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.

Established cell lines for safety assessment of food contaminants: differing furazolidone toxicity to V 79, HEp-2 and Caco-2 cells.

In vitro models, preferentially derived from human tissues, may be valuable tools to study the biotransformation and toxicity of compounds that may be present as residues in food products. Such residues may represent a risk to human health, and therefore call for increased testing. Three established cell lines were used to study the toxic effect of furazolidone (FZ), a widely used veterinary drug: HEp-2 cells, derived from a human larynx carcinoma, previously used in toxicity screening of several compounds; Caco-2 cells, derived from a human colon adenocarcinoma, able to differentiate partially in culture, and V 79, a fibroblast cell line derived from Chinese hamster lung, widely used to assess direct toxicants. Various toxicity parameters were used, primarily dealing with cell death and cell proliferation. In all cell lines FZ at a concentration of 5 micrograms/ml caused a marked decrease in cell viability and especially in cell proliferation. Inhibition of DNA synthesis has also been observed, even if at higher concentrations. However, only in V 79 cells was the decrease in cell number accompanied by a marked increase in lactate dehydrogenase leakage due to membrane damage. Moreover, the surviving V 79 cells, after removal of FZ, fully recovered from the effect of the drug, as shown by their full capacity to attach to dishes and to form colonies. Surviving cells of the other two cell lines showed much poorer colony-forming ability. Exposure of Caco-2 cells and, to a lesser extent, HEp-2 cells, caused a marked increase in oxygen consumption, that possibly was due to redox cycling of the initially formed radical nitro anion. Biotransformation of the drug by all three cell lines was accompanied by the formation of protein-bound metabolites, HEp-2 being the most active cells. The toxic effects recorded show that cell lines provide a sensitive system in toxicity assessment. Moreover, it may be suggested that a battery of cell lines, including some of human origin, as well as a battery of endpoints, may be of help in addressing further specific mechanistic investigations.

Study on the role of glutathione in the biotransformation and toxicity of furazolidone using pig hepatocytes.

Incubation of monolayer cultures of pig hepatocytes with furazolidone resulted in increased intracellular oxidized glutathione (GSSG) levels but no decreased reduced glutathione (GSH) levels. However, a clear decrease in GSH levels was observed at the higher drug concentrations when either the synthesis of GSH was blocked with buthionine-d,l-sulphoximine (BSO) or the reduction of GSSG by 1,3-bis(2-chloroethyl)-1-nitrosourea. Furthermore, when cells containing (35)S-labelled GSH were exposed to furazolidone, a dose-related loss of radiolabelled GSH was observed. The increased loss was balanced by elevated levels in the medium of GSSG and a second compound, probably the disulphide of cysteine and GSH. The biotransformation of (14)C-furazolidone by cells resulted partly in the formation of the cyano metabolite as well as a large number of more hydrophilic unknown metabolites. No evidence was obtained for the excretion by the cells of the glutathione conjugate that was previously detected in microsomal incubations. This could, however, be due to the unstable nature of the conjugate, as demonstrated by its rapid disappearance when added to the medium. In the presence but not the absence of cells, this partly resulted in the formation of the cyano metabolite, which on prolonged incubation was further metabolized into more polar metabolites. Increased LDH leakage at high drug concentrations could be observed only when GSH levels were kept at a low level with BSO. Contrary to previous observations with microsomes, decreased GSH levels did not result in increased formation of protein-bound metabolites of furazolidone and the cyano metabolite. In addition, GSH depletion did not result in increased inhibition of the pyruvate metabolism by furazolidone. No evidence was obtained for the presence of reactive thiol conjugates of furazolidone in the protein fraction of cells incubated with the drug. It is concluded that GSH has an active role in the protection of cells against the cytotoxic effects of furazolidone related to oxidative stress, but that no evidence could be obtained for the formation and excretion of reactive thiol conjugates.

Use of pig hepatocytes to study the inhibition of monoamine oxidase by furazolidone.

Primary cultures of pig hepatocytes were used to examine the irreversible inhibition of monoamine oxidase (MAO), which has been observed in tissues of a number of different animal species after oral treatment with furazolidone. The rapid biotransformation of the MAO substrate p-tyramine by intact cells could effectively and irreversibly be inhibited with the known MAO inhibitors iproniazid and clorgyline. Incubation of cells with beta-hydroxyethylhydrazine and also 3-amino-2-oxazolidinone, which were previously proposed as the metabolites of furazolidone responsible for the in vivo effect, resulted in an irreversible inhibition of the MAO activity. Incubation of cells with furazolidone also resulted in a dose-related inhibition, but this effect was completely reversible on withdrawal of the drug. A similar MAO inhibition was observed after treatment of cells with nitrofurazone and furaltadone but not with nitrofurantoin. The results obtained with intact cells were confirmed by studies with 13,000 g pellets of homogenates made from cells preincubated for 24 hr with the compounds, which showed an irreversible inhibition in the case of iproniazid and 3-amino-2-oxazolidinone, but not in the case of furazolidone. The present study shows that hepatocytes are capable of transforming 3-amino-2-oxazolidinone, but not furazolidone itself, into a potent irreversible type of MAO inhibitor.

The use of pig hepatocytes for cytotoxicity studies of veterinary drugs: A comparative study with furazolidone and other nitrofurans.

The possible use of primary cultures of pig hepatocytes for cytotoxicity studies of veterinary drugs was investigated using some nitrofurans. The substances tested were furazolidone, furaltadone, nitrofurazone, nitrofurantoin (at concentrations of 15-500 mum) and nitrovin (5-50 mum); acetaminophen (1-10 mm) was used as a positive control. The leakage of lactate dehydrogenase (LDH) from the cells into the medium could be measured at high concentrations of nitrofurantoin, nitrovin, nitrofurazone and acetaminophen. After incubation of the cells with high concentrations of any of the drugs, intracellular LDH activities were lower than in controls. The incorporation of [(14)C]leucine into proteins, especially those excreted into the medium, was shown to be a more sensitive parameter of nitrofuran toxicity than LDH leakage, and was decreased by all of the drugs at most concentrations. Intracellular levels of oxidized glutathione were increased upon exposure to any of the nitrofurans. However, only incubation with 500 mum-nitrofurantoin decreased intracellular levels of reduced glutathione (GSH). In all other cases GSH levels were unchanged or even elevated upon exposure. The most sensitive parameter measured was the accumulation of pyruvate and lactate in the medium after treatment with any of the nitrofurans except nitrovin. Experiments with furazolidone showed that this effect did not disappear immediately after the end of exposure and also that the effect accumulated upon repeated treatment of the cells, especially at low doses. The results of the study clearly demonstrate that primary cultures of pig hepatocytes can be used for cytotoxicity studies of veterinary drugs.

Determination of sulfadimethoxine, sulfamethoxazole, trimethoprim and their main metabolites in porcine plasma by column switching HPLC.

A HPLC method for the determination of sulfadimethoxine, sulfamethoxazole, trimethoprim and their main metabolites in porcine plasma is reported. The metabolites under investigation were the N4-acetyl sulfonamides and 3'- and 4'-demethyl trimethoprim. In order to obtain a sensitivity of 25-50 ng ml-1, the application of column switching HPLC was investigated. An on-line preconcentration of the drugs and metabolites was preceded by an off-line sample pre-treatment. Parent compounds and metabolites were separated by reversed-phase HPLC followed by UV-detection. The mean recoveries for 4'-demethyl trimethoprim were greater than 80% while the mean recoveries for the other compounds were greater than 90%. Application of the method for analysis of plasma samples obtained from pharmacokinetic studies is described.