Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts

https://doi.org/10.1016/j.cbi.2008.05.029Get rights and content

Abstract

Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 μg/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6, 1.2, 2.4 and 4.8 μg/mL). After 24 h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested, assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50 > 100 μg/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated, showing a genotoxic risk induced by tambjamine D.

Introduction

Marine invertebrates are an important source of bioactive natural products, many of which exhibit unique structural features not found in natural products derived from terrestrial organisms [1], [2]. As a consequence, marine organisms have attracted attention as potential sources of bioactive substances, and they continue to provide novel lead compounds for the pharmaceutical industry [3], [4], [5], [6], [7], [8].

Tambjamines are representative members of the 4-methoxypyrrolic alkaloids derived from marine sources, which exhibit cytotoxic, antimicrobial and immunosuppressive activities [9], [10]. Tambjamines A–D have been isolated from the nudibranchs Tambja eliora, T. abdere and Roboastra tigris and been shown to have antibacterial activity against Escherichia coli, Staphylococcus aureus and Vibrio anguillarum[11], [12]. Tambjamine D isolated from the nudibranch T. eliora was also found to display antibacterial activity against B. subtilis, antifungal activity against Candida albicans, antimitotic effects in the sea urchin egg development assay, and cytotoxic activity against several tumor cell lines [13].

Some of the biological effects of 4-methoxypyrrolic alkaloids may be related to their ability to intercalate DNA [14], [15]. DNA-intercalating agents such as acridines are known to cause several mutagenic events such as DNA strand breaks and frameshift mutations [16], [17]. Melvin et al. [15] also demonstrated that 4-methoxypyrrolic from natural products bind double-stranded DNA and facilitate its oxidative cleavage.

Considering the DNA-targeting properties of these compounds, the aim of the present study was to investigate the cytotoxic and genotoxic effects of the alkaloid tambjamine D isolated from the nudibranch T. eliora using V79 cells a permanent cell line derived from Chinese hamster lung fibroblasts. This study is important for determining the safety of a possible pharmacological application of this compound and for exploring other biological properties. For this purpose, we determined the cytotoxicity of tambjamine D using three different endpoints, and evaluated the oxidative stress promoted by tambjamine D by measuring nitrite/nitrate production and TBARS formation after cell exposure to tambjamine D. In order to obtain a complete evaluation of the toxic effects of this alkaloid in mammalian cells, DNA damage induced by tambjamine D was estimated using the comet assay and the micronucleus test.

Section snippets

Chemicals

Tambjamine D (Fig. 1) was isolated from T. eliora as previously described [13]. Modified Eagle's medium (MEM), fetal bovine serum, trypsin–EDTA, penicillin and streptomycin were purchased from GIBCO® (Invitrogen, Carlsbad, CA, USA). Cytochalasin-B (Cyt-B) and sulfanilamide were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). N-(1-Naphthyl)-ethylenediamine dihydrochloride was purchased Merck (USA). All other chemicals and reagents used were of analytical grade.

Cells and treatment

Chinese hamster lung

Cytotoxicity

Tambjamine D inhibited the proliferation of V79 cells when analyzed by the MTT assay. After 24 h of exposure, the alkaloid exhibited a strong cytotoxic effect on the V79 cell line with an IC50 of 1.2 μg/mL (CI 95% of 0.06–2.41). Based on the MTT results, tambjamine D was diluted to four different concentrations (0.6, 1.2, 2.4 and 4.8 μg/mL) corresponding to 1/2 × IC50, IC50, 2 × IC50 and 4 × IC50. The results in Fig. 2 show that tambjamine D exposure reduced significantly the number of viable cells at

Discussion

During our earlier investigation of bioactive metabolites from marine invertebrates [30], we isolated and evaluated the antimicrobial and cytotoxic activities of tambjamines A and D. While tambjamine A was essentially inactive in our assays, tambjamine D displayed antibacterial, antifungal and cytotoxic activities [13].

In this study, we investigated the biological effects of tambjamine D in cultured mammalian cells. V79 fibroblasts were used because they represent a well-characterized cell line

Acknowledgments

We wish to thank the Brazilian National Research Council (CNPq), the Claude Bernard Institute, the Research Support Foundation of Ceará (FUNCAP) and FAPESP (São Paulo) for financial support in the form of grants and fellowship awards. We are also grateful to Dr. A. Leyva for English editing of the manuscript.

References (50)

  • M. Fenech

    The in vitro micronucleus technique

    Mutat. Res.

    (2000)
  • A.J. McGahon et al.

    The end of (cell) line: methods for the study of apoptosis in vitro

    Methods Cell Biol.

    (1995)
  • M. Bradley et al.

    Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of literature

    Mutat. Res.

    (1981)
  • F. Fornelli et al.

    Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9)

    J. Invertebr. Pathol.

    (2004)
  • F.N. Ko et al.

    Scavenger and antioxidant properties of prenylflavones isolated from Artocarpus heterophyllus

    Free Radical Biol. Med.

    (1998)
  • H.E. Poulsen

    Oxidative DNA modifications

    Exp. Toxicol. Pathol.

    (2005)
  • A. Hartmann et al.

    Comparative study with the alkaline Comet assay and the chromosome aberration test

    Mutat. Res.

    (2003)
  • D.W. Fairbairn et al.

    The Comet assay: a comprehensive review

    Mutat. Res.

    (1995)
  • M. Kirsch-Volders

    Towards a validation of the micronucleus test

    Mutat. Res.

    (1997)
  • I. Mitchell et al.

    In vitro genotoxicity and cell transformation assessment

  • R.D. Snyder

    Assessment of atypical DNA intercalating agents in biological and in silico systems

    Mutat. Res.

    (2007)
  • V. Rizzo et al.

    Equilibrium and kinetics of rotamer interconversion in immunosuppressant prodigiosin derivatives in solution

    J. Pharm. Sci.

    (1999)
  • H.T. Suzuki et al.

    Efficient induction of chromosome-type aberrations by topoisomerase II inhibitors closely associated with stabilization of the cleavable complex in cultured fibroblastic cells

    Mutat. Res.

    (1995)
  • A.N.C. Sortibrán et al.

    Gentotoxic profile of inhibitors of topoisomerase I (camptothecin) and II (etoposide) in a mitotic recombination and sex-chromosome loss somatic eye assay of Drosophila melanogaster

    Mutat. Res.

    (2006)
  • J.X. Xiao et al.

    Morphological study on apoptosis Hela cells induced by soyasaponis

    Toxicol. In Vitro

    (2007)
  • Cited by (38)

    • Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

      2017, Biochemical Pharmacology
      Citation Excerpt :

      Emulating that characteristic, different anionophores have been designed and evaluated [15]. We have focused our attention on the bioactive marine alkaloids tambjamines, a class of natural anionophores that has shown interesting cytotoxic effects [16,17]. We have recently synthesized novel synthetic tambjamine analogues bearing aromatic substituents in the enamine moiety, as well as explored different substitution patterns on characteristic alkoxy group of the central pyrrole ring.

    • Synthesis of 2,2′-bipyrrole-5-carboxaldehydes and their application in the synthesis of B-ring functionalized prodiginines and tambjamines

      2013, Tetrahedron
      Citation Excerpt :

      D'Alessio et al. demonstrated that elaborating the ring-B methoxy group to provide other ethers has improved the therapeutic potential of certain PGs.5f The only other variation in ring B of PGs is recent work by Lubell's group who introduced alkylamine groups by a diversity-oriented strategy.14 Our recent work on the potent antimalarial activities of PGs,11 which to date have been limited to SAR studies of A- and C-ring functionalized PGs. With a few exceptions,5f,7,14 there have been no reports of a comprehensive series of B-ring functionalized PGs and tambjamines being prepared and evaluated for biological activities. A methodology for enhancing the diversity of 2,2′-bipyrrole-5-carboxaldehyde moiety is thus desirable to further advance SAR studies of interesting PPM products (PGs, tambjamines and etc.).

    • Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H<inf>2</inf>O<inf>2</inf>-induced DNA damage in cultured human leukocytes

      2012, Food and Chemical Toxicology
      Citation Excerpt :

      According to World Health Organization (2002), about 80% of the population in developing countries rely on herbal medicines at least for their primary health care. Moreover, few plants have been scientifically assessed regarding their quality, safety and efficacy (Cavalcanti et al., 2008a). In spite of this, there have been few studies of the Brazilian medicinal flora aimed at examining potential health risks.

    View all citing articles on Scopus
    View full text