Correlation between formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites determined by a comet assay, increased MDA, and decreased glutathione during long exposure to thinner inhalation

Abstract

Thinner inhalation causes toxic effects in a variety of organs, principally in the central nervous system. Some studies have shown oxidative stress effects of thinner inhalation, such as: activation of free radical processes, decrease of antioxidants, and oxidation products of proteins and lipids but not of DNA. The aim of this study is to investigate the effect of thinner inhalation on DNA. We used the comet assay in conjunction with the enzyme formamidopyrimidine glycoslyase (Fpg). Our results show a significant increase in Fpg-sensitive sites in DNA of lymphocytes from rats exposed to thinner fumes compared to lymphocytes from control rats (p < 0.05). Moreover, DNA damage detected with Fpg shows a high correlation with increased malondialdehyde (MDA) and decreased glutathione (GSH), two widely used biomarkers of oxidative stress. The most abundant base oxidation product found in DNA is 8-oxoguanine; it is the main substrate of Fpg and the most commonly used biomarker for oxidative DNA damage. This suggests that oxidative DNA damage is at least partly responsible for the DNA damage detected by Fpg. We propose the comet assay in combination with Fpg as a sensitive biomarker to monitor exposure to thinner inhalation. Limitations of this method are discussed.

Introduction

Thinners are chemical mixtures containing toluene, benzene, acetone, methanol, hexanes, and other substances. They are widely used as industrial solvents and have potential for abuse inhalation, which is a public health problem in Mexico. Data from the Mexican Survey on Addiction showed that inhaled solvents were the second most frequently used drugs among street children and teenagers (Medina-Mora et al., 2003). Glue and thinner sniffing are the most common forms of thinner abuse due to their accessibility and low cost (Villatoro et al., 2002).

Thinner sniffing causes damage to the brain, kidney, liver, lung, and reproductive system. Oxidative stress is a possible mechanism for tissue damage in individuals and animals exposed to thinner fumes. In one report, toluene increased the intensity of chemiluminescence, indicating activation of free radical processes in brain (Burmistrov et al., 2001). Increased lipid peroxidation, depletion of glutathione (GSH), and a neuroprotective effect of melatonin have been observed in brain after thinner exposure (Baydas et al., 2003). In lung, thinner inhalation caused increased lipid peroxidation, decreased superoxide dismutase activity, and GSH depletion (Zengin et al., 1998). In the plasma of people who abused inhalants, lipid peroxidation increased, and GSH levels decreased (Halifeoglu et al., 2000, Dündaröz et al., 2003).

However, the effect of thinner inhalation on DNA has not been studied. Toluene is the principal component of most thinners (accounting for 60–70% of thinner volume). Toluene is mainly metabolized to benzoic acid, but minor toluene metabolism is responsible for the toxic effects of thinner. A minor pathway of toluene metabolism yields toluene epoxide(s), which rearranges to cresol; cresol, in turn, is hydroxylated to form methylhydroquinone and methylbenzoquinone. These quinones, during redox processes generate reactive oxygen species (ROS) that can cause “oxidative stress” and oxidative DNA damage (Murata et al., 1999). The minor toluene metabolite p-cresol can also induce DNA damage by direct alkylation. Enzymatic oxidation of p-cresol results in the formation of a quinone methide, which reacts with DNA. The resulting DNA adducts have been proposed as specific biomarkers for toluene exposure in humans (Gaikwad and Bodell, 2003).

The aim of this project was to examine DNA damage in lymphocytes of rats exposed to thinner fumes over a period of 16 weeks. We examined lymphocytes, because they are routinely used to monitor DNA damage in human populations and can be obtained easily and repeatedly from the same animals during the course of exposure. We combined single cell gel electrophoresis (comet assay), a simple and sensitive technique to detect DNA damage, with a restriction enzyme formamidopyrimidine glycoslyase (Fpg), which recognizes oxidized purines (products of DNA oxidation) and some alkylated DNA products (Cadet et al., 2003, Tudek et al., 1998). In addition, we assessed two oxidative stress biomarkers (GSH and malondialdehyde (MDA)) used in previous studies that showed oxidative stress effects of thinner inhalation. We established the interrelationships between GSH depletion, MDA increase, and DNA damage. Overall, our analysis demonstrated a high correlation between the levels of these two biomarkers of oxidative stress and the number of Fpg-sensitive sites in DNA induced by thinner inhalation. Thus, we suggest that DNA damage detected by the Fpg enzyme corresponds, at least in part, to oxidative DNA damage. In addition, we showed that the comet assay, in combination with the enzyme Fpg, can be used as a rapid test for biomonitoring exposure to organic solvents.