Fumonisin-induced hepatocarcinogenesis: mechanisms related to cancer initiation and promotion.

We review the hepatocarcinogenic effects of fungal cultures of Fusarium verticillioides(= Fusarium moniliforme) strain MRC 826 in male BD IX rats. Subsequent chemical analyses of the fumonisin B (FB) mycotoxin content in the culture material used and long-term carcinogenesis studies with purified FB1 provide information about dose-response effects, relevance of hepatotoxicity during FB1-induced carcinogenesis, and the existence of a no-effect threshold. Fumonisin intake levels of between 0.08 and 0.16 mg FB/100 g body weight (bw)/day over approximately 2 years produce liver cancer in male BD IX rats. Exposure levels < 0.08 mg FB/100 g bw/day fail to induce cancer, although mild toxic and preneoplastic lesions are induced. The nutritional status of the diets used in the long-term experiments was marginally deficient in lipotropes and vitamins and could have played an important modulating role in fumonisin-induced hepatocarcinogenesis. Short-term studies in a cancer initiation/promotion model in rat liver provided important information about the possible mechanisms involved during the initial stages of cancer development by this apparently nongenotoxic mycotoxin. These studies supported the findings of long-term investigations indicating that a cytotoxic/proliferative response is required for cancer induction and that a no-effect threshold exists for cancer induction. The mechanisms proposed for cancer induction are highlighted and include the possible role of oxidative damage during initiation and the disruption of lipid metabolism, integrity of cellular membranes, and altered growth-regulatory responses as important events during promotion.

Dual functions of E2F-1 in a transgenic mouse model of liver carcinogenesis.

Deregulation of E2F transcriptional control has been implicated in oncogenic transformation. Consistent with this idea, we recently demonstrated that during hepatocarcinogenesis in c-myc/TGFalpha double transgenic mice, there is increased expression of E2F-1 and E2F-2, as well as induction of putative E2F target genes. Therefore, we generated transgenic mice expressing E2F-1 under the control of the albumin enhancer/promoter to test the hypothesis that E2F family members may contribute to liver tumor development. Overexpression of E2F-1 resulted in mild but persistent increases in cell proliferation and death during postnatal liver growth, and no increases in hepatic regenerative growth in response to partial hepatectomy. Nevertheless, from 2 months postnatally E2F-1 transgenic mice exhibited prominent hepatic histological abnormalities including preneoplastic foci adjacent to portal tracts and pericentral large cell dysplasia. From 6 to 8 months onward, there was an abrupt increase in the number of neoplastic nodules ('adenomas') with 100% incidence by 10 months. Some adenomas showed evidence of malignant transformation, and two of six mice killed at 12 months showed trabecular hepatocellular carcinoma. Endogenous c-myc was up-regulated in the early stages of E2F-1 hepatocarcinogenesis, whereas p53 was overexpressed in the tumors, suggesting that both E2F-1-mediated proliferation and apoptosis are operative but at different stages of hepatocarcinogenesis. In conclusion, E2F-1 overexpression in the liver causes dysplasia and tumors and suggests a cooperation between E2F-1 and c-myc oncogenes during liver oncogenesis.

Genomic structure and chromosome location of the mouse RelA p65 gene (Rela).

The RelA (p65) subunit of transcription factor NF-kappaB plays a critical role in development, and rela(-/-) knockout mice die in utero from massive liver apoptosis. Only partial sequences of the mouse Rela gene are available. We have determined the genomic structure of mouse Rela and promoter, and have mapped the gene to chromosome 19B1-3.

The effect of severe undernutrition, and subsequent refeeding on digestive function in human patients.

BACKGROUND: Severe undernutrition may adversely affect gut function. AIMS: To investigate the effects of severe undernutrition and subsequent refeeding on human digestive function. METHODS: Severely undernourished patients (body mass index (BMI) < 17 kg/m2) were studied before, and after a period of intensive nutritional support. Standard intestinal absorption tests (faecal fat and urinary xylose excretion), pentagastrin-stimulated acid secretion, and cholecystokinin octapeptide (CCK-8)-stimulated pancreatic enzyme secretion tests were performed. In addition, duodenal biopies were taken to assess gut mucosal morphology. Findings were evaluated in comparison to a group of normal healthy volunteers. RESULTS: Mean BMI of the patients prior to nutritional support was 13.41 kg/m2, with improvement to 16.12 kg/m2 after. Duodenal histology showed evidence of villous atrophy in six of 14 (43%) undernourished patients. Mean xylose excretion following a 5 g oral dose was 0.62 g/5 h in the group of undernourished patients prior to nutritional support (normal > 1 g/5 h), with improvement to 1.40 g/5 h (P < 0.01) after feeding. Maximal gastric acid output was significantly impaired in the undernourished group, as compared to the controls (6.94 mEq/l vs 25.53 mEq/l, P < 0.02), with a significant improvement to 12.30 mEq/l (P < 0.05) following nutritional support. Pancreatic enzyme output was significantly reduced (amylase 830.9 U/h vs 2304 U/h, P < 0.01; lipase 38.0 U/h vs 118.6 U/h, P < 0.01; trypsin 119.7 U/h vs 341.4 U/h, P < 0.01). Following a period of nutritional support there was a significant improvement in amylase and lipase outputs to 1819 U/h and 85.5 U/h, respectively (P < 0.01). These levels were not significantly different from the normal controls. Trypsin output, however, remained significantly impaired at 174.3 U/h (P < 0.01). CONCLUSIONS: Severe undernutrition is associated with significant impairment of digestive function, with improvement occurring following nutritional support. These changes may affect initial tolerance to enteral feeding, particularly in those patients with co-existent gut disease.

Injection sclerotherapy for variceal bleeding in patients with irresectable hepatocellular carcinoma.

BACKGROUND/AIMS: Patients with cirrhosis and advanced hepatocellular carcinoma are seldom cured, and have limited survival. Bleeding from esophageal varices in such patients is a major complication which, if untreated, may be a terminal event. This study evaluated the efficacy of injection sclerotherapy in controlling acute bleeding from esophageal varices and the benefit of repeated injection to eradicate varices in patients with cirrhosis and irresectable hepatocellular carcinoma. METHODOLOGY: Between 1975 and 1997, nineteen of 688 patients (2.8%) treated for bleeding esophageal varices had cirrhosis and irresectable hepatocellular carcinoma. There were 13 men and 6 women; median age, 42 years (range: 20-81). Eight patients were Child's-Pugh grade B and 11 grade C; 11 patients were Okuda stage II and 8 stage III. RESULTS: In 13 patients (68.4%) bleeding was controlled by injection sclerotherapy after a mean of 3 injections (range: 1-5), and of these esophageal varices were completely eradicated in 7 patients (53.9%), none of whom rebled. Twelve patients (63%) were discharged from hospital and had a mean survival of 100 days. Seven patients died in hospital, 5 of liver failure precipated by recurrent bleeding and 2 of hepatocellular carcinoma. Median survival for Child's-Pugh grade B patients was 80 days (range: 9-405) compared to 28 days (range: 8-117) for the grade C (P = 0.25). CONCLUSIONS: Injection sclerotherapy controlled acute variceal bleeding in most patients with hepatocellular carcinoma and provided effective palliative therapy with no further bleeding after eradication of varices.

Histopathology and gene expression changes in rat liver during feeding of fumonisin B1, a carcinogenic mycotoxin produced by Fusarium moniliforme.

Fumonisin B1 (FB1) is a carcinogenic mycotoxin produced by the fungus Fusarium moniliforme in corn. Feeding of FB1 to rats causes acute liver injury, chronic liver injury progressing to cirrhosis, and sometimes terminates in hepatocellular carcinoma or cholangiocarcinoma. This study describes the histolopathology and changes in gene expression in the rat liver during short-term feeding of FB1. Male Fischer rats were fed either FB1 250 mg/kg or control diet, and were killed weekly for 5 weeks. FB1 caused a predominantly zone 3 'toxic' liver injury, with hepatocyte death due to necrosis and apoptosis. Hepatocyte injury and death were mirrored by hepatic stellate cell proliferation and marked fibrosis, with progressive disturbance of architecture and formation of regenerative nodules. Despite ongoing hepatocyte mitotic activity, oval cell proliferation was noted from week 2, glutathione S-transferase pi-positive hepatic foci and nodules developed and, at later time points, oval cells were noted inside some of the 'atypical' nodules. Northern blot (mRNA) analysis of liver specimens from weeks 3 to 5 showed a progressive increase in gene expression for alpha-fetoprotein, hepatocyte growth factor, transforming growth factor alpha (TGF-alpha) and especially TGF-beta1 and c-myc. Immunostaining with LC(1-30) antibody demonstrated a progressive increase in expression of mature TGF-beta1 protein by hepatocytes over the 5 week feeding period. The overexpression of TGF-beta1 may be causally related to the prominent apoptosis and fibrosis seen with FB1-induced liver injury. Increased expression of c-myc may be involved in the cancer promoting effects of FB1.

The effects of dietary iron overload on fumonisin B1-induced cancer promotion in the rat liver.

The present study was performed to determine whether excess hepatic iron modulates the cancer-initiating and promoting properties of FB1. Thirty-eight male F344 rats were divided into four dietary treatment groups: (i) control diet (AIN, n = 8); (ii) FB1 250 mg/kg diet (FB1, n = 10); (iii) 1-2% carbonyl iron (CI, n = 10); or (iv) FB1 plus iron loading (FB1/CI, n = 10) for 5 weeks (2 x 2 factorial design). Hepatic iron concentrations in iron-loaded animals at 5 weeks were 444 +/- 56 (CI) and 479 +/- 80 micromol/g dry weight (FB1/CI) (mean +/- SEM). All the FB1-fed rats, in the presence or absence of CI, developed a toxic hepatitis with a 4-fold rise in serum alanine transaminase (ALT) levels. FB1 appeared to augment iron-induced hepatic lipid peroxidation, as measured by the generation of thiobarbituric acid reacting substances (TBARS) in liver homogenates (P < 0.0001). Morphometric analysis showed that FB1 caused a significantly greater mean +/- SEM number of 'enzyme-altered' foci and nodules per cm2 (5.34 +/- 1.42 vs. 1.50 +/- 0.52, P < 0.05), as well as a greater area (%) of liver occupied by foci and nodules (0.33 +/- 0.12% vs. 0.05 +/- 0.03%, P < 0.001), compared with FB1/CI. The addition of FB1 to dietary iron loading caused a shift in distribution of iron from hepatocytes to Kupffer cells, probably due to phagocytosis of necrotic iron-loaded hepatocytes. In conclusion, (i) FB1 appears to cause toxicity in the liver independently from effects on lipid peroxidation; (ii) FB1 has a potentiating effect on iron-induced lipid peroxidation; and (iii) dietary iron loading appears to protect against the cancer promoting properties of FB1, possibly due to a stimulatory effect of iron on hepatocyte regeneration.

Isolation from human fetal liver of cells co-expressing CD34 haematopoietic stem cell and CAM 5.2 pancytokeratin markers.

BACKGROUND/AIMS: Ductal plate and bile duct cells in developing human liver express haematopoietic stem cell markers, such as c-kit and CD34, in association with cytokeratin markers CAM 5.2 and CK 18. The identification of such ductal plate cells as likely progenitors for both bile duct epithelial cells and hepatocytes and their possible reappearance as oval cells in the regenerating liver have generated much interest in their pluripotential capacities. This study aimed to isolate cells from human fetal liver that co-express haematopoietic stem cell and epithelial cell markers. METHODS: Human fetal liver was harvested following legal termination of pregnancy at week 14-22. CD34+ mononuclear cells were isolated from liver cell suspensions with immunomagnetic beads. Immunofluorescent staining, using anticytokeratin CAM 5.2 against CK 8 and 18, was performed on permeabilised CD34+ cells for flow cytometry and fluorescent microscopy. CD34+ cells were also stained for other stem cell markers (HLA-DR, c-kit) and committed haematopoietic cell markers (CD33, CD38). RESULTS: Approximately 0.9% (range 0.07-4.0%) of the mononuclear cells isolated were CD34+ cells. The number of mononuclear cells isolated correlated with fetal liver weight (r=0.508). About 3-8% of these CD34+ cells stained positively for CAM 5.2. In addition, CD34+ cells were positive for HLA-DR, but only a small percentage was positive for c-kit. Staining for the committed haematological markers, CD33 and CD38, was consistently negative. CONCLUSIONS: This study describes an immunoaffinity method for the enrichment from human fetal liver of cells that co-express haematopoietic stem cell and epithelial cell markers. Such cellular subsets may correspond to pluripotential ductal plate and bile duct cells.

The influence of intravenous infusions of glucose and amino acids of pancreatic enzyme and mucosal protein synthesis in human subjects.

BACKGROUND: Animal studies have shown that the synthesis and secretion of pancreatic enzymes and the turnover of mucosal proteins is strongly influenced by diet. METHODS: To determine whether the absorbed products of digestion are responsible for these changes, we investigated in groups of five healthy volunteers, the effects of i.v. infusions of amino acids (0.08 g/kg/h) and glucose (0.3 g/kg/h) on pancreatic enzyme and mucosal protein synthesis. Proteins were labeled in vivo by a 4-hours i.v. infusion of 14C-leucine and the enteric infusion of 3H-leucine tracer, during simultaneous cholecystokinin stimulation and duodenal collection of secreted pancreatic enzymes. Labeling of mucosal proteins was measured by endoscopic biopsy. RESULTS: The amino acid infusions elevated plasma amino acid levels, and the glucose infusions increased both glucose and insulin concentrations. The rates for amylase and trypsin secretion were significantly lower during the first 2 hours of glucose infusion and the rate of synthesis of trypsin was delayed by i.v. amino acid infusions from 52.1 +/- 4.1 to 77.6 +/- 8.5 minutes. Mucosal protein turnover rates were unaffected. 3H-labeling via the enteral route showed similar enzyme synthesis rates but variable mucosal incorporation rates. CONCLUSIONS: I.v. nutrients do not appear to stimulate the synthesis of pancreatic and mucosal proteins in human subjects.

Surgical resection for hepatocellular carcinoma in Cape Town--a clinical and histopathological study.

OBJECTIVE: Review of surgical resections performed for hepatocellular carcinoma (HCC) at our institution between 1990 and 1996, histology of resected specimens, and clinical outcome. DESIGN: Retrospective and prospective study of 14 patients who underwent resection for HCC. SETTING: The Hepatobiliary Unit and Liver Clinic, Groote Schuur Hospital, Cape Town. PATIENTS: Fourteen patients who underwent liver resections for HCC. INTERVENTIONS: Hepatic resections using prolonged vascular inflow occlusion. OUTCOME MEASURES: Clinical outcome and disease-free survival following resection. RESULTS: Fourteen patients (5.6% of the total number presenting with HCC) underwent liver resection for HCC at our institution between 1990 and 1996. There were 7 men, median age 40 years (range 18-74 years). Only 2 patients were black, and only 1 of these patients had evidence of hepatitis B virus (HBV) infection in the liver. Extensive liver resections were often required. The mean (SD) ischaemic time was 81 (26) minutes and mean estimated blood loss was 938 (649) ml. During hospital admission, 1 patient developed a minor bile leak that settled spontaneously, and 1 patient suffered a stroke and died. The mean hospital stay following operation was 12 days (range 7-21 days). Disease-free patient survival at 1, 2 and 3 years was 85%, 75%, and 62%, respectively. Histopathology of the resected specimens showed that 10 of 14 tumours had arisen in non-cirrhotic livers. Mean tumour size was 10.6 (4.6) cm. Only 1 specimen showed the fibrolamellar variant of HCC. CONCLUSIONS: Only a small proportion of patients with HCC seen at Groote Schuur Hospital were eligible for resection, and only a minority of these had HBV-associated 'African' HCC. The results of hepatic resection at our institution compare favourably with literature reports, despite the relatively large size of the tumours. It is of interest that most tumours arose in non-cirrhotic livers. There was no evidence of proliferation of 'oval-like' cells in non-neoplastic liver tissue.

The management of biliary complications following orthotopic liver transplantation.

OBJECTIVE: Review of the biliary complications following orthotopic liver transplantation (OLT) at our institution, and their management and outcome. DESIGN: Retrospective study of medical records of 63 patients who underwent 68 transplant operations. SETTING: The Liver Transplant Unit, Groote Schuur Hospital and Red Cross War Memorial Children's Hospital, Cape Town PATIENTS: Six patients treated for 9 biliary complications. INTERVENTIONS: Reoperation with biliary reconstruction, or non-operative measures by either endoscopic retrograde pancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). OUTCOME MEASURES: Clinical outcome and survival following treatment for biliary complications. RESULTS: Biliary complications occurred in 8.8% of patients who underwent transplantation at our institution. These consisted of strictures in 4 patients (with leak in 2), bile leak in 1 patient, and unsuspected primary sclerosing cholangitis (PSC) of the recipient duct in 1 patient. The mean time interval of biliary complications following OLT was 8 weeks (range 3-16). Biliary reconstruction was required in 4 patients while 2 patients were treated by endoscopic stenting. After a mean follow-up period of 30 months (range 1-64), 4 patients remained stable, 1 patient developed progressive stricturing of the intrahepatic ducts requiring repeated PTC dilatations, and 1 patient experienced stent blockages requiring endoscopic stent changes (died of unrelated causes). CONCLUSIONS: The rate of biliary complications following OLT at our institution compares favourably with literature reports. While biliary reconstruction is usually needed, endoscopic stenting appears effective in selected cases of biliary stricture and leak. PSC should be excluded prior to transplantation in young patients with cryptogenic cirrhosis.

Hydatid cyst in the head of the pancreas with obstructive jaundice.

We report a rare case of obstructive jaundice caused by an intrapancreatic hydatid cyst in a 17-year-old black girl. Ultrasonography and computed tomography demonstrated the obstructing cyst in the head of the pancreas. Cyst aspiration produced clear fluid with a low amylase content and no hydatid hooklets or protoscolices. Pancreaticoduodenectomy was performed for a presumed cystic neoplasm of the pancreas, but histology showed the true diagnosis. Pancreatic hydatidosis should be considered in the differential diagnosis of obstructing pancreatic cysts in the appropriate epidemiological setting.

Severe hypophosphataemia in anorexia nervosa.

In addition to well-described acid-base and electrolyte disturbances, anorexia nervosa may be complicated by severe hypophosphataemia. We report a case of anorexia nervosa complicated by life-threatening hypophosphataemia manifesting as generalized muscle weakness and bulbar muscle dysfunction, resulting in an aspiration pneumonia and cardiorespiratory arrest.

Primary sclerosing cholangitis. Requiem for biliary drainage operations?

OBJECTIVE: To review the outcome of a consecutive number of patients with primary sclerosing cholangitis (PSC) treated at one institution to define prognostic variables and determine the influence of surgery on outcome. DESIGN: Case series of patients with PSC seen in Cape Town, South Africa, between 1981 and 1991. SETTING: Tertiary referral center. PATIENTS: Thirty-six patients with PSC were studied. Diagnosis was based on cholangiographic findings of multiple strictures of the bile ducts together with compatible clinical and biochemical features. Thirty-two patients were followed up prospectively for up to 9 years. MAIN OUTCOME MEASURES: Patient outcome was defined as good (stable or slowly progressive disease) or poor (death or liver transplantation). RESULTS: During the follow-up period, seven patients with PSC died and two underwent liver transplantation. Actuarial survival at 5 years was 52%. An increased serum bilirubin concentration was the only variable at presentation that independently predicted a poor outcome. Cholangiography was unhelpful in predicting patient outcome. Six patients who developed obstructive jaundice associated with advanced liver disease underwent biliary drainage operations for surgically correctable strictures, but this did not seem to prevent progression of the disease. Two patients who progressed to end-stage liver disease went on to have liver transplantation and were alive with functioning grafts at 7 and 14 months, respectively. CONCLUSIONS: Symptomatic PSC is a progressive disorder with a poor prognosis. Our experience suggests that patients with advanced liver disease caused by PSC should be considered directly for liver transplantation rather than biliary bypass operations.