Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
JCR Impact Factor (IF) – 2.1 (5-Year IF – 2.0)
Journal Citation Indicator (JCI) (2023) – 0.4
Scopus CiteScore – 3.7 (CiteScore Tracker 3.3)
Index Copernicus  – 161.11; MNiSW – 70 pts

ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

Download original text (EN)

Advances in Clinical and Experimental Medicine

2010, vol. 19, nr 1, January-February, p. 65–75

Publication type: original article

Language: English

The Influence of Organic Nitrogen Compounds on Melanoma, Bacterial, and Fungal Cells

Wpływ organicznych związków azotowych na komórki czerniaka, bakterii i grzybów

Ewa Obłąk1,, Anna Krasowska2,

1 Institute of Genetics and Microbiology, University of Wrocław, Poland

2 Faculty of Biotechnology, University of Wrocław, Poland

Abstract

Background. The influence of the quaternary ammonium salt (QAS) N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride (IM) and the lysosomotropic aminoester N-N-dimethylaminoethyl dodecanoate hydrochloride (DM-11) on bacterial, fungal, and melanoma cell viability and plasma membrane H+-ATPase activity in Saccharomyces cerevisiae was investigated.
Objectives. The aim was to determine the tested compounds’ activities as potential disinfectants.
Material and Methods. The minimal inhibitory concentration (MIC) was determined in the fungal and bacterial cells (Gram positive and negative strains) on solid YPD and LB medium with different concentrations of the tested drugs (20, 40, 80, 120, 140, 160, 320 μM). Critical micelle concentrations (CMC) were measured at room temperature with a tensiometer using a 24-cm2 Teflon vessel and Wilhelm’s plates. Proton extrusion was determined by pH measurements (computer-linked pH-meter) in distilled water to which a yeast cell suspension (2 mg dry weight), glucose (final concentration 200 mM), either IM or DM-11 (final concentrations of 20, 80 or 140 μM), and KCl (final concentration 100 mM) were successively added. Determination of the ATP concentration in melanoma cells was carried out in 24-well plates at a density of 0.05 × 106 cells per well. ATP determination was performed using the ATP determination kit with luciferase. The MTT (methylthiazole tetrazolium) assay was used to determine the cytotoxicity of the tested agents. This test quantifies the conversion of water-soluble MTT to insoluble purple formazan, which is catalyzed by mitochondrial dehydrogenase of only living cells.
Results. Both tested agents (IM and DM-11) inhibited the growth of Saccharomyces cerevisiae, Candida albicans, Staphylococcus aureus, and Escherichia coli. Moreover, IM influenced the mitochondrial metabolism of the melanoma cells. The inhibitory activity of the aminoester was only temporary, in contrast to the QAS. Both compounds inhibited proton extrusion by plasma membrane H+-ATPase. This inhibitory effect was concentration dependent. IM strongly reduced the ATP level in melanoma cells.
Conclusion. These results suggest that the modes of action of QASs and aminoesters differ. The QAS influenced the plasma membrane, in contrast to the aminoester, which passed through the plasma membrane and accumulated in vacuoles.

Streszczenie

Wprowadzenie. Badano wpływ czwartorzędowej soli amoniowej chlorku dodecyloksykarboksymetylo-N,N,N-trimetyloamoniowego (IM) i aminoestru chlorowodorku estru 2-(dimetyloamino)etylowego kwasu laurynowego (DM-11) na przeżywalność komórek czerniaka, bakterii, grzybów oraz aktywność H+-ATPazy błony komórkowej Saccharomyces cerevisiae.
Cel pracy. Określenie wpływu testowanych związków na drobnoustroje i wykorzystanie ich jako potencjalnych dezynfektantów.
Materiał i metody. Minimalne inhibicyjne stężenie (MIC) wyznaczono dla grzybów i bakterii (Gram+ i Gram-), na podłożu stałym YPD lub LB bez związku (badanie kontrolne) i w jego obecności (stosowano IM lub DM-11 w końcowych stężeniach 20, 40, 80, 120, 140, 160, 320 μM). Pomiaru krytycznego stężenia micelizacji (CMC) dokonano w temperaturze pokojowej na tensometrze w teflonowym naczyniu o rozmiarze 24 cm2, używając bibuły Wilhelma. Aktywność H+-ATPazy błony komórkowej drożdży in vivo określano przez pomiar pH. W tym celu do zawiesiny głodzonych komórek drożdży (2 mg/ml) dodawano kolejno glukozę (200 mM), która indukuje wyrzucanie protonów przez ATPazę i odpowiednie stężenia badanych związków (IM lub DM-11) oraz 100 mM KCl. Stężenie ATP w komórkach czerniaka mierzono metodą chemiluminescencyjną z wykorzystaniem testu z lucyferazą. Pomiary wykonywano w 24-dołkowych płytkach, gęstość hodowli wynosiła 0,05 × 106 komórek na dołek. Do określania cytotoksyczności badanych związków użyto testu ze związkiem MTT (methylthiazole tetrazolium). W teście tym następuje konwersja MTT do czerwonego formazanu, która jest katalizowana przez mitochondrialną dehydrogenazę tylko w żywych komórkach.
Wyniki. Obydwa testowane związki hamowały wzrost Saccharomyces cerevisiae, Candida albicans, Staphylococcus aureus i Escherichia coli. Związek IM wpływał na mitochondrialny metabolizm w komórkach czerniaka. Inhibicyjne oddziaływanie aminoestru było tylko czasowe w przeciwieństwie do aktywności czwartorzędowej soli amoniowej. Obydwa związki hamowały wyrzucanie protonów przez H+-ATPazę i ten proces był zależny od stężenia związków. Związek IM zmniejszał stężenie ATP w komórkach czerniaka.
Wnioski. Wyniki badań własnych sugerują odmienny mechanizm działania testowanych związków na drobnoustroje. Czwartorzędowa sól amoniowa oddziaływuje na błonę komórkową w przeciwieństwie do aminoestru, który przenika przez błonę i akumuluje się w wakuolach (Adv Clin Exp Med 2010, 19, 1, 65–75).

Key words

quaternary ammonium salt, aminoester, fungi, bacteria, H+-ATPase

Słowa kluczowe

czwartorzędowa sól amoniowa, aminoester, grzyby, bakterie, H+-ATPaza

References (46)

  1. Xiao Y, Chen J, Fang M, Xing X, Wang H, Wang Y. Li F: Antibacterial effects of three experimental quaternary ammonium salt (QAS) monomers on bacteria associated with oral infections. J Oral Sci 2008, 50, 323–327.
  2. Thorsteinsson T, Masson M, Kristinsson KG, Hjalmarsdottir MA, Hilmarsson H, Loftsson T: Soft antimicrobial agents: Synthesis and activity of labile environmentally friendly long chain quaternary ammonium compounds. J Med 2003, 46, 4173–4181.
  3. Massi L, Guittard F Geribaldi S: Quaternary bisammonium fluorosurfactants for antimicrobial devices. Prog Colloid Polym Sci 2004, 124, 190–193.
  4. Kourai H, Yabuhara T, Shirai A, Maeda T, Nagamune H: Syntheses anitimicrobial activities of a series of new bis-quaternary ammonium compounds. Eur J Med Chem 2006, 41, 437–444.
  5. Denyer SP: Mechanism of action of antibacterial biocides. Int Biodeterior Biodegrad 1995, 36, 227–245.
  6. Ohta Y, Kondo Y, Kawada K, Teranaka T, Yoshino N: Synthesis and antibacterial activity of quaternary ammonium salt-type antibacterial agents with a phosphate group. J Oleo Sci 2008, 57, 8, 445–452.
  7. Ohkawa K, Kim H, Lee K: Biodegradation of Electrospun Poly(e-caprolactone) Non-woven Fabrics by PureCultured Soil Filamentous Fungi. J Polym Env 2004, 12, 211–218.
  8. Lee C: Structure, conformation and action of neuromuscular blocking drugs. Br J Anaesth 2001, 8, 7755–7769.
  9. Koyama K, Shimazu Y: Benzalkonium chlorides. Drugs and Poisons in Humans 2005, 23, 407–413.
  10. Rodrigo G, Rodrigo C, Burschtin O: A meta-analysis of the effects of ipratropium bromide in adults with acute asthma. Am J Med 1999, 107, 363–370.
  11. Debbash C, de Saint Jean M, Pisella PJ, Warnet J, Baudouin C: Quaternary ammonium cytotoxicity in a human conjunctival cell line. J Fr Ophtalmol 1999, 22, 950–958.
  12. Russell AD: Bacterial spores and chemical sporicidal agents. Clin Microbiol Rev 1990, 3, 99–119.
  13. Russell AD: Activity of biocides against mycobacteria. J Appl Bacteriol Symp Suppl 1996, 81, 87–101.
  14. Resnick L, Varen K, Salahuddin Sz, Tondreau S, Markham PD: Stability and inactivation of HTLV–III/LAV under clinical and laboratory environments. JAMA 1986, 255, 1887–1891.
  15. Mitchell, BA, Paulsen, IT, Brown, MH, Skurray, RA: Bioenergetics of the staphylococcal multidrug export protein QacA: identification of distinct binding sites for monovalent and divalent cations. J Biol Chem 1999, 274, 3541–3548.
  16. DeDuve C, De Barsy T, Poole B, Trout A, Tulkens P, van Hoff F: Lysosomotropic agents. Biochem Pharmacol 1974, 23, 2495–2519.
  17. Lachowicz TM, Obłąk E, Piatkowski J: Auxotrophy-stimulated sensitivity to quaternary ammonium salt and its relation to active transport in yeast. Bul Pol Acad Sci 1992, 40, 173–182.
  18. Lachowicz TM, Witek S, Łuczynski J, Witkowska R, Krasowska A, Kleszczynska H: Aminoethyl esters of fatty acids as model lysosomotropic substances. Folia Microbiol 1996, 41, 102–105.
  19. Witek S, Goffeau A, Nader J, Łuczynski J, Lachowicz TM, Kuta B, Obłąk E: Lysosomotropic aminoesters act as H+-ATPase inhibitors in yeast. Folia Microbiol 1997, 42, 252–254.
  20. Obłąk E, Lachowicz TM, Łuczyński J, Witek S: Lysosomotropic N,N-dimethyl aminoesters and their quaternary ammonium salts as plasma membrane and mitochondrial ATPase inhibitors. Cell Mol Biol Lett 2002, 7, 1121–1129.
  21. Obłąk E, Ułaszewski S, Morawiecki A, Witek S, Witkowska R, Majcher K: Quaternary ammonium salt mutants in yeast Saccharomyces cerevisiae. Yeast 1989, 5, 273–278.
  22. Obłąk E, Witkowska R, Witek S, Lachowicz TM: Respiratory deficiency and sensitivity to a quaternary ammonium salt in yeast Saccharomyces cerevisiae. Genetics of Respiratory Enzymes in Yeast. Wrocław University Press, Wrocław 1990, 96–102.
  23. Lachowicz TM, Witkowska R, Obłąk E: Amino acid auxotrophy increases sensitivity of yeast to a quaternary ammonium salt IM. Acta Microbiol Pol 1990, 39,157–162.
  24. Lachowicz TM, Piatkowski J, Witek S: Quaternary ammonium salts and arginine are inhibitors of general amino acid permease. Pestic Sci 1995, 43, 169–171.
  25. Obłąk E, Lachowicz TM, Witek S: DL-leucine transport in a Saccharomyces cerevisiae mutant resistant to quaternary ammonium salts. Folia Microbiol 1996, 41, 116–118.
  26. Dubowchik GM, Padilla L, Edinger K, Firestone RA: Reversal of doxorubicin resistance and catalytic neutralization of lysosomes by a lipophilic imidazole. Biochim Biophys Acta 1994, 1191, 103–108.
  27. Obłąk E, Adamski R, Lachowicz TM: pH dependent influence of a quaternary ammonium salt and aminoester on ultrastructure of yeast Saccharomyces cerevisiae. Cell Mol Biol Lett 2003, 8, 105–110.
  28. Krasowska A, Chmielewska L, Łuczynski J, Witek S, Sigler K: The dual mechanism of the antifungal effect of new lysosomotropic agents on the Saccharomyces cerevisiae RXII strain. Cell Mol Biol Lett 2003, 8, 111–120.
  29. Słonimski PP, Perrodin G, Croft JH: Ethidium bromide-induced mutation of yeast mitochondria: complete transformation of cells into respiratory deficient nonchromosomal petites. Biochem Biophys Res Comm 1968, 30, 232–239.
  30. Thompson R: Amphiphilic glycine-based esters as soft antimicrobial agents. PhD Thesis, Goeteborg 1992.
  31. Rucka M, Oswiecimska M, Witek S: New biocides for cooling water treatment. III. Quaternary ammonium salts derivatives of glycine esters. Envir Protec Eng 1983, 9, 25–31.
  32. Devinsky F, Masarova J, Lacko J: Surface activity and micelle formation of some new bisquaternary ammonium salt. J Colloid Interf Sci 1985, 105, 235–239.
  33. Sigler K, Höfer M: Mechanisms of acid extrusion in yeast. Biochim Biophys Acta 1991, 1071, 375–391.
  34. Sigler K, Knotkova A, Kotyk A: Factors governing substrate-induced generation and extrusion of protons in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1981, 643, 572–582.
  35. Kotyk A, Dvorakova M: Are proton symports in yeast directly linked to H+-ATPase acidification? Biochim Biophys Acta 1992, 1104, 293–298.
  36. Radomski R, Radomska M, Szajnowska K, Wisialski Z: Microcomputer controlled electrochemical universal meter. Comput Chem Res 1995, 7, 40–46.
  37. Matsuka Y, Neubert JK, Maidment NT, Spigelman I: Concurrent release of ATP and substance P within guinea pig trigeminal ganglia in vivo. Brlain Res 2001, 915, 248–255.
  38. Luo L, MacLean DB: Effects of thyroid hormone on food intake, hypothalamic Na/K ATPase activity and ATP content. Brain Res 2003, 973, 233–239.
  39. Hattori N, Sakakibara T, Kajiyama N, Igarashi T, Maeda M, Murakami S: Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride. Anal Biochem 2003, 319, 287–295.
  40. Obłąk E, Lachowicz TM, Łuczyński J, Witek S: The aminoesters as inhibitors of plasma membrane H+-ATPase in the yeast Saccharomyces cerevisiae. Cell Mol Biol Lett 2004, 9, 755–763.
  41. Krasowska A, Chmielewska L, Adamski R, Łuczynski J, Witek S, Sigler K: The sensitivity of yeast and yeast-like cells to new lysosomotropic agents. Cell Mol Biol Lett 2004, 9, 675–683.
  42. Lichtenberg D, Robson RJ, Dennis EA: Solubilization of phospholipids by detergents structural and kinetic aspects. Biochim Biophys Acta 1983, 737, 285–304.
  43. Krasowska A, Stasiuk M, Oswiecimska M, Kozubek A, Bien M, Witek S: Suppression of radical-induced lipid peroxidation In a model system by alkyl ester of cinnamate quaternary ammonium salts. Z Naturforsch 2001, 56c, 878–885.
  44. Isomaa B, Hagerstrand H, Makela JH: Effects of amphiphiles on cell membranes and membrane connected events. 11th School on Biophysics of Membrane Transport Poland, Zakopane 1992, 274–289.
  45. Walker GM: Yeast Physiology and Biotechnology. John Wiley & Sons Ltd Chichester, England 1968.
  46. Parkkinen E, Oura E, Soumalainen H: The esterases of baker’s yeast. I. Activity and localization in the yeast cell. J Inst Brew 1978, 84, 5–8.
© Wroclaw Medical University