The hepato-steatogenic compound ethionine has been used to investigate the correlations between in vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and beta-oxidation (liver specific functions). Ethionine (0-30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10-30 mM ethionine and reduced after 20 h in cultured hepatocytes (18-30 mM). Protein synthesis was reduced and beta-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo.

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The aim of this study was to compare the in vitro toxicities of two hepatotoxins in hepatocyte cultures and in liver slices from both rats and dogs. Hepatocytes and liver slices were pre-incubated for 2 hours and then exposed to galactosamine or paracetamol, both of which mainly induce liver necrosis in vivo. Following exposure to the compounds for 20 hours, neutral red uptake (NRU [hepatocyte cultures only]), MTT reduction, and reduced glutathione (GSH), adenosine triphosphate (ATP) and protein content, were used to measure the toxicity induced. In general, galactosamine and paracetamol exposure caused comparable levels of toxicity in hepatocyte cultures and in liver slices. For galactosamine, no consistent differences were seen between hepatocyte cultures and liver slices. With paracetamol, the toxic effects were generally slightly more pronounced in hepatocyte cultures than in liver slices, and the preparations from dog liver were more sensitive than those from rat liver to paracetamol exposure. These results are in agreement with previously described species differences in vitro. NRU and GSH content were more sensitive and more consistent endpoints than MTT reduction, ATP content or protein content. Liver slices appeared to lose viability over the 20 hours in culture. Therefore, it can be concluded that liver slices should only be used in relatively short-term investigations.

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Toxic nephropathy is one of the major diseases that cause renal failure in humans. It can be caused by drugs such as cephalopsporins, aminoglycosides, cyclosporins, and certain anti-cancer drugs. Therefore, drug-induced toxic nephropathy is a relevant and important issue for the pharmaceutical industry.An in vim screen than can identify potential nephrotoxins &y in drug development would therefore be very usehl.Within the kidney, proximal tubule cells are a major target for toxins, and therefore, provide an appropriate cell system for an in vitro model. Cultures of primary renal tubule epithelial cells (RTC) have been established in wed a n i d species (1). It would be extremely valuable for the understanding of species differences and for the predictive value of such in vitro screens if human RTC could also be used.It is possible to isolate human RTC from nephrwtomy specimens removed during tumour resection (Z), but the availability of normal human renal tissue is very poor. A possible source of such n o d RTC could be kidneys donated for transplantation but which cannot be used because of technical or logistical problems. Such kidneys would have been stored for several hours at hypothermia before they could be considered for RTC isolationI1 has so far been uncertain whether viable RTC could be isolated and cultured from such tissues. To investigate this, we have used porcine kidneys, which are similar in size and anatomy to human organs, to develop a method for RTC isolation and culture after hypothermic preservation. Kidneys were. rem6ved fkom Large White pigs under terminal anaesthesia, and flushed via the renal artery with 25Oml of icecold citrate preservation solution (3). The organs were packed in fresh citrate solution in sterile bags and placed in ice for storage. M e r approximately l8h, the kidneys were removed and a 2-step digestion method was used to isolate RTC. The kidneys were bisected laterally, the capsule removed and they were injected randomly with Hanks buffer containing 25mM EGTA to remove any remaining blood. The cortex was then removed from the medullary tissue, chopped and placed into the digestion solution of Hanks buffer with 0.2%w/v dispase and 0.125% w/v collagenase. This mixture was incubated at 37°C on an orbital shaker (1 50rpm). After 70min the digested cortex was washed twice in Hanks (centrihgation at 350rpm for 2 min). This procedure leads to isolated tubular fragments and glomeruli. These were then re-incubated with Hanks containing EGTA for 15 min, followed by 2 washes in Hanks buffer. The second digestion step was canied out in fresh digestion solution (as described above) for 2.31, after which time the tubular fragments were broken down into cells and small clumps of cells. The cells were washed twice as above and the final pellet resuspended in Dh4EMlFlZ media. The cells were then seeded in collagencoated 6. 12 and 24 well plates at 5x10' celldml, and incubated at 37°C for 2 days. These cultures of porcine RTC were assessed by both light and scanning electron microscopy (...