Ethanol-induced apoptosis in vitro

doi:10.1016/S0009-9120(99)00054-5

Clinical Biochemistry

Volume 32, Issue 7, October 1999, Pages 547-555

https://doi.org/10.1016/S0009-9120(99)00054-5 Get rights and content

Abstract

Objectives: The aim is to study the apoptotic process in a human hepatocyte model for ethanol (EtOH)-induced apoptosis.

Design and methods: Normal human primary hepatocytes (HPH) and Hep G2 cells were exposed to increasing EtOH. 6000 cells/sample were analyzed by transmission electron microscopy.

Results: Apoptotic cells were observed (mmol/L EtOH): 40: 6 ± 0.5%, 60: 13 ± 2% (p < 0.05), 80: 26 ± 1% (p < 0.001) (vs. control). Two consecutive doses of 80 mmol/L for 24 h each additionally increased apoptosis 55 ± 3% (p < 0.0001 vs. control and p < 0.001 vs. single dose). In response to this exposure, there is a stronger apoptotic activity in HPH when compared to Hep G2 (p < 0.05).

Conclusions: In vitro, EtOH-induced apoptosis is regulated by dose level and the frequency of exposure.

Introduction

R egulation of organ size and liver function is the result of a dynamic balance between stimulatory and inhibitory signals 1, 2, 3, 4, 5, 6, 7, 8. Under physiological conditions, senescent or damaged cells are removed by apoptosis, whereas the response to acute cell injury mostly involves a necrotic process 9, 10. After EtOH ingestion, rat and mice models showed subcellular changes compatible with apoptosis 11, 12.

We have been able to reproduce a model of EtOH-induced liver damage in human Hep G2 cells in vitro (13). Biochemical and morphological features in our model present the same pattern observed in the serum of patients with alcohol-induced liver damage and human biopsies from the same category of patients (14). Previously, we reported that in Hep G2 cells (15) and Hep G2 in co-culture with normal human stellate cells, apoptosis can be detected and measured when exposed to 80 mmol/L EtOH (16). Hep G2 cells have inducible cytochrome P450 2E1 activity but low content of CYP 2E1 (17) and a small amount of alcohol dehydrogenase activity (ADH) (18). CYP 2E1 and ADH are associated with EtOH-induced liver damage and are thought to be critical in the hepatocellular regulation of apoptotic processes.

When comparing the inducibility of cytochrome P450 2E1 and ADH activity in Hep G2 cells versus HPH treated with 80 mmol/L EtOH a significant difference was observed 17, 18. Therefore, we decided to parallel our observations in Hep G2 line with studies in HPH. Cells in these HPH cultures retain morphological features of hepatocytes by both light and electron microscopy. They also retain glucose-6-phosphatase activity and secrete proteins characteristic of hepatocytes, such as albumin, alpha-fetoprotein and transferrin (19).

This work deals with the differences and similarities in an apoptotic-induced cascade by EtOH in HPH in comparison with the Hep G2 cells. Detection of cell death by apoptosis, expressed by normal hepatocytes that retain liver-specific functions of differentiated hepatocytes, can be considered a new application of studying human hepatocyte reactions under EtOH stress.

Section snippets

Primary human hepatocytes culture

The hepatocyte cultures were developed from normal liver tissue obtained after lobectomy from organ donors where only a part of the liver was used for transplantation, by cultivating in a highly enriched medium. Our method for preparing hepatocytes suspension was based originally on that of Ballet et al. (20). The liver graft was perfused with ice-cold University of Wisconsin solution (21) and kept on ice (2–10 h) until cell isolation. The material was washed with HEPES media. The hepatocytes

Light and electron microscopy

Cells incubated with plain media show normal organelles: abundant mitochondria, rough and smooth endoplasmic reticulum and only occasional small lipid vesicles (Figure 1). Bile canaliculus-like structure can be observed at the confluence of 3 cells suggesting that the cells are functional.

In previous publications 13, 24, we reported that exposure to 80 mmol/L EtOH produces changes in mitochondria, endoplasmic reticulum, and in the number of lipid vesicles per cell, as well as changes in lipid

Discussion

The mechanism whereby EtOH produces liver damage is still a matter of considerable debate. The current study was designed to test the hypothesis that chronic EtOH exposure promotes changes in hepatocytes that lead to apoptosis. Hepatocytes contain plasma membranes as well as many other membrane-bound subcellular organelles including mitochondria, lysosomes and endoplasmic reticulum which have been as targets of injurious effects of EtOH and/or its toxic metabolites (26).

There is a growing body

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InvitedEthanol-induced apoptosis in vitro

Abstract

Introduction

Section snippets

Primary human hepatocytes culture

Light and electron microscopy

Discussion

J Hepatol

Gastroenterology

In Vitro Toxicol

Hepatology

Dev Brain Res

J Lab Clin Med

Shrinkage necrosisA distinct mode of cellular death

J Path

ApoptosisA basic biological phenomenon with wide ranging implications in tissue kinetics

Br J Cancer

Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes

Nature

Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor α1

Proc Natl Acad Sci USA

Apoptosis is induced by transforming growth factor-α1 within 5 hours in regressing liver without significant fragmentation of the DNA

Hepatology

Hepatocytes apoptosisIs transforming growth factor-β1 the kiss of death?

Hepatology

Mechanisms and genes of cellular suicide

Science

Apoptosis in the pathogenesis and treatment of disease

Science

Unbalanced transmethylation and the perturbation of the differentiated state leading to cancer

Bioassays

Life (and death) in malignant tumor

Nature

Apoptotic bodies in a murine model of alcoholic liver diseaseReversibility of ethanol-induced changes

J Pathol

In vitro, assessment of the ethanol-induced hepatotoxicity on Hep G2 cell line

Biochem Biophys Res Commun

Molecular pathological and electron microscopic changes in ethanol-induced apoptotic cells, in vitro

Hepatology

Ethanol-induced apoptosis in a co-culture of Hep G2 and normal human stellate cells

Cell Biol Toxicol

Invited
Ethanol-induced apoptosis in vitro