Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Molecular mechanisms of DNA damage induced by procarbazine in the presence of Cu(II)
Introduction
Hydrazine derivatives have been implicated as mutagens and carcinogens [1], [2], [3]. Procarbazine [N-isopropyl-α-(2-methylhydrazino)-p-toluamide], a hydrazine derivative, has effective antineoplastic activity in Hodgkin’s disease, lung tumor, brain tumor, malignant melanoma and skin tumor [4], [5]. Procarbazine can induce tumor including leukemia, lung tumor and nasal tumor in animals [6], [7], [8], [9]. MOPP chemotherapy including procarbazine was found to induce lung tumor and leukemia in humans [10], [11], [12], [13]. The International Agency for Research on Cancer (IARC) has assessed that procarbazine is probably carcinogenic to humans (group 2A) [14], [15].
It is reported that several enzymes, including horseradish peroxidase, prostaglandin synthetase and cytochrome P450, catalyze oxidation of procarbazine, leading to the formation of carbon-centered free radicals [16], [17]. Several studies suggest the importance of azoxy derivatives in metabolism and toxicity of procarbazine, which would lead to alkylation [18]. Therefore, generation of alkyl radicals and alkylation under some enzymatic systems play a role in both antineoplastic action and carcinogenesis.
In order to clarify a new molecular mechanism of procarbazine-induced antineoplastic and carcinogenic activity through non-enzymatic activation, we examined procarbazine-induced site-specific DNA damage using -5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. We also investigated the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage [19], in calf thymus DNA with an electrochemical detector coupled to an HPLC (HPLC-ECD). Moreover, we analyzed the formation of free radicals, especially carbon-centered radicals, by electron spin resonance (ESR) spin-trapping experiments.
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Materials
Restriction enzymes (HindIII, AvaI and PstI) and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). Restriction enzymes (EcoRI, StyI and ApaI) and calf intestine alkaline phosphatase were from Boehringer Mannheim GmbH (Mannheim, Germany). [γ-]-ATP (222 TBq/mmol) was from New England Nuclear (Boston, MA). Procarbazine hydrochloride was a gift from Roche (Mannheim, Germany). 2-Methyl-2-nitrosopropane (MNP), catalase (45,000 units/mg from bovine liver) and
Damage to -labeled DNA fragments by procarbazine in the presence of Cu(II)
Fig. 1 shows an autoradiogram of DNA fragments treated with procarbazine plus Cu(II). Oligonucleotides were detected on the autoradiogram as a result of DNA damage. In the presence of Cu(II), procarbazine-induced DNA damage in dose-dependent (Fig. 1) and time-dependent (data not shown) manners. Piperidine treatment enhanced DNA damage, suggesting that not only strand breakage but also base modification and/or liberation were induced by procarbazine plus Cu(II). Procarbazine did not cause DNA
Discussion
We demonstrated that procarbazine-induced DNA damage non-enzymatically. Procarbazine plus Cu(II) induced piperidine-labile lesions preferentially at thymine, cytosine and guanine residues and Fpg-sensitive lesions at guanine residues. Fpg protein catalyzes the excision of 8-oxodG, a relevant indicator of oxidative base damage [29], [30], [31]. It is noteworthy that procarbazine caused double-base lesions at the 5′-A-3′ sequence, complementary to a hotspot of the p53 gene, and the 5′-TG-3′
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The first two authors contributed equally to this work.