Evaluation of the 13C-octanoate breath test as a surrogate marker of liver damage in animal models.

BACKGROUND: Octanoate (also known as sodium octanoate), a medium-chain fatty acid metabolized in the liver, is a potential substrate for non-invasive breath testing of hepatic mitochondrial beta-oxidation. METHODS: We evaluated the 13C-octanoate breath test (OBT) for assessing injury in acute hepatitis and two rat models of liver cirrhosis, first testing octanoate absorption (per os or intraperitoneally (i.p.)) in normal rats. We then induced acute hepatitis with thioacetamide (300 mg/kg/i.p., 24-h intervals). Liver injury end points were serum aminotransferase levels and 13C-OBT (24 and 48 h following initial injection). Thioacetamide (200 mg/kg/i.p., twice per week, 12 weeks) was used to induce liver cirrhosis. OBT and liver histological assessment were performed every 4 weeks. Bile duct ligation (BDL) was used to induce cholestatic liver injury. We completed breath tests with 13C-OBT and 13C-methacetin (MBID), liver biochemistry, and liver histology in BDL and sham-operated rats (baseline, 6, 14, 20 days post-BDL). RESULTS: Octanoate absorbs well by either route. Peak amplitudes and cumulative percentage dose recovered at 30 and 60 min (CPDR30/60), but not peak time, correlated with acute hepatitis. Fibrosis stage 3 at week 8 significantly correlated with each OBT parameter. Cholestatic liver injury (serum bilirubin, ALP, gamma-GT, liver histology) was associated with significant suppression of the maximal peak values and CPDR30/60, respectively (P<0.05),using MBID but not 13C-octanoate. CONCLUSIONS: OBT is sensitive for potentially evaluating liver function in rat models of acute hepatitis and thioacetamide-induced liver cirrhosis but not in cholestatic liver injury. The MBID test may be better for evaluation of cholestatic liver disease in this model.

Utility of a 13C-methacetin breath test in evaluating hepatic injury in rats.

BACKGROUND AND AIM: Methacetin is thought to be a good substrate for the evaluation of different cytochrome P450 enzymatic systems of liver microsomes because of its rapid metabolism and lack of toxicity in small doses. Recent studies indicate that a methacetin breath test may be a non-invasive alternative for the evaluation of liver function since it correlates well with the severity of liver damage. It may also discriminate between different stages of liver cirrhosis and correlates with the Child-Pugh score. The application of this test in experimental liver damage in animal models has not yet been examined. This study aimed to evaluate the efficacy of the (13)C-methacetin breath test in assessing the extent of hepatic injury in models of acute liver failure, liver cirrhosis, and fatty liver in rats. METHODS: Absorption of methacetin given per os or intraperitoneally in normal rats was evaluated. The association between liver mass and (13)C-methacetin breath test results was assessed in a 70% hepatectomy rat model. Fulminant hepatic failure was induced by three consecutive intraperitoneal injections of thioacetamide, 300 mg/kg, at 24 h intervals. For induction of liver cirrhosis, rats were given intraperitoneal injections of thioacetamide, 200 mg/kg, twice a week for 12 weeks. A methionine-choline deficient diet was used for the induction of fatty liver. Rats were analyzed for (13)C-methacetin by BreathID (MBID) using molecular correlation spectrometry. BreathID continuously sampled the animal's breath for 60 min and displayed the results on the BreathID screen in real-time. RESULTS: Methacetin was absorbed well irrespective of the administration method in normal rats. Liver mass was associated with peak amplitude, complete percent dose recovery (CPDR) at 30 and 60 min and MBID peak time. A high degree of association was also demonstrated with MBID results in acute hepatitis (peak amplitude, 19.6 +/- 3.4 vs 6.3 +/- 1.63.4; CPDR30, 6.0 +/- 3.3 vs 1.2 +/- 0.5; CPDR60, 13.3 +/- 4.5 vs 3.2 +/- 1.4; and peak time, 31.0 +/- 14.9 vs 46.9 +/- 10.8 min) and liver cirrhosis (peak amplitude, 24.4 +/- 2.3 vs 15.6 +/- 6.4; CPDR30, 7.9 +/- 1.2 vs 2.7 +/- 1.0; CPDR60, 17.8 +/- 2.6 vs 8.8 +/- 2.1; and peak time, 30.2 +/- 1.5 vs 59.6 +/- 14.5 min), but not with grade of liver steatosis. CONCLUSIONS: Methacetin is well absorbed and exclusively metabolized in the liver. MBID is a sensitive test and may be a useful tool for the evaluation of functional liver mass in animal models of acute liver failure and cirrhosis. However, MBID could not distinguish between fatty liver and normal liver in rats.

The cyclin-dependent kinase inhibitor, p27, has no correlation with the malignant potential of GIST.

BACKGROUND: Tumor size and mitotic activity are characteristically associated with the malignant potential and prognosis of gastrointestinal stromal tumors (GIST). However, since neither small tumor size nor low mitotic activity can rule out malignancy, additional factors that may predict malignant behavior have been suggested. AIM: To evaluate the correlation between the cyclin-dependent kinase inhibitor (CDI), p27kip1, expression and the malignant potential of GIST. METHODS: Serial sections were evaluated by immunohistochemistry after staining with antibodies against p27/Kip1 and Ki-67 in surgical material obtained from 36 patients with GIST. p27kip1 staining intensity and the percentage of positive cells were investigated for association with the malignancy risk, pathological features, overall survival, and disease-free survival. Histologic grade was assigned by spindle vs. epithelioid cell histology, mucosal invasion, tumor cell necrosis or atypia and the number of mitoses. RESULTS: In the multivariate model, p27kip1 expression was not significantly associated with any of the variables examined except Kit protein-CD117 (r = 0.37, p = 0.03). Comparative evaluation of p27kip1 expression in GIST cells revealed higher levels of p27kip1 in patients who died compared to those who survived (2.04 +/- 0.54 vs. 1.3 +/- 0.99, p = 0.1). CONCLUSION: The CDI p27kip1 was not associated with malignancy of GISTs and did not serve as a predictor of survival.

Curcumin ameliorates acute thioacetamide-induced hepatotoxicity.

BACKGROUND AND AIM: Increased production of reactive oxygen species and nitric oxide and activation of nuclear factor kappa B are implicated in the pathogenesis of various liver diseases, including fulminant hepatic failure. Curcumin is a naturally occurring anti-oxidant that reduces oxidative stress and inhibits nuclear factor kappa B and nitric oxide formation. The aim of the present study is to assess curcumin's therapeutic potential in acute thioacetamide hepatotoxicity, a rat model of fulminant hepatic failure. METHODS: Fulminant hepatic failure was induced by two intraperitoneal (i.p.) injections of 300 mg/kg thioacetamide (TAA) at 24-h intervals. The experimental groups received a low-dose (200 mg/kg per day, i.p.) or a high-dose (400 mg/kg per day) of curcumin, initiated 48 h prior to the first TAA injection. A fourth group was administered neither TAA nor curcumin and served as a control. RESULTS: The survival rate was higher in both curcumin-treated groups compared to the TAA only treated group. Biochemical parameters of liver injury, blood ammonia and hepatic necroinflammation were lower in the low-dose curcumin group compared to TAA controls, and were further reduced in the high-dose group (P < 0.05 and P < 0.01, respectively). Curcumin treatment also reduced the TAA-induced elevated hepatic levels of thiobarbituric acid-reactive substances (TBARS), and inhibited the nuclear binding of nuclear factor kappa B (NFkappaB) and inducible nitric oxide (iNOS) protein expression. CONCLUSIONS: Curcumin improved survival and minimized oxidative stress, hepatocellular injury and hepatic necroinflammation, NFkappaB binding and iNOS expression in a rat model of FHF. These findings support the role of ROS, NFkappaB and iNOS in mediating liver insult due to TAA, and that of curcumin as a hepato-protectant.

p27kip1 expression is inversely related to the grade of gastric MALT lymphoma.

BACKGROUND: Decreased or lost expression of the cyclin-dependent kinase inhibitor p27kip1 protein has been found to be a poor prognostic factor in many cancers, including gastric cancer. AIM: To evaluate p27kip1 expression in gastric mucosa-associated lymphoid tissue (MALT) and gastric B-cell lymphoma. METHODS: Fifty-two cases of gastric lymphoma, mean age 68.7 yr (range 23-90 yr), 11 of chronic Helicobacter pylori-associated gastritis, and 5 of normal gastric mucosa were studied. Patients were classified into two groups. Stage IE gastric lymphomas were defined as local gastric lymphoma of MALT and more advanced stages as advanced gastric lymphoma. Twenty-three patients diagnosed as stage IE, 13 of these were low-grade and 10 diffuse large B-cell lymphoma (DLBL). Twenty-nine patients were at stage IIE or above, 18 with low-grade and 11 with DLBL. Serial sections were evaluated by immunohistochemistry after staining with antibodies against p27/Kip1 and Ki-67. RESULTS: The proliferative index was higher in gastric DLBL than in low-grade MALT lymphomas, 57.1+/-31.2 vs 17.3+/-20.6 (p=0.0001). The mean p27kip1 expression score for high-grade patients was significantly lower compared with that of low-grade patients, 0.5+/-0.4 and 1.6+/-0.8, respectively (p=0.001). Comparative evaluation of p27kip1 expression in malignant lymphoid cells revealed that B cells of the localized gastric DLBL patients expressed the least p27kip1, 0.36+/-0.32. This value was lower than that of malignant lymphoid cells of patients with advanced DLBL, 0.64+/-0.53, advanced low-grade MALT lymphoma, 1.59+/-0.79, and localized low-grade MALT lymphoma, 1.59+/-0.84. In the multivariate model in which all p27kip1 variables were entered, the expression of p27kip1 in malignant lymphoid cells was inversely correlated with the grade of the lymphoma irrespective of the stage of the disease (p=0.0001), and significantly predicted grade: OR:0.07, 95% CI 0.07-0.31, p=0.0001. CONCLUSION: p27kip1 may be a putative distinct molecular marker to differentiate between low-grade and high-grade gastric lymphoma.

Thioacetamide-induced hepatic damage in a rat nutritional model of steatohepatitis.

BACKGROUND:: Nonalcoholic steatohepatitis is most often attributed to the effects of obesity, hyperlipidemia, diabetes mellitus and drugs. It is still unknown whether livers with steatohepatitis are more vulnerable to toxic damage. AIM:: To determine the effect of the hepatotoxicant thioacetamide in a rat nutritional model of hepatic steatohepatitis. METHODS:: Steatohepatitis was induced in rats by placing them on a methionine-choline deficient diet for 1 month. Thioacetamide was administered by three consecutive intraperitoneal injections (300mg/kg) at 24h intervals. RESULTS:: Following treatment with thioacetamide, the elevated serum levels of liver enzymes and blood ammonia, liver necroinflammation and the survival rate after 48h were not different between rats with normal or fatty liver. However, those parameters were significantly worse when steatohepatitis regressed after return to normal diet for 1 month (P < 0.01). Western blot analysis of hepatic extracts revealed no difference in cytochrome P4502E1 levels between livers with steatohepatitis and steatohepatitis after regression, suggesting that the enhanced hepatotoxicity after regression of steatohepatitis could not be attributed to increased cytochrome P4502E1. CONCLUSIONS:: In a nutritional model of steatohepatitis, rats with fatty liver were not more vulnerable than normal rats to liver damage induced by thioacetamide. However, liver damage was significantly more severe in rats with steatohepatitis after 1 month regression.

Melatonin inhibits nuclear factor kappa B activation and oxidative stress and protects against thioacetamide induced liver damage in rats.

BACKGROUND/AIMS: Free radical-mediated oxidative stress has been implicated in the pathogenesis of acute liver injury. The aim of our study was to investigate whether melatonin, a potent free radical scavenger could prevent fulminant hepatic failure in rats. METHODS: Liver damage was induced by two consecutive injections of thioacetamide (TAA, 300 mg/kg/i.p.) at 24 h intervals. Treatment with melatonin (3 mg/kg/daily, i.p) was initiated 24 h prior to TAA. RESULTS: Twenty-four h after the second TAA injection, serum liver enzymes and blood ammonia were lower in rats treated with TAA+melatonin compared to TAA (P<0.001). Liver histology was significantly improved and the mortality in the melatonin-treated rats was decreased (P<0.001). The increased nuclear binding of nuclear factor kappa B in the livers of the TAA-treated rats, was inhibited by melatonin. The hepatic levels of thiobarbituric acid reactive substances, protein carbonyls and inducible nitric oxide synthase were lower in the TAA+melatonin-treated group (P<0.01), indicating decreased oxidative stress and inflammation. CONCLUSIONS: In a rat model of TAA-induced fulminant hepatic failure, melatonin improves survival and reduces liver damage and oxidative stress. The results suggest a causative role of oxidative stress in TAA-induced hepatic damage and suggest that melatonin may be utilized to reduce liver injury associated with oxidative stress.

Glycine modulates cytokine secretion, inhibits hepatic damage and improves survival in a model of endotoxemia in mice.

BACKGROUND AND AIM: There is substantial experimental evidence that the amino acid glycine may have a role in protecting tissues against insults such as ischemia, hypoxia and reperfusion. Our aim was to investigate the ability of the amino acid glycine to prevent hepatic damage induced by injection of lipopolysaccharide and d-galactosamine (d-Gal), to modulate pro- and anti-inflammatory cytokine levels, and to improve survival. METHODS: Mice were challenged with an intraperitoneal injection of d-Gal (16 mg/mouse) and lipopolysaccharide (LPS, 1 microg/mouse). The intervention group also received an intraperitoneal injection of glycine (150 mg/kg) in two doses: 24 h before and just after LPS challenge. Serum cytokine levels were measured 2 h after challenge, and liver enzymes and histology were determined 16 h after LPS. Separate groups of mice received the same treatment and the survival rate was determined 24 h and ten days after endotoxin administration. In in vitro experiments, cultured mononuclear cells were stimulated by LPS, and TNF-alpha and IL-10 secretion were measured, in the presence or absence of glycine. RESULTS: In the glycine-treated mice, the serum levels of liver enzymes and TNF-alpha, the histologic necroinflammation score and the mortality rate were significantly reduced compared to control mice (P<0.001). Serum IL-10 levels in the glycine-treated mice were increased (P<0.01). In vitro studies in cultured lymphocytes isolated from either normal or glycine pretreated mice, demonstrated a significant and dose-dependent inhibition of LPS-induced TNF-alpha secretion and increase in IL-10 response after treatment with glycine (P<0.01). In conclusion, glycine reduces hepatic damage and improves survival rate in this mouse model of endotoxemia. The protective effect of glycine is associated with modulation of TNF-alpha and IL-10 secretion.

Chronic cholestasis associated with Turner's syndrome: 12 years of clinical and histopathological follow-up.

Clinically significant liver disease is probably a rare condition in Turner's syndrome and only a few cases of severe liver disease have been described. The natural history of patients with Turner's syndrome and elevated cholestatic liver enzymes is unclear. We report a case with a long history of intrahepatic cholestasis and without clinical or histopathological progression in 12 years of follow-up. Like some other reports our case suggests, also histologically, that the liver disease in these patients runs a benign course.

Thioacetamide-induced hepatic damage in a rat nutritional model of steatohepatitis.

BACKGROUND: Nonalcoholic steatohepatitis is most often attributed to the effects of obesity, hyperlipidemia, diabetes mellitus and drugs. It is still unknown whether livers with steatohepatitis are more vulnerable to toxic damage. AIM: To determine the effect of the hepatotoxicant thioacetamide in a rat nutritional model of hepatic steatohepatitis. METHODS: Fatty liver was induced in rats by placing them on a methionine-choline deficient diet for one month. Thioacetamide was administered by 3 consecutive intraperitoneal injections (300 mg/kg) at 24 h intervals. RESULTS: Following treatment with thioacetamide, the elevated serum levels of liver enzymes and blood ammonia, liver necrotic inflammation and the survival rate after 48 h were not different between rats with normal or fatty liver. However, those parameters were significantly worse when fatty liver regressed after return to normal diet for one month (p < 0.01). Western blot analysis of hepatic extracts revealed no difference in cytochrome P4502E1 levels between fatty livers and fatty livers after regression, suggesting that the enhanced hepatotoxicity after regression of fatty liver could not be attributed to increased cytochrome P4502E1. CONCLUSIONS: In a nutritional model of steatohepatitis, rats with fatty liver were not more vulnerable than normal rats to liver damage induced by thioacetamide. However, liver damage was significantly more severe in rats with fatty livers after one month regression of steatosis.