Molecular & Cellular Toxicology

The Korean Society of Toxicogenomics and Toxicoproteomics (대한독성유전 단백체학회)
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Genotoxicity Study on Khal, a Halocidin Derivative, in Bacterial and Mammalian Cells

  • Kim, Youn-Jung (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology) ;
  • Kim, Mi-Soon (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology) ;
  • Jeon, Hee-Kyoung (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology) ;
  • Ryu, Jae-Chun (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology)
  • Published : 2006.09.30
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Abstract

Khal was a synthetic congener of halocidin, a heterodimeric peptide consisting of 19 and 15 amino acid residues detected in Halocynthia aurantium. This compound was considered a candidate for the development of a novel peptide antibiotic. The genotoxicity of Khal was subjected to high throughput toxicity screening (HTTS) because they revealed strong antibacterial effects. Mouse lymphoma thymidine kinase ($tk^{+/-}$) gene assay (MOLY), single cell gel electrophoresis (Comet) assay and chromosomal aberration assay in mammalian cells and Ames reverse mutation assay in bacterial system were used as simplified, inexpensive, short-term in vitro screening tests in our laboratory. These compounds are not mutagenic in S. typhimurium TA98 and TA100 strains both in the presence and absence of metabolic activation. Before performing the comet assay, $IC_{20}$ of Khal was determined the concentration of $25.51\;{\mu}/mL\;and\;21.99\;{\mu}g/mL$ with and without S-9, respectively. In the comet assay, Khal was not induced DNA damage in mouse lymphoma cell line. Also, the mutation frequencies in the Khal-treated cultures were similar to the vehicle controls. It is suggests that Khal is non-mutagenic in MOLY assay. And no clastogenicity was observed in Khal-treated Chinese hamster lung cells. The results of this battery of assays indicate that Khal has no genotoxic potential in bacterial or mammalian cell systems. Therefore, we suggest that Khal, as the optimal candidates with both no genotoxic potential and antibacterial effects must be chosen.

Keywords

References

  1. Lupetti, A. et al. Antimicrobial peptides: therapeutic potential for the treatment of Candida infections. Expert. Opin. Investig. Drugs 11, 309-318 (2002) https://doi.org/10.1517/13543784.11.2.309
  2. Alcouloumre, M.S. et al. Fungicidal properties of defensin NP-1 and activity against Cryptococcus neoformans in vitro. Antimicrob. Agents Chemother. 37, 2628-2632 (1993) https://doi.org/10.1128/AAC.37.12.2628
  3. Lopez-Garcia, B., Lee, P.H., Yamasaki, K. & Gallo, R.L. Anti-fungal activity of cathelicidins and their potential role in Candida albicans skin infection. J. Invest. Dermatol. 125, 108-115(2005) https://doi.org/10.1111/j.0022-202X.2005.23713.x
  4. Viejo-Diaz, M., Andres, M.T. & Fierro, J.F. Different anti-Candida activities of two human lactoferrinderived peptides, Lfpep and kaliocin-1. Antimicrob. Agents Chemother. 49, 2583-2588 (2005) https://doi.org/10.1128/AAC.49.7.2583-2588.2005
  5. Bulet, P., Stocklin, R. & Menin, L. Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198, 169-184 (2004) https://doi.org/10.1111/j.0105-2896.2004.0124.x
  6. Devine, D.A. & Hancock, R.E. Cationic peptides: distribution and mechanisms of resistance. Curr. Pharm. Des. 8, 703-714 (2002) https://doi.org/10.2174/1381612023395501
  7. Jang, W.S. et al. Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium. FEBS Lett. 521, 81-86 (2002) https://doi.org/10.1016/S0014-5793(02)02827-2
  8. Jang, W.S. et al. Biological activities of synthetic analogs of halocidin, an antimicrobial peptide from the tunicate Halocynthia aurantium. Antimicrob. Agents Chemother. 47, 2481-2486 (2003) https://doi.org/10.1128/AAC.47.8.2481-2486.2003
  9. Ames, B.N., Durston, W.E., Yamasaki, E. & Lee, F.D. Carcinogens are mutagens :a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci USA. 70, 2281-2285(1973)
  10. Ames, B.N., McCann, J. & Yamasaki, E. Method for detecting carcinogens and mutagens with Salmonella/ mammalian-microsome mutagenicity test. Mutation Res. 31, 347-364 (1975) https://doi.org/10.1016/0165-1161(75)90046-1
  11. Maron, D.M. & Ames, B.N. Revised methods for the Salmonella mutagenicity test. Mutation Res. 113, 173-215(1983) https://doi.org/10.1016/0165-1161(83)90010-9
  12. Singh, N.P., McCoy, M.T., Tice, R.R. & Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184-191 (1988) https://doi.org/10.1016/0014-4827(88)90265-0
  13. Singh, N.P., Stephens, R.E. & Schneider, E.L. Modifications of alkaline microgel electrophoresis for sensitive detection of DNA damage. Int. J. Radiat. Biol. 66, 23-28 (1994) https://doi.org/10.1080/09553009414550911
  14. Ryu, J.C., Kim, H.J., Seo, Y.R. & Kim, K.R. Single Cell Gel Electrophoresis (Comet Assay) to detect DNA damage and Apoptosis in cell level. Environ. Mutagens & Carcinogens. 17(2), 71-77 (1997)
  15. Ostling, O. & Johanson, K.J. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophy. Res. Commun, 123, 291-298 (1984) https://doi.org/10.1016/0006-291X(84)90411-X
  16. Ryu, J. C. et al. Recent advanced toxicological methods for environmental hazardous chemicals. Kor. J. Environ. Toxicol. 14, 1-12 (1999) https://doi.org/10.1002/(SICI)1522-7278(199902)14:1<1::AID-TOX1>3.0.CO;2-I
  17. Tice, R.R. et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206-221 (2000) https://doi.org/10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J
  18. Honma, M. et al. Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test. Mutagenesis. 14, 5-22 (1999) https://doi.org/10.1093/mutage/14.1.5
  19. Clements, J. Gene mutation assays in mammalian cells, In S. O'Hare and C. K. Atterwill (Ed.), Methods in Molecular Biology, Vol. 43: in vitro Toxicity Testing Protocols. Humana Press Inc. Totowa, NJ. 277-286 (1990)
  20. Robinson, W.D. et al. Statistical evaluation of bacterial/ mammalian fluctuation tests, in Statistical Evaluation of Mutagenicity Test Data (Kirkland, D. J., ed.). Cambridge University Press. Cambridge, UK, 102-140 (1990)
  21. OECD. OECD guideline for the testing of chemicals, Documents 473, Genetic toxicology: in vitro mammalian cytogenetic test. Organization for Economic Cooperation and Development. Paris, France (1993)
  22. Ishidate, M. & Odashima, S. Chromosome test with 134 compounds on chinese hamster cells in vitro-A screening for chemical carcinogens. Mutation Res. 48, 337-354 (1977) https://doi.org/10.1016/0027-5107(77)90177-4
  23. Ryu, J.C. et al. A study on the clastogenicity of trichothecene mycotoxins in chinese hamster lung cells. Korean J. Toxicol. 9(1), 13-21 (1993)
  24. Ryu, J.C., Lee, S., Kim, K.R. & Park, J. Evaluation of the genetic toxicity of synthetic chemicals (I). Chromosomal aberration test on chinese hamster lung cells in vitro. Environ. Mutagens & Carcinogens 14 (2), 138-144 (1994)
  25. Ryu, J.C. et al. Evaluation of the genetic toxicity of synthetic chemicals (II). a pyrethroid insecticide, fenpropathrin. Arch. Pharm. Res. 19(4), 251-257 (1996a) https://doi.org/10.1007/BF02976235
  26. Ryu, J.C. et al. Chromosomal aberration assay of taxol and 10-deacetyl baccatin III in chinese hamster lung cells in vitro. Environ. Mutagens & Carcinogens 16 (1), 6-12 (1996b)
  27. Ryu, J.C., Youn, J.Y., Cho, K.H. & Chang, I.M. Mutation spectrum in lacI gene of transgenic Big Blue cell line following to short term exposure 4- nitroquinoline-N-oxide. Environ. Mol. Mutagen. 31 (Suppl. 29), 16 (1998a)
  28. Ryu, J.C. et al. Genotoxicity Study of Bojungchisuptang, an oriental herbal decoction-in vitro chromosome aberration assay in chinese hamster lung cells and in vitro supravital-staining micronucleus assay with mouse peripheral reticulocytes. Arch. Pharm. Res. 21(4), 391-397 (1998b) https://doi.org/10.1007/BF02974632
  29. Ryu, J.C., Kim, K.R., Lee, S. & Park, J. Evaluation of the genetic toxicity of synthetic chemicals (III), Chromosomal aberration assay with 28 chemicals in chinese hamster lung cells in vitro. Environ. Mutagens & Carcinogens. 21(1), 14-22 (2001)
  30. JEMS-MMS. Atlas of chromosome aberration by chemicals. Japanese Environmental Mutagen Society- Mammalian Mutagenicity Study Group, Tokyo, Japan (1988)
  31. Altman, D.G. Practical statistics for medical research, in Chapter 10. Comparing groups-categorical data, London, Chapman & Hall. 229-276, (1993)