Resistance of three immortalized human hepatocyte cell lines to acetaminophen and N-acetyl-p-benzoquinoneimine toxicity
Section snippets
Materials and Methods
Acetaminophen, reduced glutathione (GSH), oxidised glutathione (GSSG) o-phthaldialdehyde, n-ethylmaleimide, dicumarol, 1,2:5,6-di-benzanthracene (DBA), Hoechst dye 33258 and buthionine-sulfoximine were from Sigma-Aldrich (Poole UK). 7-Ethoxyresorufin, 7-methoxyresorufin and resorufin were from Cambridge Bioscience (Cambridge UK). β-Glucuronidase/arylsulphatase was from Boehringer Mannheim UK (Lewis, Sussex, UK).
Willam's E medium, (MEM) alpha, fetal bovine serum (FBS), antibiotics, trypsin and
Results
There were marked differences in the cytotoxicity of APAP in isolated rat hepatocytes as compared with three human hepatocyte cell lines. Rat hepatocytes were susceptible to the effects of APAP over a relatively short incubation period of 2 h Fig. 1, whereas the three hepatocyte cell lines were resistant to any deleterious effects of APAP in incubations of up to 16 h (Fig. 2) using APAP concentrations that produce cytotoxicity in vivo. A significant decrease in the viability of the rat
Discussion
In this study we have investigated the hepatotoxic effects of APAP and its active metabolite NAPQI on rat hepatocyte cultures and on three human hepatocyte lines: HH25, HH29 and HHY41. Our results on rat hepatocytes are in agreement with the published data 18., 32.. We have shown that rat hepatocytes are susceptible to increasing concentrations of APAP, leading to a decrease in both GSH and GSSG values with a corresponding increase in cytotoxicity as measured by LDH release. Using higher
Acknowledgements
We thank The Liver Group Charity for supporting this work.
References (46)
- et al.
Evidence for the involvement of NABQI in acetaminophen metabolism
Biochem Pharmacol
(1979) - et al.
Varying effects of sulfhydryl nucleophiles on acetaminophen oxidation and sulfhydryl adduct formation
Biochem Pharmacol
(1979) - et al.
The toxicity of acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cytosolic Ca2+
J Biol Chem
(1985) - et al.
Reversal of acetaminophen toxicity in isolated hamster hepatocytes by dithiothreitol
Toxicol Appl Pharmacol
(1986) - et al.
Establishment of a human hepatocyte line derived from primary culture in a collagen gel sandwich culture system
Exp Cell Res
(1995) - et al.
Species differences in the hepatotoxicity of paracetamol are due to differences in the rate of conversion to its toxic metabolite
Biochem Pharmacol
(1987) - et al.
Further characterisation of 'hepatotropin', a high molecular weight hepatotrophic factor in rat serum
J Hepatol
(1989) - et al.
A fluorometric method for determination of oxidised and reduced glutathione in tissues
Anal Biochem
(1976) - et al.
A microassay for measuring Cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96 well plates
Anal Biochem
(1993) - et al.
Measurement of Cytochrome P-450 dependent dealkylation of alkoxyphenoxazones in hepatic S9s and hepatocyte homogenates: effects of dicumarol
Mutat Res
(1985)