Toxicity of atractyloside in precision-cut rat and porcine renal and hepatic tissue slices.

Atractyloside (ATR) causes acute fatal renal and hepatic necrosis in animals and humans. Precision-cut renal cortical and hepatic slices (200 +/- 15 microns) from adult male Wistar rat and domestic pigs, incubated with ATR (0.2-2.0 mM) for 3 h at 37 degrees C, inhibited pyruvate-stimulated gluconeogenesis in a concentration- and time-dependent manner. p-Aminohippurate accumulation was significantly inhibited in both rat and pig renal cortical slices from 0.2 mM ATR (p < 0.05). There was a small decrease in mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium to formazan in both rat and pig kidney slices, which was significant at > or = 2 mM, but no changes in liver slices from either species. However, cellular ATP was significantly depleted at > or = 0.2 mM ATR in kidney and in liver slices from both species. ATR also caused a marked leakage of lactate dehydrogenase and alkaline phosphatase from both pig and rat kidney slices at all concentrations, but only lactate dehydrogenase was significantly elevated in liver slices from both species. ATR > or = 0.5 mM caused a significant increase in lipid peroxidation, but only in liver slices of both species, and > or = 0.2 mM ATR caused a marked depletion of reduced glutathione and significant increase in oxidized glutathione in both kidney and liver slices of both species. However, GSH to GSSG ratio was only significantly altered in the liver slices, indicating that oxidative stress may be the cause of toxicity in this organ. Both rat and pig tissue slices from the same organ responded similarly to ATR, although their basal biochemistry was different. ATR toxicity to both kidney and liver showed similar patterns but it appears that the mechanisms of toxicity are different. While cytotoxicity of ATR in kidney is only accompanied with GSH depletion, that of the liver is linked to both lipid peroxidation and GSH depletion. Striated muscle slices from both species were not affected by the highest ATR concentration. This further strengthens the argument that the molecular basis of ATR, target selective toxicity, is not a measure of the interaction between ATR and mitochondria and that other factors such as selective uptake are involved. Precision-cut tissue slices show organ-specific toxicity in kidney and liver from both rat and pig and suggest different mechanisms of injury for each organ.

Optimizing preincubation conditions for precision-cut rat kidney and liver tissue slices: effect of culture media and antioxidants.

Tissue slices are commonly 'preincubated' before use, but optimal conditions to ensure their maximal viability have not been systematically investigated. The effects of serum-free Dulbecco's minimum Eagle's medium and Ham's nutrient mixture (DMEM/F12) (1:1) culture media with and without phenol red (+/-PR), or RPMI-1640 and six different antioxidants on the viability of precision-cut rat kidney and liver slices (200+/-5mum) were investigated. Slice viability was assessed every 30 minutes over a 2-hour preincubation period and after 24 hours of incubation in a multiwell plate culture system maintained at 37 degrees C. In all cases, preincubation produced a time-dependent significant reduction of ethidium bromide positive nuclei stained in each medium and in both kidney and liver slices. Based on lactate dehydrogenase (LDH) leakage, there are viability differences between the media. In contrast, alkaline phosphatase (ALP) leakage and MTT reduction were less sensitive and did not differentiate between slice viability in each incubation medium. Preincubation of kidney and liver slices in DMEM/F12 medium containing antioxidants, indicated an enhanced viability which was specific for each tissue. Extension of the culture period to 24 hours after 1 hour of preincubation showed up to a further 4-13% leakage of ALP or LDH in DMEM/F12 (+/-PR) media for both kidney and liver slices and with a further 5-15% decline in MTT viability assay. RPMI-1640 medium on its own was not a suitable medium for maintaining the viability of either kidney or liver slices. However, kidney or liver slices preincubated with DMEM/F12 medium in the presence of some of the antioxidants were satisfactorily maintained for 24 hours. Exposure of slices to atractyloside (ATR) at concentrations of 0.2-2.0mm in the different media for 24 hours showed a significant increase in enzyme leakage, decline of MTT reductive capacity and increased oxidative damage, with toxicity more elaborate in RPMI-1640 medium. Preincubation of kidney slices with either reduced glutathione (GSH) or alpha-tocopherol (TOC) and liver slices with either GSH or deferoxamine (DEF) followed by 24 hours of exposure to ATR showed a similar decline in toxicity profile. The antioxidants provided partial protection of slices from ATR toxicity. The results demonstrate the importance of slice preincubation and indicate that slices could be maintained in culture using an appropriate medium, thus providing slices that could serve as a useful alternative in vitro system for assessing novel compounds for toxicity.