Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations.
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.
Waterfield
C J
CJ
Department of Pharmacy, King's College London, UK.
Westmoreland
C
C
Asker
D S
DS
Murdock
J C
JC
George
E
E
Timbrell
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000410:Alanine Transaminase / Q000378:metabolism; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D005001:Ethionine / Q000633:toxicity*; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D051381:Rats; D018675:Toxicity Tests / Q000379:methods*; D014280:Triglycerides / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D005227:Fatty Acids; D014280:Triglycerides; D002956:Citrulline; D000255:Adenosine Triphosphate; D014508:Urea; D007770:L-Lactate Dehydrogenase; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D005978:Glutathione; D005001:Ethionine