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 test as a marker for viability. When possible, drugs were tested in the absence and presence of dimethyl sulfoxide (DMSO), to study the possible effect of this solvent when used in the case of less hydrophilic compounds. IC(50) values calculated from the dose-response curves for acetylsalicylic acid (7 mm) and acetaminophen (paracetamol; 10 mm) in rat hepatocytes were similar to those reported by other groups. IC(50) values for acetylsalicylic acid (8.7 mm), erythromycin (0.7 mm), chloramphenicol (8.1 mm), stilboestrol (diethylstilboestrol; 0.16 mm and propranolol (0.17 mm) in pig hepatocytes were similar to those reported in the literature for rat hepatocytes. In comparison to rat hepatocytes, clenbuterol was about equally cytotoxic in pig hepatocytes (IC(50) of 2.1 nu. 1.6 mm), whereas paracetamol was much more cytotoxic (IC(50) of 2.8 nu. 10 mm). Unlike chloramphenicol, the related drug thiamphenicol showed no signs of decreased MTT formation in pig hepatocytes at the highest possible test concentration of 10 mm, as was the case for furazolidone, oxytetracycline, carbadox and the putative furazolidone and furaltadone metabolites 3-amino-2-oxazolidinone and 3-amino-5-morpholinomethyl-2-oxazolidinone tested at concentrations up to (respectively) 500 mum, 3 mm, 100 mum, 5 mm and 5 mm. iC(50) values of 22 mum 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 acetylsalicylic acid, erythromycin, propranolol and clenbuterol in pig hepatocytes. In the case of acetaminophen, DMSO significantly reduced its toxicity in pig hepatocytes (IC(50) of 5.1 nu. 2.8 mm), but not in rat hepatocytes. DMSO also significantly reduced the cytotoxicity of furaltadone in pig hepatocytes (IC(50) of 0.87 nu. 0.25 mm). Following incubation with 0.5 mm furaltadone in the absence of 1% DMSO, intracellular GSH levels were decreased (38 nu. 49 nmol/mg protein), whereas in the presence of DMSO a slight increase (59 nu. 52 nmol/mg protein) was observed. DMSO had no effect on the overall degradation of the related drug furazolidone, or the formation of protein-bound metabolites. It is hypothesized that DMSO is involved in the detoxification of reactive oxygen species generated during the degradation of nitrofuran drugs, either directly or through a stimulation of the synthesis of glutathione. It is concluded that pig hepatocytes are a valuable tool to study the cytotoxicity of veterinary drugs and possible interactions with other xenobiotics, and to reveal possible species differences between farm animals and laboratory animals used to study the toxicology of these compounds.
Huveneers-Oorsprong
M B
MB
State Institute for Quality Control of Agricultural Products (RIKILT-DLO), Bornsesteeg 45, 6708 PD Wageningen, The Netherlands.
Hoogenboom
L A
LA
Kuiper
H A
HA