DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique

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Abstract

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with β-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.

Introduction

Several plant extracts and their components exhibit toxic side effects in addition to their desired pharmacological activities. Many adverse effects are due to selective interaction of specific structures (e.g. ion pumps, intracellular receptors) or as a result of metabolic activation to reactive intermediates, which are capable of covalent binding to cellular macromolecules. Covalent binding to proteins can cause cell death by disturbing essential biochemical processes or immunological reactions. Furthermore, a large body of evidence in experimental systems suggests that DNA adduct formation is a critical event in the initiation stage of carcinogenesis [1]. Indeed, some of the most potent known carcinogens are natural products [2]. Among those identified in plants, safrole, aflatoxins, cycasin, aristolochic acids and pyrrolizidine alkaloids have attracted considerable attention [2], [3], [4].

Quaternary benzo[c]phenanthridine alkaloids (QBA) represent another group of natural products exhibiting a wide spectrum of pharmacological activities and numerous toxic side effects [5], [6], [7], [8]. QBA were found in plants of Caprifoliaceae, Fumariaceae, Meliaceae, Papaveraceae, and Rutacea families [6]. They belong to the elicitor-inducible secondary metabolites and are called phytoallexines because of their anti-microbial and anti-fungal activities [5], [6], [7], [8]. Sanguiritrin, a QBA extract from Macleya cordata (a mixture of sanguinarine, chelerythrine (Fig. 1) and three minor QBAs), is used as an anti-microbial agent and for treatment of myopathy [5], [9]. Sanguinaria, extracted from Sanguinaria cadanensis (a mixture of sanguinarine, chelerythrine and four minor QBAs), is used as the antiplaque component in toothpaste and oral rinses manufactured in Europe and USA [10]. Sanguinarine may have an antitumor potential [11]. Another QBA, fagaronine (Fig. 1), demonstrates antileukemic activity [5], inhibits HIV-1 and HIV-2-reverse transcriptase [12], and DNA topoisomerase I and II [13]. Fagaronine is considered a potential novel antitumor drug [5].

A well-known toxic effect of QBA is the epidemic dropsy syndrome, which is associated with consumption of plant oil contaminated by alkaloids of Argemone mexicana[14]. Damm et al. [15] have reported that the long-term use of oral products containing sanguinaria appears to be associated with an increased prevalence of leukoplakia of maxillary vestibule. Furthermore, sanguinarine and chelerythrine have demonstrated toxicity in rat hepatocytes [16], cultured human and mouse fibroblasts [17] and/or in the porcine and human hepatocyte primary cultures [18]. The sanguinarine-mediated cytotoxicity in mice was reduced by pretreatment with 3-methylcholanthrene, an inducer of cytochrome P450 enzymes [19]. In addition to cytotoxicity, sanguinarine elicited positive mutagenic responses in the Salmonella mutagenicity test after metabolic activation [20] and chelerythrine induced respiration-deficient mutants in Saccharomyces cerevisieae[21]. However, neither teratogenicity nor increases in preneoplastic or neoplastic lesions have been shown following treatment with a mixture of sanguinarine and chelerythrine in long-term rat bioassays [5].

Sanguinarine forms a molecular complex with DNA by intercalation [22], [23], [24], [25]. Whereas, DNA intercalation of this alkaloid is well understood [22], [23], [24], [25], other types of DNA modification by QBA remain to be explored. Therefore, the objective of this study was to investigate whether other types of in vitro DNA modification by sanguinarine, chelerythrine and fagaronine are generated. The detection of DNA adducts derived from sanguinarine and chelerythrine by 32P-postlabeling analysis [26] is reported herein for the first time.

Section snippets

Alkaloids and chemicals

Sanguinarine and chelerythrine were isolated from the alkaloid extract of Macleaya cordata (Papaveraceae) using column chromatography on alumina [27]. Sanguinarine in 98.1% purity, MP 279–282 °C (Ref. 277–280 °C [28]) and chelerythrine in 95% purity, MP 200–204 °C [27] were obtained. Fagaronine in 96.2% purity was synthesized by Šmidrkal [29], MP 203–206 °C (Ref. 202 °C [28]). IR, UV, MS and NMR spectra were consistent with the structures of the above alkaloids.

Chemicals were obtained from the

Results

The 32P-postlabeling technique [26] represents a suitable method for the analysis of different types of DNA modification generated with toxic compounds in vivo and in vitro, and is used mainly for detection of covalent DNA adducts [37]. Therefore, this technique was employed for investigation of in vitro interactions between DNA and alkaloids sanguinarine, chelerythrine and fagaronine.

In the presence of microsomes of uninduced rats and NADPH, one major and two minor DNA adduct spots were

Discussion

The results presented in this paper clearly show that DNA adducts are formed by sanguinarine and chelerythrine in the presence of rat hepatic microsomes. These results are in favor of a potential genotoxic effect of both alkaloids, after metabolic conversion, confirming previous finding [20], [21]. Sanguinarine elicits positive mutagenic response in Salmonella typhimurium (Ames) assay after metabolic activation [20] and chelerythrine has been reported to induce respiration-deficient mutants in

Acknowledgements

The financial support of German Cancer Research Center, the Grant Agency of the Czech Republic (203/01/0996, 203/00/0633), the Grant Agency of the Ministry of Health (NL 5267-3/1999) and Ministry of Education of the Czech Republic (MSM 1131 00001 and 1511 00003) is greatly acknowledged.

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