Modulation of hepatocyte thiol content by medium composition: implications for toxicity studies.
Toxicity of compounds requiring glutathione for detoxification, thiol content and synthesis were determined in 24-h rat hepatocytes cultured in medium containing different concentrations of the sulphur amino acids. Glutathione synthesis was determined following prior depletion of glutathione with diethylmaleate. L-15 medium, which has high levels of cysteine and methionine (1 mM of each), provided some protection against dichloroacetone, dibromopropanol and dichloropropanol toxicity, and had a small effect on increasing glutathione content and synthesis, relative to Williams' medium E (WE) which has low levels (less than 0.5 mM) of both amino acids. However, WE containing N-acetylcysteine (NAC) (1 mM final cysteine concentration), with or without methionine (final concentration 1 mM), was a better cytoprotectant medium than L-15, markedly reducing toxicity of all three compounds, and rapidly (within 1.5 h) increasing cellular glutathione content. WE supplemented with methionine alone stimulated glutathione synthesis after an initial lag phase, and protected cultures against dichloropropanol, but not dibromopropanol or dichloroacetone, both of which are highly reactive in these cultures. There was a clear association between glutathione content at early time points in culture and toxicity observed at later time points, and overall these results indicate that differences in culture medium composition can alter intracellular glutathione content and xenobiotic toxicity.
Hammond
A H
AH
School of Biomedical Sciences, E Floor, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. alison.hammond@nottingham.ac.uk
Garle
M J
MJ
Sooriakumaran
P
P
Fry
J R
JR
D000096:Acetone / Q000031:analogs & derivatives* / Q000633:toxicity; D000603:Amino Acids, Sulfur / Q000032:analysis / Q000494:pharmacology; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008298:Maleates / Q000633:toxicity; D020005:Propanols / Q000633:toxicity; D051381:Rats; D017208:Rats, Wistar; D018675:Toxicity Tests / Q000379:methods*; D000517:alpha-Chlorohydrin / Q000031:analogs & derivatives* / Q000633:toxicity
D000603:Amino Acids, Sulfur; D003470:Culture Media; D004338:Drug Combinations; D008298:Maleates; D020005:Propanols; C033489:1,3-dichloro-2-propanol; D000096:Acetone; C098109:1,3-dibromo-2-propanol; D000517:alpha-Chlorohydrin; C014476:diethyl maleate; D005978:Glutathione; C047675:1,3-dichloroacetone