Inhibition of alcohol dehydrogenase blocks enhanced Gi-protein expression following ethanol treatment in experimental hepatocellular carcinoma in vitro.

OBJECTIVE: Chronic alcohol abuse is one of the major contributors to the onset and progression of hepatocellular carcinoma (HCC). We have previously identified increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in primary human and animal models of HCC. Stimulation of Gi-proteins in HCC stimulates cell mitogenesis, an effect not observed in hepatocytes. The aim of this study was to determine the effect of ethanol and ethanol metabolism on Gi-protein expression in an experimental model of HCC. DESIGN: Pharmacological agents that inhibit alcohol metabolism were used in conjunction with ethanol or ethanol metabolites. We were also able to assess the relative contribution of alcohol and acetaldehyde, the major metabolite of alcohol, on Gi-protein expression in HCC and hepatocytes. METHODS: These studies used the rat hepatic tumorigenic H4IIE cell line in conjunction with isolated rat hepatocytes. Cells were cultured in vitro and exposed to ethanol, ethanol in the presence of an alcohol dehydrogenase (ADH) inhibitor, or acetaldehyde for varying lengths of time. Ethanol metabolism and changes in Gi-protein expression were subsequently determined by assay. RESULTS: Exposure to ethanol alone led to significant dose and time dependent increases in Gialpha1/2 and Gialpha3 protein and mRNA expression in HCC cells. In contrast, ethanol failed to alter Gialpha1/2, and only moderately affected Gialpha3 protein expression in isolated cultured hepatocytes. Pretreatment of HCC cells and hepatocytes with 4-methyl pyrazole (4-MP, 10 microm) significantly inhibited alcohol metabolism. Treatment of HCC cells with 4-MP inhibited changes in Gi-protein expression following exposure to ethanol (25 mm, 24 h). In addition, the increased expression of Gi-proteins observed after exposure to ethanol in HCC were mimicked by direct exposure of HCC cells to acetaldehyde in a dose and time dependent manner. CONCLUSIONS: These data suggest that alcohol metabolites, not alcohol, lead to increased Gi-protein expression in HCC in vitro. Ethanol and ethanol metabolites, in contrast, fail to significantly alter Gialpha1/2 protein expression in hepatocytes. These data may have significant implications in HCC progression in vivo.     

Demonstration of increased concentrations of circulating glycated insulin in human Type 2 diabetes using a novel and specific radioimmunoassay

AIMS/HYPOTHESIS: Glycation of insulin, resulting in impaired bioactivity, has been shown within pancreatic beta cells. We have used a novel and specific radioimmunoassay to detect glycated insulin in plasma of Type 2 diabetic subjects. METHODS: Blood samples were collected from 102 Type 2 diabetic patients in three main categories: those with good glycaemic control with a HbA(1c) less than 7%, moderate glycaemic control (HbA(1c) 7-9%) and poor glycaemic control (HBA(1c) greater than 9%). We used 75 age- and sex-matched non-diabetic subjects as controls. Samples were analysed for HbA(1c), glucose and plasma concentrations of glycated insulin and insulin. RESULTS: Glycated insulin was readily detected in control and Type 2 diabetic subjects. The mean circulating concentration of glycated insulin in control subjects was 12.6+/-0.9 pmol/l ( n=75). Glycated insulin in the good, moderate and poorly controlled diabetic groups was increased 2.4-fold ( p<0.001, n=44), 2.2-fold ( p<0.001, n=41) and 1.1-fold ( n=17) corresponding to 29.8+/-5.4, 27.3+/-5.7 and 13.5+/-2.9 pmol/l, respectively. CONCLUSION/INTERPRETATION: Glycated insulin circulates at noticeably increased concentrations in Type 2 diabetic subjects.     

Comparison of serological tests for detection of antibodies to infectious laryngotracheitis virus

Four serological tests i.e. ELISA, serum neutralisation (SN), fluorescent antibody (FA), and agar gel immunodiffusion (AGID) were compared for sensitivity using several criteria, for detection and titration of infectious laryngotracheitis (ILT) virus antibodies in chicken sera. In the ELISA test, sera were tested in parallel on virus positive and negative control antigens with results expressed as positive-negative difference. Non-specific binding was not a problem in this test. SN tests were performed in microtitre plates using chick embryo liver cells, while sera for FA tests were titrated on multispot slides on which were fixed ILT virus-infected chick embryo liver cell cultures. The AGID test was the standard test still widely used for serological diagnosis of ILT. The four tests were compared using (1) sera from experimentally inoculated birds bled regularly at intervals from 4 to 35 days post-inoculation, (2) convalescent sera from a natural outbreak of ILT, and (3) serial dilutions of an ILT positive serum. In all experiments the ELISA test was of slightly greater sensitivity than SN and was comparable to the FA test. In the experimentally infected birds ELISA and FA test detected sero-conversion in more birds at 7 days than SN. In tests with the serially diluted hyperimmune ILT serum, ELISA, FA and SN tests were comparable. SN however was the most useful test for quantification of ILT antibodies. ILT-SN titres in birds were never high, the highest titre recorded in experimental birds and in convalescent sera was 1/48. AGID was found to be less sensitive than ELISA, FA or SN test but was considered useful for detection of antibodies on a flock basis, since, with the convalescent sera AGID detected a significant proportion of positives.     

Alcohol induces liver neoplasia in a novel alcohol-preferring rat model

Background: Alcohol is a significant risk factor for the development of hepatocellular carcinoma (HCC). To date, no rodent model has demonstrated the formation of hepatic neoplasia in the setting of chronic alcohol consumption alone. Methods: We investigated whether rats selectively bred for high alcohol preference (P rats), allowed free access to water, or water and 10% (v/v) alcohol, for 6, 12, or 18 months, develop hepatic neoplasia. Results: At necropsy, liver tumor incidence and multiplicity were significantly increased in 18‐month alcohol‐consuming versus water‐consuming P rats. These data were confirmed histologically by glutathione‐S‐transferase pi‐class (GSTp) staining. Phosphorylated mitogen‐activated protein kinase/extracellular signal‐regulated kinase 1/2 (MAPK/ERK) staining was also increased in the sinusoidal lining cells within livers of alcohol‐consuming versus water only P rats. In addition, cytochrome p450IIE1 (CYP2E1) mRNA, protein expression/activity, and intrahepatic oxidative stress were significantly increased in alcohol‐consuming P rat livers versus water only. In contrast, acetaldehyde dehydrogenase expression decreased in alcohol‐consuming versus water only P rats. No significant difference in alcohol dehydrogenase expression was detected. Conclusions: These data demonstrate that chronic alcohol consumption is associated with hepatic neoplasia, MAPK/ERK activation, increased CYP2E1 activity, and intrahepatic oxidative stress in P rats. As these rats are well characterized as a model of alcoholism, these findings identify a novel rodent model of alcohol or “alcoholism”‐induced liver neoplasia.     

Gastrin-releasing peptide in normal and neoplastic human lung: measurement and biochemical characterization

Levels of gastrin-releasing peptide (GRP) were determined by radioimmunoassay in human normal main and lobar bronchus and parenchymal lung tissue extracts. It was found that the level of GRP differed significantly between all 3 areas. The concentration of GRP was statistically higher in main bronchus (median 6.74 ng/g) compared to both lobar bronchus (median 4.79 ng/g) and parenchymal lung (median 1.73 ng/g), and also statistically higher in lobar bronchus compared to parenchymal lung. Chromatographically, GRP-immunoreactivity in both main and lobar bronchial extracts corresponded to GRP1-27 and GRP18-27, while in lung tissue only one major species was identified which corresponded in retention time to GRP18-27. No significant difference was detected when the levels of GRP in normal lobar bronchus and normal lung tissue were compared to the levels in lobar bronchus and lung taken from patients with lung carcinoma, at a site adjacent to the carcinoma. However, a significant difference was observed between the GRP content of normal main bronchus compared to main bronchus from patients with carcinoma. GRP was measured in 26/56 lung carcinomas examined. The levels ranged from 42,000 ng/g in a carcinoid tumour to 0.18 ng/g in a squamous-cell carcinoma, though only in 6 tumours were the levels outside the range determined for normal pulmonary tissue. Chromatography of selected tumour extracts of different histopathologies showed that there were differences in the GRP products present.     

Avian encephalomyelitis following oral vaccination

Outbreaks of clinical avian encephalomyelitis (AE) which developed after vaccination of 14-week-old birds by the oral route are reported. Mortality during weeks 2 to 5 following vaccination reached 2%. Experimental studies showed that, in contrast to popular opinion, AE vaccine virus given orally can spread to the central nervous system and produce encephalomyelitis (mild). The severity of the vaccine induced lesions was not affected by chicken anaemia virus (CAV) infection 2 weeks before AE vaccination. It is postulated that non-CAV-induced immunosuppression allowed vaccine virus to produce the severe lesions and deaths occurring during the disease outbreaks, and that in one case, Marek's disease virus was the immunosup-pressive agent.     

'Apathetic' thyrotoxicosis presenting with hypercalcaemia and spurious normalization of serum thyroid hormone levels

A patient with thyrotoxicosis presented with weight loss and hypercalcaemia, leading to an erroneous diagnosis of occult malignant disease. Intercurrent illness and drug treatment of hypercalcaemia in this patient caused a depression of circulating thyroid hormone levels, leading to a delay in diagnosis. Radionuclide studies of thyroid function, in contrast, consistently suggested a thyrotoxic state. It is suggested that in this situation, radionuclide studies may give a more accurate assessment of thyroid status than biochemical tests, which may be difficult to interpret in the presence of non-thyroidal illness.