Formation of DNA adducts by microsomal and peroxidase activation of p-cresol: role of quinone methide in DNA adduct formation

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Abstract

We have investigated the activation of p-cresol to form DNA adducts using horseradish peroxidase, rat liver microsomes and MnO2. In vitro activation of p-cresol with horseradish peroxidase produced six DNA adducts with a relative adduct level of 8.03±0.43×10−7. The formation of DNA adducts by oxidation of p-cresol with horseradish peroxidase was inhibited 65 and 95% by the addition of either 250 or 500 μM ascorbic acid to the incubation. Activation of p-cresol with phenobarbital-induced rat liver microsomes with NADPH as the cofactor; resulted in the formation of a single DNA adduct with a relative adduct level of 0.28±0.08×10−7. Similar incubations of p-cresol with microsomes and cumene hydroperoxide yielded three DNA adducts with a relative adduct level of 0.35±0.03×10−7. p-Cresol was oxidized with MnO2 to a quinone methide. Reaction of p-cresol (QM) with DNA produced five major adducts and a relative adduct level of 20.38±1.16×10−7. DNA adducts 1, 2 and 3 formed by activation of p-cresol with either horseradish peroxidase or microsomes, are the same as that produced by p-cresol (QM). This observation suggests that p-cresol is activated to a quinone methide intermediate by these activation systems. Incubation of deoxyguanosine-3′-phosphate with p-cresol (QM) resulted in a adduct pattern similar to that observed with DNA; suggesting that guanine is the principal site for modification. Taken together these results demonstrate that oxidation of p-cresol to the quinone methide intermediate results in the formation of DNA adducts. We propose that the DNA adducts formed by p-cresol may be used as molecular biomarkers of occupational exposure to toluene.

Introduction

Benzene, toluene, xylene and styrene are monocyclic aromatic hydrocarbons. These compounds are widely used in the chemical industry as intermediates for synthesis and as solvents and dyes [1], [2]. These compounds are also present in gasoline and a variety of other products such as paints and adhesives. With recognition of benzene as a human leukemogen [3], its use in many of these products has been replaced by toluene and xylene.

Epidemiology studies have demonstrated that occupational exposure to benzene results in a increased risk for acute myelogenous leukemia in humans [3]. Similar studies with toluene, xylene and styrene have suggested an association between exposure to these agents and a increased risk for certain cancers [4], [5], [6], [7]. However, the analysis of the association(s) between exposure to a monocyclic hydrocarbon such as toluene, xylene or styrene and risk for cancer is complicated by the fact that in most occupational settings exposure is to a mixture of these hydrocarbons with previous exposure to benzene also a possibility [2].

Following inhalation exposure, these agents are metabolized in the liver. In the case of benzene and styrene, this metabolism results in the formation of reactive intermediates which bind to DNA and protein [8], [9], [10], [11]. The DNA adducts formed by the metabolites of benzene [12], [13], [14] and styrene [15], [16], [17], [18] have been identified and these DNA adducts have been used as biomarkers for exposure in both rodent models [19], [20] and in exposed populations [21], [22], [23]. These results with benzene and styrene suggest that identification of the DNA adducts formed by toluene and xylene may provide molecular biomarkers to evaluate human exposure to these agents.

A minor pathway of toluene metabolism is to toluene epoxide(s) which rearrange to either ortho- or p-cresol. Previous studies have demonstrated that enzymatic oxidation of p-cresol results in the formation of a quinone methide [24]. The formation of quinone methides from various 4-alkylphenols has been established by the isolation and characterization of glutathione conjugates produced by oxidation of alkylphenols with PB-induced rat liver microsomes and NADPH [24]. Our laboratory has been interested in studying DNA adduct formation by quinones and quinone methides. We have demonstrated that p-benzoquinone [25], ortho-phenyl-benzoquinone [26] and the quinone methides generated from eugenol [27] and tamoxifen metabolites [28], [29] can react with DNA to form adducts. These results suggest that the quinone methide of p-cresol may react with DNA to form adducts.

The purpose of this study was to determine if oxidation of p-cresol results in a intermediate which can bind to DNA. The results of the study demonstrate that p-cresol can be activated by either rat liver microsomes, HRP or MnO2 to form a quinone methide intermediate which reacts with DNA to form adducts. These results suggest that the DNA adducts formed by p-cresol may be used as molecular biomarkers to evaluate occupational exposure to toluene.

Section snippets

Reagents

p-Cresol, calf thymus DNA, HRP (Type VI), and H2O2 were purchased from Sigma Chemical Co. (St. Louis, MO). Rat liver microsomes prepared from female Sprague–Dawley rats given daily ip injections of PB (80 mg kg−1) for 5 days were purchased from Molecular Toxicology, Inc. (Boone, NC). All other chemicals were of the highest analytical grade available.

Microsomal activation of p-cresol

The incubation mixture consisted of PB-induced rat liver microsomal protein (2 mg), 100 μM p-cresol, 500 μg of purified calf thymus DNA and either

Results

DNA adduct formation by microsomal activation of p-cresol was investigated. Microsomes prepared from the livers of PB-treated rats were used for these studies. In control experiments in which no cofactor was added to the incubation mixture no DNA adducts were detected (data not shown). When NADPH was used as the cofactor for microsomal activation of p-cresol, one DNA adduct (1) was formed. The relative adduct level was 0.28±0.08×10−7 (Fig. 1A). When CuOOH was used as the cofactor for microsomal

Discussion

These studies have investigated the activation of p-cresol with rat liver microsomes, HRP and MnO2 to form DNA adducts. The results of this study demonstrate that p-cresol can be activated to form multiple DNA adducts. Three of the DNA adducts formed by p-cresol (1, 2 and 3) were common to all of the activation systems. The other DNA adducts detected were unique to the activation method.

PB-induced rat liver microsomes were used as one of the activation methods. We chose PB-induced microsomes for

Acknowledgements

These studies have been supported by grants CA 78237 from NCI and ES09500 from NIEHS.

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