An in vitro based investigation into the cytotoxic effects of D-amino acids

Open access


In the present study, cytotoxic effects of D-Ala, D-Pro and D-Lys are demonstrated. In an effort to study the possible mechanisms of the observed cytotoxicity, catalase activity, H2O2 generation, and apoptotic activity were measured in HeLa and MCF-7 cell lines. Although D-Lys is a poor substrate for DAO and therefore low H2O2 was detected, it was shown to provoke severe impairment of cellular integrity and survival. Interestingly, a very good substrate for DAO, such as D-Pro, did not substantially reduce cell viability. On the other hand, a moderate substrate for DAO, represented by D-Ala, was shown to moderately trigger toxicity in the tested cell lines. Although a correlation between the in vitro cytotoxicity of D-amino acids and the amount of H2O2 produced was absent, there was a good agreement between the ability of D-amino acids to trigger apoptosis and to provoke toxicity. Our results indicate that the toxicity of D-amino acids does not appear to be solely mediated by H2O2. Therefore, we hypothesize that other possible contributing apoptosis-mediated pathways might cause the observed toxicity.

1. D. L. Kirschner and T. K. Green, Separation and sensitive detection of D-amino acids in biological matrices, J. Sep. Sci. 32 (2009) 2305-23018; DOI: 10.1002/jssc.200900101.

2. R. Konno, H. Bruckner, A. D’Aniello, G. Fisher, N. Fujii and H. Homma, D-Amino Acids: A New Frontier in Amino Acid and Protein Research - Practical Methods and Protocols, Nova Science Publishers, New York 2008.

3. K. Hamase, A. Morikawa and K. Zaitsu, D-amino acids in mammals and their diagnostic value, J. Chromatogr. B 781 (2002) 73-91; DOI: 10.1016/S1570-0232(02)00690-6.

4. Y. Miyoshi, K. Hamase, Y. Tojo, M. Mita, R. Konno and K. Zaitsu, Determination of D-serine and D-alanine in the tissues and physiological fluids of mice with various D-amino-acid oxidase activities using two-dimensional high-performance liquid chromatography with fluorescence detection, J. Chromatogr. B 877 (2009) 2506-2512; DOI: 10.1016/j.jchromb.2009.06.028.

5. A. Morikawa, K. Hamase and K. Zaitsu, Determination of D-alanine in the rat central nervous system and periphery using column-switching high-performance liquid chromatography, Anal. Biochem. 312 (2003) 66-72; DOI: 10.1016/S0003-2697(02)00432-3.

6. S. H. Snyder and C. D. Ferris, Novel neurotransmitters and their neuropsychiatric relevance, Am. J. Psychiatr. 157 (2000) 1738-1751; DOI: 10.1176/appi.ajp.157.11.1738.

7. H. Mori and R. Inoue, Serine racemase knockout mice, Chem. Biodivers. 7 (2010) 1573-1578; DOI: 10.1002/cbdv.200900293.

8. H. Wolosker, E. Dumin, L. Balan and V. N. Foltyn, D-amino acids in the brain: D-serine in neurotransmission and neurodegeneration, FEBS J. 275 (2008) 3514-3526; DOI: 10.1111/j.1742-4658.2008.06515.x.

9. H. Wolosker, A. D’Aniello and S. H. Snyder, D-aspartate disposition in neuronal and endocrine tissues: ontogeny, biosynthesis and release, Neuroscience 100 (2000) 183-189; DOI: 10.1016/ S0306-4522(00)00321-3.

10. A. D’Aniello, M. M. Di Fiore, G. H. Fisher, A. Milone, A. Seleni, S. D’Aniello, A. F. Perna and D. Ingrosso, Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release, FASEB J. 14 (2000) 699-714.

11. S. Ishio, H. Yamada, M. Hayashi, S. Yatsushiro, T. Noumi, A. Yamaguchi and Y. Moriyama, D-aspartate modulates melatonin synthesis in rat pinealocytes, Neurosci. Lett. 249 (1998) 143-146; DOI: 10.1016/S0304-3940(98)00414-5.

12. Y. Nagata, H. Homma, J. A. Lee and K. Imai, D-Aspartate stimulation of testosterone synthesis in rat Leydig cells, FEBS. Lett. 444 (1999) 160-164; DOI: 10.1016/S0014-5793(99)00045-9.

13. Y. Nagata, T. Fujiwara, K. Kawaguchi-Nagata, Y. Fukumori and T. Yamanaka, Occurrence of peptidyl D-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes, Biochim. Biophys. Acta. 1379 (1998) 76-82.

14. H. Wang, H. Wolosker, J. F. Morris, J. Pevsner, S. H. Snyder and D. J. Selkoe, Naturally occurring free D-aspartate is a nuclear component of cells in the mammalian hypothalamo-neurohypophyseal system, Neuroscience 109 (2002) 1-4; DOI: 10.1016/S0306-4522(01)00545-0.

15. L. Pollegioni, B. Langkau, W. Tischer, S. Ghisla and M. S. Pilone, Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis, J. Biol. Chem. 268 (1993) 13850-13857.

16. B. Curti, S. Ronchi and M. Pilone Simonetta, D- and L-amino acid oxidases, in Chemistry and Biochemistry of Flavoenzymes (Ed. F. Müller), CRC Press, Boca Raton (FL) 1992.

17. A. D’Aniello, A. Vetere and L. Petrucelli, Further study on the specificity of D-amino acid oxidase and D-aspartate oxidase and time course for complete oxidation of D-amino acids, Comp.Biochem. Physiol. B 105 (1993) 731-734; DOI: 10.1016/0305-0491(93)90113-J.

18. A. D’Aniello, G. Donofrio, M. Pischetola, G. Daniello, A. Vetere, L. Petrucelli and G. H. Fisher, Biological role of D-amino-acid oxidase and D-aspartate oxidase - Effects of D-amino acids, J. Biol. Chem. 268 (1993) 26941-26949.

19. N. Ercal, X. Luo, R. H. Matthews and D. W. Armstrong, In vitro study of the metabolic effects of D-amino acids, Chirality 8 (1996) 24-29.

20. K. B. Beckman and B. N. Ames, Oxidative decay of DNA, J. Biol. Chem. 272 (1997) 19633-19636; DOI: 10.1074/jbc.272.32.19633.

21. B. S. Berlett and E. R. Stadtman, Protein oxidation in aging, disease, and oxidative stress, J. Biol.Chem. 272 (1997) 20313-20316; DOI: 10.1074/jbc.272.33.20313.

22. B. Halliwell and J. M. Gutteridge, Free radicals, lipid peroxidation, and cell damage, Lancet 2 (1984) 1095-1100; DOI: 10.1016/S0140-6736(84)91530-7.

23. T. Matsura, M. Kai, Y. Fujii, H. Ito and K. Yamada, Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation, Free Radic. Res. 30 (1999) 73-83; DOI: 10.1080/10715769900300081.

24. J. A. Barltrop, T. C. Owen, A. H. Cory and J. G. Cory, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-dime- thylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple watersoluble formazans as cell-viability indicators, Bioorg. Med. Chem. Lett. 1 (1991) 611-614.

25. H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 105-121; DOI: 10.1016/S0076-6879(84) 05016-3.

26. K. J. Davies, Oxidative stress: the paradox of aerobic life, Biochem. Soc. Symp. 61 (1995) 1-31.

27. M. Sundaresan, Z. X. Yu, V. J. Ferrans, K. Irani and T. Finkel, Requirement for generation of H2O2 for platelet-derived growth factor signal transduction, Science 270 (1995) 296-299.

28. J. M. Mates, C. Perez-Gomez and I. Nunez de Castro, Antioxidant enzymes and human diseases, Clin. Biochem. 32 (1999) 595-603; DOI: 10.1016/S0009-9120(99)00075-2.

29. J. Soutourina, P. Plateau and S. Blanquet, Metabolism of D-aminoacyltRNAs in Escherichia coli and Saccharomyces cerevisiae cells, J. Biol. Chem. 275 (2000) 32535-32542.

30. O. Soutourina, J. Soutourina, S. Blanquet and P. Plateau, Formation of D-tyrosyl-tRNATyr accounts for the toxicity of D-tyrosine toward Escherichia coli, J. Biol. Chem. 279 (2004) 42560-42565; DOI: 10.1074/jbc.M402931200.

31. M. L. Ferri-Fioni, M. Fromant, A. P Bouin, C. Aubard, C. Lazennec, P. Plateau and S. Blanquet, Identification in archaea of a novel D-Tyr-tRNA(Tyr) deacylase, J. Biol. Chem. 281 (2006) 27575-27585.

32. L. M. Dedkova, N. E. Fahmi, S. Y. Golovine and S. M. Hecht, Enhanced D-amino acid incorporation into protein by modified ribosomes, J. Am. Chem. Soc. 125 (2003) 6616-6617; DOI: 10.1021/ ja035141q.

Acta Pharmaceutica

The Journal of Croatian Pharmaceutical Society

Journal Information

IMPACT FACTOR 2017: 1.071
5-year IMPACT FACTOR: 1.623

CiteScore 2017: 1.46

SCImago Journal Rank (SJR) 2017: 0.362
Source Normalized Impact per Paper (SNIP) 2017: 0.642

Cited By


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 102 102 14
PDF Downloads 22 22 2