The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes

Abstract

The toxicity and carcinogenicity of formaldehyde (HCHO) has been attributed to its ability to form adducts with DNA and proteins. A marked decrease in mitochondrial membrane potential and inhibition of mitochondrial respiration that was accompanied by reactive oxygen species formation occurred when isolated rat hepatocytes were incubated with low concentrations of HCHO in a dose-dependent manner. Hepatocyte GSH was also depleted by HCHO in a dose-dependent manner. At higher HCHO concentrations, lipid peroxidation ensued followed by cell death. Cytotoxicity studies were conducted in which isolated hepatocytes exposed to HCHO were treated with inhibitors of HCHO metabolising enzymes. There was a marked increase in HCHO cytotoxicity when either alcohol dehydrogenase or aldehyde dehydrogenase was inhibited. Inhibition of GSH-dependent HCHO dehydrogenase activity by prior depletion of GSH markedly increased hepatocyte susceptibility to HCHO. In each case, cytotoxicity was dose-dependent and corresponded with a decrease in hepatocyte HCHO metabolism and increased lipid peroxidation. Antioxidants and iron chelators protected against HCHO cytotoxicity. Cytotoxicity was also prevented, when cyclosporine or carnitine was added to prevent the opening of the mitochondrial permeability transition pore which further suggests that HCHO targets the mitochondria. Thus, HCHO-metabolising gene polymorphisms would be expected to have toxicological consequences on an individual's susceptibility to HCHO toxicity and carcinogenesis.

Introduction

Humans are exposed to formaldehyde (HCHO) from both direct environmental sources as well as from the metabolism of xenobiotics. HCHO is commonly used as a tissue preservation agent and is produced in large quantities industrially. Everyday exposure to HCHO includes building materials (e.g. paint, plywood), cosmetics, cigarette smoke, photochemical smog and even various fruits [1], [2]. HCHO is also formed during P450-catalysed dealkylation reactions (e.g. of nitrosamines), aspartame hydrolysis and the oxidation of methanol [1], [2], [3]. Physiological HCHO can be formed by the metabolism of l-methionine, histamine or methylamine and can contribute to biological methylation by folic acid [4].

HCHO has been implicated as a cause of carcinomas, especially in the nasal passage, due to its highly reactive nature with proteins and DNA. The carcinogenicity of hydrazine has also been attributed to DNA reactive formaldehyde hydrazone formation from physiological HCHO [5]. In addition, some studies have linked chronic HCHO exposure in humans to neurodegenerative disorders [6]. Teratogenicity induced in mice by HCHO has been attributed to folic acid deficiency in the embryo [7].

However, the toxicity of HCHO as a consequence of chronic exposure to HCHO-generating xenobiotics has been largely overlooked because HCHO is rapidly metabolised and removed from the organism. As shown in Fig. 1, HCHO could be reduced to methanol by cytosolic alcohol dehydrogenase (ADH1) or oxidised to formate by mitochondrial aldehyde dehydrogenase (ALDH2) or cytosolic GSH-dependent formaldehyde dehydrogenase (ADH3) [8], [9]. It has also been shown that HCHO is a substrate for CYP 2E1 and can thus be oxidised by the endoplasmic reticulum [10].

The kinetics of HCHO metabolism by ADH1, ALDH2 and ADH3 have been studied as have the effects of various enzyme inhibitors on its rate of metabolism [8], [9], [11], but few metabolism studies have been correlated with HCHO-induced cytotoxicity. Since methanol or HCHO given orally to rodents causes hepatotoxicity and decreases antioxidant enzyme levels [12], we have used isolated hepatocytes to compare the cytoprotective activity of these metabolising enzymes and determine the molecular mechanisms involved in HCHO-induced cell lysis.