Attenuation of endogenous oxidative stress–induced cell death by cytochrome P450 inhibitors in primary cultures of rat hepatocytes

doi:10.1016/S0891-5849(99)00150-1

Free Radical Biology and Medicine

Volume 27, Issues 9–10, November 1999, Pages 1019-1026

https://doi.org/10.1016/S0891-5849(99)00150-1 Get rights and content

Abstract

This study reports an examination of the effects of endogenous oxidative stress on primary cultures of rat hepatocytes. To produce endogenous oxidative stress, 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), which are known to inhibit catalase and glutathione peroxidase activities, respectively, were used. When ATZ or MS was used alone, the extent of cell injuries was negligible, but a combination of the two agents resulted in cell death as assessed by trypan blue exclusion after 24 h of incubation. Cell death was accompanied by an approximately 5.8-fold the increase in the levels of thiobarbituric acid reactive substances, and showed chromatin condensation and DNA fragmentation. These deleterious effects were time dependent in that no significant change was detected up to 6 h. Treatment with SKF or 1-aminobenzotriazole, which are inhibitors of cytochrome P450, greatly attenuated this cell death as well as prevented chromatin condensation and DNA fragmentation. N^G-monomethyl-l-arginine at 1 mM had no inhibitory effects on these changes. These findings suggest that endogenous oxidative stress under these conditions induced cell death that resembles apoptosis and that endogenous oxidative stress was directly related to the cytochrome P450 enzyme system in this system.

Introduction

A cell is generally able to maintain an appropriate balance between oxidants and antioxidants in normal conditions. When this balance is perturbed and shifts toward an oxidative state by either an increase in oxidants or decrease in antioxidants, the cell becomes more susceptible to injury and, if this condition exists for an extended time, death ensues [1], [2], [3]. In vitro studies of oxidative stress could be classified as endogenous and exogenous, depending on the method used to induce oxidative stress. The relationship between oxidative stress and cell injuries in hepatocytes has been the subject of extensive study and has largely involved studies using exogenous oxidative stress, which includes a wide variety of chemicals [4], [5], [6] as well as both low [7], [8], [9] and high [10], [11], [12], [13] molecular weight biological substances. In terms of cell injuries by oxidative stress, it is generally accepted that the site of generation of oxidative stress must be taken into consideration, in addition to species and the amounts of reactive oxygen species produced. The reason for this is that each reactive oxygen species, such as superoxide anion, hydrogen peroxide, and hydroxyl radical, has different characteristics with regard to chemical reactivity or stability. In this regard, cell injury induced by endogenous oxidative stress would be expected to be different from that induced by exogenous approaches. In addition, it is generally accepted that two modes of cell death, apoptosis and necrosis, exist that are thought to be determined by the type of oxidative stress, the level of oxidative stress, or both [14]. Low doses of oxidants induce apoptosis, whereas higher doses provoke necrosis [4], [15], [16]. Several growth factors, when applied exogenously, have been observed to induce apoptosis [10], [11] or necrosis [12], [13]. In either case, the involvement of oxidative stress was presumed. Furthermore, cytochrome P450 has long been known to be involved in oxidative stress [17], [18]. As mentioned above, numerous reports have appeared relative to exogenous oxidative stress, but studies concerning the relationship between endogenous oxidative stress and cell injuries are relatively few. Thus this study was conducted to examine cell injuries, including mode of death, by endogenous oxidative stress in primary cultures of rat hepatocytes in relation to participation of the cytochrome P450 enzyme system. To produce direct, intracellular oxidative stress, we used 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), which are known to inhibit catalase (Cat) and glutathione peroxidase (GPx) activities, respectively.

Section snippets

Chemicals

Williams’ E medium, 3-amino-1,2,4-triazole (ATZ), mercaptosuccinic acid (MS), SKF (proadifen), and N^G-monomethyl-l-arginine (l-NMMA) were obtained from Sigma Ltd. (St. Louis, MO, USA). Hoechst 33342 was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). 1-Aminobenzotriazole (ABT) was obtained from Fluka Ltd. (Buchs, Switzerland). All other chemicals were obtained from Sigma Ltd. or Wako (Osaka, Japan) and were of the highest quality commercially available.

Isolation and culture of rat hepatocytes

Male Wistar rats (150–190

Cell death induced by treatment with ATZ and MS

The viability of untreated hepatocytes at 24 h was 92.8 ± 0.8% (Fig. 1). Exposure of primary cultures of rat hepatocytes for 24 h to ATZ or MS alone had almost no effect on cell viability (85.4 ± 2.2% and 91.1 ± 2.3%, respectively). A combination of ATZ and MS, however, caused statistically significant lethal cell injuries as revealed by a decrease in viability to 13.7 ± 5.2%. The effect of combined treatments with ATZ and MS was significant as compared with that of either ATZ or MS treatment.

Discussion

In studies using hepatocyte cultures, experimental conditions are thought to be one of the key determinants [21]. In this study almost all cells were mono- and binuclear cells which ratio remained unchanged, the ratio of viability was maintained at a high level, and the ratio of chromatin condensation and TBARS production remained very low during the experimental periods herein. Thus the experimental system is considered to be relevant to the current study.

In primary cultures of rat

Acknowledgements

We thank Dr. S. Asahi and Dr. T. Nakashima for their helpful discussion, and Dr. N. Yamazaki for statistical analysis. We are also grateful to Drs. M. Fujino, K. Meguro, O. Nishimura, and S. Terao for their encouragement throughout this study.

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Original ContributionsAttenuation of endogenous oxidative stress–induced cell death by cytochrome P450 inhibitors in primary cultures of rat hepatocytes

Abstract

Introduction

Section snippets

Chemicals

Isolation and culture of rat hepatocytes

Cell death induced by treatment with ATZ and MS

Discussion

Acknowledgements

Toxicol. Lett.

Arch. Biochem. Biophys.

Gastroenterology

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Gastroenterology

Immunol. Today

Methods. Cell. Biol.

Biochem. Med.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

Hepatology

Biochem. Pharmacol.

Free Radic. Biol. Med.

Biochem. Pharmacol.

Kidney Int.

Arch. Biochem. Biophys.

J. Biol. Chem.

Gastroenterology

Original Contributions
Attenuation of endogenous oxidative stress–induced cell death by cytochrome P450 inhibitors in primary cultures of rat hepatocytes