THE USE OF THE MTT TEST FOR DETERMINING THE CYTOTOXICITY OF VETERINARY DRUGS IN PIG HEPATOCYTES

Pig hepatocytes were used for determining the cytotoxicity of a number of veterinary drugs and known hepatotoxic compounds, using the MTT te st as a marker for viability. When possible, drugs were tested in the absence and presence of dimethyl sulfoxide (DMSO), to study the possib le effect of this solvent when used in the case of less hydrophilic co mpounds. IC50 values calculated from the dose-response curves for acet ylsalicylic acid (7 mM) and acetaminophen (paracetamol; 10 mM) in rat hepatocytes were similar to those reported by other groups. IC50 value s for acetylsalicylic acid (8.7 mM), erythromycin (0.7 mM), chloramphe nicol (8.1 mM), stilboestrol (diethylstilboestrol; 0.16 mM and propran olol (0.17 mM) in pig hepatocytes were similar to those reported in th e literature for rat hepatocytes. In comparison to rat hepatocytes, cl enbuterol was about equally cytotoxic in pig hepatocytes (IC50 of 2.1 v. 1.6 mM), whereas paracetamol was much more cytotoxic (IC50 of 2.8 v . 10 mM). Unlike chloramphenicol, the related drug thiamphenicol showe d no signs of decreased MTT formation in pig hepatocytes at the highes t possible test concentration of 10 mM, as was the case for furazolido ne, oxytetracycline, carbadox and the putative furazolidone and furalt adone metabolites 3-amino-2-oxazolidinone and 3-amino-5-morpholinometh yl-2-oxazolidinone tested at concentrations up to (respectively) 500 m u M, 3 mM, 100 mu M, 5 mM and 5 mM. IC50 values of 22 mu M and 0.25 mM were obtained for menadione and furaltadone, respectively. DMSO, used at a concentration of 1%, had no effect on the toxicity of acetylsali cylic acid, erythromycin, propranolol and clenbuterol in pig hepatocyt es. In the case of acetaminophen, DMSO significantly reduced its toxic ity in pig hepatocytes (IC50 of 5.1 v. 2.8 mM), but not in rat hepatoc ytes. DMSO also significantly reduced the cytotoxicity of furaltadone in pig hepatocytes (IC50 of 0.87 v. 0.25 mM). Following incubation wit h 0.5 mM furaltadone in the absence of 1% DMSO, intracellular GSH leve ls were decreased (38 v. 49 nmol/mg protein), whereas in the presence of DMSO a slight increase (59 v. 52 nmol/mg protein) was observed. DMS O had no effect on the overall degradation of the related drug furazol idone, or the formation of protein-bound metabolites. It is hypothesiz ed that DMSO is involved in the detoxification of reactive oxygen spec ies generated during the degradation of nitrofuran drugs, either direc tly or through a stimulation of the synthesis of glutathione. It is co ncluded that pig hepatocytes are a valuable tool to study the cytotoxi city of veterinary drugs and possible interactions with other xenobiot ics, and to reveal possible species differences between farm animals a nd laboratory animals used to study the toxicology of these compounds. (C) 1997 Elsevier Science Ltd.