Elsevier

Journal of Hepatology

Volume 31, Issue 5, November 1999, Pages 841-851
Journal of Hepatology

Resistance of three immortalized human hepatocyte cell lines to acetaminophen and N-acetyl-p-benzoquinoneimine toxicity

https://doi.org/10.1016/S0168-8278(99)80285-2Get rights and content

Abstract

Background/Aims: Acetaminophen toxicity in hepatocytes is attributed to generation of the toxic metabolite N-acetyl-p-benzoquinoneimine, leading to depletion of intracellular glutathione, alteration of redox potential and ultimately, cellular necrosis. We aimed to determine the effect of acetaminophen and N-acetyl-p-benzoquinoneimine on three human hepatocyte cell lines HH25, HH29 and HHY41, and for comparison, on primary rat hepatocytes, a cell type that is relatively resistant to acetaminophen-induced toxicity.

Methods: We investigated the effect of incubation of rat hepatocytes and 3 hepatocyte cell lines with acetaminophen or N-acetyl-p-benzoquinoneimine on LDH release, glutathione status, mitochondrial function, CYP1A activity, albumin synthesis and DNA content. Results: We demonstrated that HH25, HH29 and HHY41 are resistant to the toxic effects of acetaminophen under conditions that induce cytotoxicity in rat primary hepatocytes, as indicated by maintenance of glutathione levels and basal LDH release. Incubation with N-acetyl-p-benzoquinoneimine caused a dose-dependent cytotoxicity in rat hepatocytes. Under comparable conditions N-acetyl-p-benzoquinoneimine had no effect on any of the hepatocyte cell lines. Nevertheless, when culturing the cells for a further 48 h, a decrease in glutathione levels, albumin synthesis, CYP1A activity, DNA content and mitochondrial function was apparent.

Conclusion: HH25, HH29 and HHY41 cells are highly resistant to acetaminophen and N-acetyl-p-benzoquinoneimine-induced toxicity. They tolerate a much higher concentration of both toxins for a longer period of time compared to rat primary hepatocytes. These results are of relevance in the use of these cell lines to investigate acetaminophen hepatotoxicity, and may be of importance in the choice of cells for use in bioartificial liver support systems.

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.

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