Tissue Pathologl Unit , , ' , The , Roehampton Institute, West Hill, London, Departments of Clinical Biochemistry and Ilmmunology, 3The Institute of Liver Studies, Kin 's College School of Medicine and Dentistry, Bessenier Road, London %acuity of Medicine, University of Southampton, Southampton, 4Department of Gastroenterology and Nutrition, Central Middlesex Hospital, Park Royal, London and 5Department of Pathology, The Maudsley Hospital, Denmark Hill, London. Acute ethanol administration reduces the synthesis rates of both, smooth and skeletal muscle proteins [ 1-31. Chronic ethanol feeding studies have been shown to lead to reductions in the relative amounts of smooth and skeletal muscle proteins [4-61. I t is possible that mediating factors may include cytokines, derived from macrophages and other mononuclear cells, in response to both acute and chronic ethanol toxicity. Tumour necrosis factor alpha (TNF-a) and some interleukins, induce a variety of cellular and biochemical changes, including loss of skeletal muscle protein [7] and increased nitrogen excretion, which are also characteristics of chronic ethanol toxicity. Changes in protein synthesis in response to cytokines have also been reported. As cytokine production has been reported to be influenced by acute and chronic ethanol toxicity, some of the pathological changes observed in our rat models of alcohol toxicity, may be mediated by cytokine production. To test this hypothesis we measured plasma levels of interleukin-6 (IL-6) and TNF-a in rats acutelyand chronically-treated with ethanol. However, chronic ethanol intake characteristically leads to a retardation in normal growth rates and growth hormone (GH) levels have been shown to be suppressed by acute and chronic ethanol administration. The receptor for GH is a member of the cytokine receptor superfamily and it is possible that tissue responsiveness to cytokines is modulated by changes in receptor expression. In this regard we also measured plasma GH-BP levels, since GH-BP is derived from the extracellular portion of the GH receptor and should therefore provide an indirect measure of GH receptor expression. Male Wistar rats (100 g body weight) were subjected to either acute or chronic ethanol treatment. In acute studies rats were injected with an intraperitoneal (i.p.) dose of ethanol ( I m1/100 g body weight; 75 mmol/kg body weight). Controls were injected with isovolumetric saline (0. I5 mol/l NaCl i.p.). Rats were killed by decapitation after 2.5 hr and the blood collected into heparinised tubes and plasma extracted after centrifugation. In chronic studies, treated rats were fed a nutritionally complete liquid diet, containing 35% of total calories as either ethanol or glucose. as previously described 161. Controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. At 3 weeks rats were killed by decapitation, and the blood collected as for acute studies. Levels of 11,-6 were measured in a bioassay using specific cell target. the B9 cell line. Cell proliferation was assessed using a rapid and sensitive modified colorimetric method [ S ] , which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2.5diphenyl tetrazolium bromide. IL-6 activity was expressed in U/ml, defined as the dilution giving half maximal proliferation of B9 cells. Plasma TNF-a was measured using a bioassay. Cultures of L-929 tumorigenic murine fibroblasts were seeded at 4 x 10 cells/well in 50 pl of RPMl (Gibco. U.K.) supplemented with 10% fetal calf serum (Gibco. I1.K.) in 96-well flat-bottomed plates. After 4 h r of culture at 37°C i n 5% carbon dioxide samples and three-fold dilutions of rccombinant human TNF standard (Eurocetus, USA) were added to L.929 cells at a volume of 50 pliwell with 2 pg actinomycin D. Standard curve range was from 1.000 pg/ml to 4 pgiml. The specificity of the assay was checked by neutralisation o f the bioactivity with anti-serum to TNF. After 20 hours of culture, medium was removed and the monolayers stained for 10 min with crystal violet (0.5% in methano1:water). The plate was washed with water and the cells solubilised in 20% acetic acid and absorbance measured at 570 nm using a Dynatech MR5000 microtitrc plate reader. Plasma GH-BP activity was assayed by gel chromatography csscntially as described previously 191. Various analytes, as indicated in Table I were assayed by standard laboratory tests. MICHAEL NORM AN^, ADRIAN B. BONNER, MUNTHER J.