS: Significant copper accumulation and liver disease were observed in 5-month-old LEC rats, with occasional treated rats showing increased bile copper excretion. The liver was repopulated extensively in 10 of 14 treated LEC rats (71%) 20 months after cell transplantation. In these 10 rats, hepatic copper content was virtually normal in 6 rats (53 +/- 12 microg/g liver) and substantially less in 4 others (270 +/- 35 microg/g) compared with elevated liver copper levels in untreated LEC rats (1090 +/- 253 microg/g) (P < 0.001). Changes in serum ceruloplasmin levels, bile copper excretion capacity, and liver histology were in concordance with decreases in liver copper levels. CONCLUSIONS: Transplanted cells proliferated subsequent to the onset of liver injury, and the liver was repopulated over an extended period. Cell transplantation eventually restored copper homeostasis and reversed liver disease without hepatic preconditioning in LEC rats.     

Regional Distribution and Localization of Zinc and Metallothionein in the Intestine of Rats Fed Diets Differing in Zinc Content

Background: Zinc (Zn) is protective and enhances epithelial repair in gut diseases. In this study we investigate the localization and distribution of Zn and its binding protein, metallothionein (MT), in the gut of rats fed diets varying in Zn content. Methods: Male-Sprague Dawley rats were fed low, normal, high, or excess Zn in their diets (10, 100, 400, or 1000 mg Zn/kg, respectively) and killed 7 days later. Blood, liver, and gut tissues were collected. Tissue Zn was determined with atomic absorption spectrophotometery and MT with a Cd/haem affinity assay. Zn and MT were immunohistochemically localized in the small-intestinal wall with zinquin and an anti-MT antibody. Results: Most Zn in the intestinal wall was present in the mucosal scrapings, with 94% membrane-bound and 6% cytosolic, irrespective of dietary Zn. MT levels increased in all gut regions at dietary Zn levels above 100 mg Zn/kg. MT was 40% higher in the ileum than in other gut regions in rats fed low- and normal-Zn diets. The Zn content of the ileum was also 20% higher than that of other gut regions in rats fed low-, normal-, or high-Zn diets. Zn and MT were co-localized in the base of the intestinal crypts, most visibly in the ileum. Conclusion: Mucosal cytosolic Zn and MT concentrations are increased only at high or excessive Zn intakes in all gut regions except the ileum, which can respond to a lower Zn intake. As the cytosolic Zn pool most likely influences mucosal protection and repair mechanisms, it is proposed that an increased MT may indicate the adequacy of oral Zn therapy in gut disease.     

Regional distribution and localization of zinc and metallothionein in the intestine of rats fed diets differing in zinc content.

BACKGROUND: Zinc (Zn) is protective and enhances epithelial repair in gut diseases. In this study we investigate the localization and distribution of Zn and its binding protein, metallothionein (MT), in the gut of rats fed diets varying in Zn content. METHODS: Male-Sprague Dawley rats were fed low, normal, high, or excess Zn in their diets (10, 100, 400, or 1000 mg Zn/kg, respectively) and killed 7 days later. Blood, liver, and gut tissues were collected. Tissue Zn was determined with atomic absorption spectrophotometery and MT with a Cd/haem affinity assay. Zn and MT were immunohistochemically localized in the small-intestinal wall with zinquin and an anti-MT antibody. RESULTS: Most Zn in the intestinal wall was present in the mucosal scrapings, with 94% membrane-bound and 6% cytosolic, irrespective of dietary Zn. MT levels increased in all gut regions at dietary Zn levels above 100 mg Zn/kg. MT was 40% higher in the ileum than in other gut regions in rats fed low- and normal-Zn diets. The Zn content of the ileum was also 20% higher than that of other gut regions in rats fed low-, normal-, or high-Zn diets. Zn and MT were colocalized in the base of the intestinal crypts, most visibly in the ileum. CONCLUSION: Mucosal cytosolic Zn and MT concentrations are increased only at high or excessive Zn intakes in all gut regions except the ileum, which can respond to a lower Zn intake. As the cytosolic Zn pool most likely influences mucosal protection and repair mechanisms, it is proposed that an increased MT may indicate the adequacy of oral Zn therapy in gut disease.     

Lamivudine treatment failure in preventing fatal outcome of de novo severe acute hepatitis B in patients with haematological diseases

BACKGROUND: Patients with malignant haematological diseases administered or no longer receiving immunosuppressive therapy are at high risk of reactivation or de novo hepatitis B infection and fulminant hepatitis. Despite promising results in the treatment of chronic hepatitis and its use in selected patients with acute hepatitis B, there is no consensus on lamivudine treatment in severe acute hepatitis portending a fatal clinical outcome. CASE REPORTS: Of the ten patients with malignant haematological disorders who became infected with the same strain of hepatitis B virus during hospitalisation in a haematology ward, five received lamivudine (and in some cases, ganciclovir and famciclovir). The other patients received only supportive therapy, since deteriorating clinical conditions hampered specific treatment efforts. Eight patients died from acute liver failure and one from a fatal course of the haematological disease; one had a favourable outcome from the therapy. There was no significant difference in terms of survival between the treated and untreated patients. CONCLUSIONS: Although lamivudine has proved promising in the therapy of chronic hepatitis B and of recurrent hepatitis after liver transplantation, its use in de novo severe acute hepatitis should be investigated further, particularly in immunocompromised patients.     

Zinc supplementation at the time of ethanol exposure ameliorates teratogenicity in mice

BACKGROUND: We have previously demonstrated that ethanol teratogenicity in mice is related to the maternal expression of metallothionein (MT), a zinc (Zn)-binding protein. Ethanol induces maternal liver MT, which causes plasma Zn concentrations to decrease as Zn moves into the liver. During pregnancy it is suggested that this change decreases fetal Zn supply and contributes to abnormal development. Here we investigated whether maternal Zn supplementation at the time of ethanol exposure reduces teratogenicity. METHODS: Mice were injected with 25% ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) and ZnSO4 (2.5 microg Zn/g subcutaneously at 0 hr) and were killed over 16 hr to ascertain changes in plasma Zn. Plasma Zn concentrations peaked at 2 hr, where levels were 5-fold normal and then returned toward normal over 14 hr. Pregnant mice were treated in a similar manner on gestation day 8 with saline, saline + Zn, ethanol + Zn, or ethanol alone, and fetal abnormalities were assessed on gestation day 18. RESULTS: External abnormalities were most prevalent in offspring from dams treated with ethanol. Zn treatment at the time of ethanol exposure reduced the incidence of fetal abnormalities to basal levels. Litters from dams treated with ethanol + Zn contained more fetuses and fewer fetal resorption sites compared with those from ethanol-treated dams. CONCLUSIONS: These findings demonstrate that Zn supplementation at the time of ethanol exposure significantly negates the deleterious effects of ethanol on the fetus.     

Androgen dependency of hepatocarcinogenesis in TGFalpha transgenic mice

AIMS/BACKGROUND: Sex difference has been shown to play a major role in susceptibility to the development of hepatocellular carcinoma in humans and rodents. In order to clarify the necessity of androgens in hepatic tumorigenesis in transgenic mice overexpressing transforming growth factor (TGF) alpha (MT42), androgen supplement after castration and the LH-RH analogue, leuprolerin acetate, were tested in an experimental model in MT42. METHODS: Male MT42 mice were castrated and supplemented with dihydrotestosterone (DHT) every three months up to 15 months and hepatic tumorigenesis was observed. Leuprolerin acetate was administered to both male and female MT42 mice once a month from 2 months after birth to 15 months to observe the effect on hepatic tumorigenesis. Northern hybridization was performed to detect messenger RNA (mRNA) of TGFalpha expression and the rate of proliferative cell nuclear antigen (PCNA) staining compared with the castrated and non-treated mice. RESULTS: Castration tended to decrease both body and liver weight in MT42 mice which was then restored by DHT. Untreated MT42 males developed 11 liver tumors in 6 mice. Hormonal treatment including castration and DHT supplementation did not change the expression of TGFalpha-mRNA. Castrated transgenic mice developed 2 liver tumors in 2 out of 6 mice and DHT supplementation after castration restored the number of liver tumors to 9 in 5 of 6 mice. PCNA labelling indexes of liver tumors and adjacent non-tumorous-liver were 7.1% (p<0.05): 0.6% in untreated MT42, 3.2%: 0.2% in castrated MT42 and 10.1% (p<0.05): 0.5% in MT42 with castration and DHT supplementation (significant difference compared with castrated mice). Leuprolerin acetate-treated MT42 males developed one liver tumor in 6 mice compared to MT42 administered with saline as a vehicle control in which group 7 liver tumors in 6 male MT42 were observed. Tumors in castrated-MT42 and leuprolerin treated-MT42 were smaller than those in control MT42 mice. CONCLUSION: TGFalpha related hepatocarcinogenesis and hepatocyte proliferation are increased by androgenic stimulation. Suppression of androgens may be useful for the treatment of TGFalpha related liver tumors.