Abstract
We investigated the toxic effects on Prototheca zopfii of indole-3-acetic acid (IAA) and 2,4-pentanedione (PD) combined with horseradish peroxidase (HRP) alongside the oxidation products of 3-methyl-2-oxindole (MOI) and indole-3-carbinol (I3C) from the IAA/HRP system and methylglyoxal (MGO) from the PD/HRP system. The microorganism was incubated in the absence (control) or presence of IAA, PD, IAA/HRP, PD/HRP, MOI, I3C and MGO and determined: (1) cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay; (2) growth inhibitory concentration by resazurin assay and (3) antioxidant enzymes activities of: catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD). P. zopfii was more susceptible to IAA at 40 mM than PD at the same concentration, which seems to indicate that IAA was more effective at initiating cell death. These data corroborate results from the resazurin assay. Concentrations of 40 mM of IAA, IAA/HRP and PD/HRP, 20 mM of PD/HRP, 10 mM of MOI, 2 mM of I3C and 8 mM of MGO inhibited the growth of P. zopfii. With sub-inhibitory concentrations of IAA and IAA/HRP at 30 mM, MOI at 8 mM and I3C at 1 mM, the activities of CAT and GR increased, whereas no statistical difference was observed for CAT activity with IAA/HRP. Thus, PD at 30 mM and MGO at 6 mM increased the activities of CAT and GR, whereas PD/HRP system at 15 mM decreased CAT activity and PD/HRP and MGO showed no statistical difference for SOD activity. In conclusion, IAA/HRP or PD/HRP systems and their oxidation products exert cytotoxic effects on P. zopffi; however, I3C and MGO appear to exert greater microbicidal effect on P. zopfii.
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References
Mayorga J, Barba-Gómez JF, Verduzco-Martínez AP, Muñoz-Estrada VF, Welsh O. Protothecosis. Clin Dermatol. 2012;30(4):432–6.
Todd JR, King JW, Oberle A, Matsumoto T, Odaka Y, Fowler M, Pore RS, Shahan TA, Yin L, Sanusi ID. Protothecosis: report of a case with 20 year follow-up and review of previously published cases. Med Mycol. 2012;50(7):673–89.
Costa EO, Ribeiro MG, Ribeiro AR, Rocha NS, Nardi G Jr. Diagnosis of clinical bovine mastitis by fine needle aspiration followed by staining and scanning electron microscopy in a Prototheca zopfii outbreak. Mycopathologia. 2004;158(1):81–5.
Zaini F, Kanani A, Falahati M, Fateh R, Salimi-Asl M, Saemi N, Farahyar SH, Kargar KA, Nazeri M. Identification of Prototheca zopfii from bovine mastitis. Iran J Public Health. 2012;41(8):84–8.
Pore RS. 2011, Prototheca Krüger. In: Kurtzman CP, Fell JW, Boekhout T, editors. The yasts, a taxonomic study. Amsterdam, Netherlands: Elsevier; 1984;3(5):2071–80.
Marques S, Silva E, Kraft C, Carvalheira J, Videira A, Huss VAR, Thompson G. Bovine mastitis associated with the Prototheca blaschkeae. J Clin Microbiol. 2008;46(6):1941–5.
Jagielski T, Buzzini P, Lassa H, Malinowski E, Branda E, Turchetti B, Polleichtner A, Roesler U, Lagneau PE, Marques S, Silva E, Thompson G, Stachowiak R, Bielecki J. Multicentre Etest evaluation of in vitro activity of conventional antifungal drugs against European bovine mastitis Prototheca spp. isolates. J Antimicrob Chemother. 2012;67(8):1945–7.
Roesler U, Scholz H, Hensel A. Emended phenotypic characterization of Prototheca zopfii: a proposal for three biotypes and standards for their identification. Int J Syst Evol Microbiol. 2003;53(4):1195–9.
Osumi T, Kishimoto Y, Kano R, Maruyama H, Onozaki M, Makimura K, Ito T, Matsubara K, Hasegawa A. Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan. Vet Microbiol. 2008;131(3–4):419–23.
Ricchi M, Goretti M, Branda E, Cammi G, Garbarino CA, Turchetti B, Moroni P, Arrigoni N, Buzzini P. Molecular characterization of Prototheca strains isolated from Italian dairy herds. J Dairy Sci. 2008;93(10):4625–31.
Jagielski T, Lassa H, Arhholdt J, Malinowski E, Roesler U. Genotyping of bovine Prototheca mastitis isolates from Poland. Vet Microbiol. 2001;149(1–2):283–7.
Sobukawa H, Yamaguchi S, Kano R, Ito T, Suzuki K, Onozaki M, Hasegawa A, Kamata H. Short communication: molecular typing of Prototheca zopfii from bovine mastitis in Japan. J Dairy Sci. 2012;95(8):4442–6.
Onozaki M, Makimura K, Satoh K, Hasegawa A. Detection and identification of genotypes of Prototheca zopfii in clinical samples by quantitative PCR analysis. Jpn J Infect Dis. 2013;66(5):383–90.
Lassa H, Jagielski T, Malinowski E. Effect of different heat treatments and disinfectants on the survival of Prototheca zopfii. Mycopathologia. 2011;171(3):177–82.
Melville PA, Benites NR, Sinhorimi IL, Costa EO. Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate, silver nitrate and chlorexidine. Mycophatologia. 2002;156(1):1–7.
Lopes MM, Ribeiro R, Carvalho D, Freitas G. In vitro antimicrobial susceptibility of Prototheca spp. isolated from bovine mastitis in a Portugal dairy herd. J Mycol Med. 2008;18(4):205–920.
Krukowski H, Lisowski A, Nowakowicz-Dębek B, Wlazlo L. Susceptibility of Prototheca zopfii strains isolated from cows with mastitis to chlorhexidine and iodine. Turk J Vet Anim Sci. 2013;37(1):106–8.
Buzzini P, Turchetti B, Branda E, Goretti M, Amici M, Lagneau PE, Scaccabarozzi L, Bronzo V, Moroni P. Large-scale screening of the in vitro susceptibility of Prototheca zopfii towards polyene antibiotics. Med Mycol. 2008;46(5):511–4.
Lee J-H, Cho MH, Lee J. 3-Indolylacetonitrile decreases Escherichia coli O157:H7 biofilm formation and Pseudomonas aeruginosa virulence. Environ Microbiol. 2011;13(1):62–73.
Kim DS, Jeon SE, Jeong YM, Kim SY, Kwon SB, Park KC. Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis. FEBS Lett. 2006;580(5):1439–46.
Greco O, Folkes LK, Wardman P, Tozer GM, Dachs GU. Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid. Cancer Gene Ther. 2000;7(11):1414–20.
Park KC, Kim SY, Kim DS. Experimental photodynamic therapy for liver cancer cell-implanted nude mice by an indole-3-acetic acid and intense pulsed light combination. Biol Pharm Bull. 2009;32(9):1609–13.
Kim SY, Ryu JS, Li H, Park WJ, Yun HY, Baek KJ, Kwon NS, Sohn UD, Kim DS. UVB-activated indole-3-acetic acid induces apoptosis of PC-3 prostate cancer cells. Anticancer Res. 2010;30(11):4607–12.
Folkes LK, Wardman P. Oxidative activation of indole-3- acetic acids to cytotoxic species—a potential new role for plant auxins in cancer therapy. Biochem Pharmacol. 2001;61(2):129–36.
Cunha LT, Pugine SMP, Silva MRM, Costa EJX, De Melo MP. Microbicidal action of indole-3-acetic acid combined with horseradish peroxidase on Prototheca zopfii from bovine mastitis. Mycopathologia. 2010;169(2):99–105.
Rodrigues AP, Fonseca LM, Oliveira OMMF, Brunetti IL, Ximenes VF. Oxidation of acetylacetone catalysed by horseradish peroxidase in the absence of hydrogen peroxide. Biochim Biophys Acta. 2006;1760(12):1755–61.
Varadarajan A, Utekar SS, Malve SP. Synthesis, structural characterization and antimicrobial studies of 2,4-pentanedione derivatives. Acta Pol Pharm. 1998;55(2):137–41.
Sena CM, Matafome P, Crisóstomo J, Rodrigues L, Fernandes R, Pereira P, Seiça RM. Methylglyoxal promotes oxidative stress and endothelial dysfunction. Pharmacol Res. 2012;65(5):497–506.
Matafome P, Santos-Silva D, Crisóstomo J, Rodrigues T, Rodrigues L, Sena CM, Pereira P, Seiça R. Methylglyoxal causes structural and functional alterations in adipose tissue independently of obesity. Arch Physiol Biochem. 2012;118(2):58–68.
Semchyshyn HM. Reactive carbonyl species in vivo: generation and dual biological effects. Sci World J. 2014;1–10.
Verma A, Prasad KN, Singh AK, Nyati KK, Gupta RK, Paliwal VK. Evaluation of the MTT lymphocyte proliferation assay for the diagnosis of neurocysticercosis. J Microbiol Methods. 2010;81(2):175–8.
Pereira DH, Kitagawa RR, Raddi MSG, Fonseca LM, Ximenes VF. The triad indole-3-acetic acid ethyl ester/esterase/horseradish peroxidase as a new cytotoxic prodrug/enzyme combination. Appl Cancer Res. 2010;30(1):204–9.
Fotakis G, Timbrell JA. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett. 2006;160(2):171–7.
Alejo CJB, Fasciani C, Grenier M, Netto-Ferreira JC, Scaiano JC. Reduction of resazurin to resorufin catalyzed by gold nanoparticles: dramatic reaction acceleration by laser or LED plasmon excitation. Catal Sci Technol. 2011;1(8):1506–11.
Hudman DA, Sargentini NJ. Resazurin-based assay for screening bacteria for radiation sensitivity. Springerplus. 2013;2(1):55.
Pugine SMP, Brito P, Alba-Loureiro TC, Costa EJX, Curi R, De Melo MP. Effect of indole-3-acetic acid administration by gavage and by subcutaneous injection on rat leukocytes. Cell Biochem Funct. 2007;25(6):723–30.
Folkes LK, Wardman P. Oxidative activation of indole-3-acetic acids to cytotoxic species—a potential new role for plant auxins in cancer therapy. Biochem Pharmacol. 2001;61(2):129–36.
Kim DS, Kim SY, Jeong YM, Jeon SE, Kim MK, Kwon SB, Park KC. Indole-3-acetic acid/horseradish peroxidase-induced apoptosis involves cell surface CD95 (Fas/APO-1) expression. Biol Pharm Bull. 2006;29(8):1625–9.
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This paper was supported by Grants from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).
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Cunha, L.T., Pugine, S.M.P., Lins, P.G. et al. Induction of Oxidative Stress in Prototheca zopfii by Indole-3-Acetic Acid/HRP or 2,4-Pentanedione/HRP Systems and Their Oxidation Products. Mycopathologia 179, 73–79 (2015). https://doi.org/10.1007/s11046-014-9807-8
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DOI: https://doi.org/10.1007/s11046-014-9807-8