Original Contribution
Mechanism of oxidative DNA damage induced by carcinogenic allyl isothiocyanate

https://doi.org/10.1016/S0891-5849(00)00168-4Get rights and content

Abstract

Several isothiocyanates have been proposed as promising chemopreventive agents for human cancers. However, it has been reported that allyl isothiocyanate exhibit carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumor-promoting activities. We investigated whether these isothiocyanates could cause DNA damage, using 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Allyl isothiocyanate caused Cu(II)-mediated DNA damage and formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) more strongly than benzyl and phenethyl isothiocyanates. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited Cu(II)-mediated DNA damage by these isothiocyanates, suggesting involvement of H2O2 and Cu(I). Isothiocyanates induced DNA damage frequently at thymine and cytosine residues in the presence of Cu(II). A UV-visible spectroscopic study revealed an association between the generation of superoxide and the yield of SH group from isothiocyanates. Furthermore, the yield of 8-oxodG formation was correlated with their superoxide-generating ability. Allyl isothiocyanate significantly induced 8-oxodG formation in HL-60 cells, but not in H2O2-resistant HP100 cells, suggesting the involvement of H2O2 in cellular DNA damage. We conclude that oxidative DNA damage may play important roles in carcinogenic processes induced by allyl isothiocyanate.

Introduction

Organic isothiocyanates (R-N=C=S), also known as mustard oils, are widely distributed in plants, many of which are consumed by humans. Vegetables belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (ITCs), mostly in the form of their glucosinolate precursors [1]. Epidemiological studies have shown a marked reduction in the risk of developing a variety of malignancies by large consumption of vegetables [2], [3]. In addition, extracts of broccoli sprouts were effective in reducing tumors in carcinogen-treated rats [4]. In relation to these findings, several studies revealed that preventive effects of ITCs against chemical carcinogen-induced carcinogenesis in vivo and in vitro [5], [6], [7], [8], [9], [10], [11]. Therefore, it has been expected that some kinds of ITCs can be promising chemopreventive agents for human neoplasia.

The National Toxicology Program [12] has evaluated that allyl isothiocyanate (AITC) is carcinogenic to rats. That is, AITC caused transitional-cell papillomas and epithelial hyperplasia in the urinary bladders of male rats, and fibrosarcomas in the subcutaneous tissue in female rats in two-year bioassay [12], [13]. It has been reported that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) exhibit promotion potential during the postinitiation stage [14], [15]. BITC has been shown to induce both chromosome aberrations and sister chromatid exchanges in cultured cells in the absence of an exogenous metabolic activation system and also to induce DNA strand breaks as measured by single-cell gel electrophoresis assay [16].

To clarify the mechanism of carcinogenic activity by ITCs, we investigated whether ITCs can cause DNA damage, using 32P-5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. We analyzed 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in calf thymus DNA treated with ITCs in the presence of Cu(II). To clarify the mechanism of oxidative DNA damage, we measured the amounts of the SH group and superoxide (O2•−) using UV-visible spectroscopy. Furthermore, inductions of 8-oxodG formation were investigated in human leukemia cell lines HL-60 cells and its hydrogen peroxide (H2O2)-resistant clone HP100 cells.

Section snippets

Materials

Restriction enzymes (Sma I, Eco RI, Apa I and Sty I), calf intestine phosphatase, and proteinase K were purchased from Boehringer Mannheim (Mannheim, Germany). Restriction enzymes (HindIII, Ava I, and Xba I) and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA, USA). [γ-32P]-ATP (222 TBq/mmol) was from New England Nuclear (Boston, MA, USA). The chemical structures of the three ITCs used in this study are shown in Fig. 1. AITC and copper (II) chloride dihydrate were

Damage of 32P-labeled DNA fragments by ITCs in the presence of Cu(II)

Figure 2 shows an autoradiogram of DNA fragments treated with ITCs in the presence and absence of Cu(II). Oligonucleotides were detected on the autoradiogram as a result of DNA damage. In the absence of ITCs, DNA damage was not observed with Cu(II) alone under the conditions used. ITCs alone did not cause DNA damage. ITCs induced DNA damage in the presence of Cu(II). The intensity of DNA damage increased with increasing concentration of ITCs (Fig. 2) and incubation time (data not shown).

Discussion

The present study has demonstrated that three representative ITCs have abilities to cause site-specific DNA damage in the presence of Cu(II). In addition, these ITCs induced 8-oxodG formation, a marker of oxidative DNA damage. The extent of DNA damage was dependent on the yield of O2, of which generation is associated with formation of the SH group. Relevantly, reduced glutathione, which has an SH group, was found to cause DNA damage in the presence of Cu(II) [28], [29], [30], [31]. A

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