Sulforhodamine B Assay to Determine Cytotoxicity of Vibrio vulnificus Against Human Intestinal Cells

  • Lee, Byung-Cheol (College of Pharmacy, Chonmam National University) ;
  • Choi, Sang-Ho (Department of Food science and Technology, School of Agricultural Biotechnology, and Center for Agricultural Biomaterials Seoul National University) ;
  • Kim, Tae-Sung (College of Pharmacy, Biotechnology Research Center, Chonnam National University)
  • Published : 2004.04.01

Abstract

Sulforhodamine B (SRB) assay is a rapid, sensitive, and inexpensive method for measuring cell proliferation and chemosensitivity. However, the lactate dehydrogenase (LDH) release assay is generally used to measure cytototoxicity of infectious microorganisms against host cells. In this study, we investigated the possibility of applying the SRB assay to determine cytotoxicity for infectious microorganisms, and compared the results with those obtained by the LDH release assay. We used Vibrio vulnificus as a model of infectious microorganisms. The SRB assay showed that V vulnificus strongly induced cytotoxic activity against human intestinal cells, Caco-2 and INT-407 cells. The degree of cytotoxicity closely correlated with infection time and number ratios of V. vulnificus to intestinal cells (MOI, multiplicity of infection). Furthermore, cytotoxicity values obtained by SRB assay correlated well with results obtained by the LDH release assay, and both assays gave a linear response with respect to MOI Heat-inactivation of V. vulnificus for 35 min at $60^{\circ}C$ did not induce cytotoxic activity, indicating that viability of V. vulnificus is crucial for cytotoxic activity against intestinal cells. Although both assays are suitable as cytotoxicity endpoints, the SRB assay is recommended for measuring cytotoxicity of infectious microorganisms against host cells because of its significantly lower cost and more stable endpoint than the LDH release assay.

Keywords

References

  1. J. Microbiol. Immunol. Infect. v.36 Vibrio vulnificus infection: Clinical manifestations, pathogenesis, and antimicrobial therapy Chiang,S.R.;Y.C.Chuang
  2. Biochem. Biophys. Res. Commun. v.26 Involvement of in vivo induced icmF gene of Vibrio cholerae in motility, adherence to epithelial cells, and conjugation frequency Das,S.;A.Chakrabortty;R.Banerjee;K.Chaudhuri
  3. Anticancer Res. v.16 Comparison of two colorimetric assays as cytotoxicity endpoints for an intro screen for antitumor agents Fricker,S.P.:R.G.Buckley
  4. J. Infect. Dis. v.163 Cytotoxicity of Borrelia burgdorferi for cultured rat glial cells Garcia-Monco,J.C.;B. Fernandez Villar;A.Szczepanski;J.L.Benach https://doi.org/10.1093/infdis/163.6.1362
  5. J. Microbiol. Biotechnol. v.13 Identification of the Vibrio vulnificus lrp and its influence on survival under various stresses Jeong,H.S.;J.E.Rhee;J.H.Lee;H.K.Choi;S.J.Park;S.H.Choi
  6. Infect. Immun. v.68 Construction and phenotypic evaluation of a Vibrio vulnificus vvpE mutant for elastolytic protease Jeong,K.C.;H.S.Jeong;J.H.Rhee;S.E.Lee;S.S.Chung;A.M.Starks;G.M.Escudero;P.A..Gulig;S.H.Choi https://doi.org/10.1128/IAI.68.9.5096-5106.2000
  7. Br. J. Pharmacol. v.135 Enhancement of 1α,25-dihydroxyvitamin D₃-induced differentiation of human leukemia HL-60 cells into monocytes by parthenolide via inhibition of NF-kB activity Kang,S.N.;S.H.Kim;S.W.Chung;M.H.Lee;H.J.Kim;T.S.Kim https://doi.org/10.1038/sj.bjp.0704573
  8. Infect. Immun. v.71 Strain-specific association of cytotoxic activity and virulence of clinical Staphylococcus aureus isolates Krut,O.;O.Utermohlen;X.Schlossherr;M.Kronke
  9. J. Microbiol. Biotechnol. v.13 Identification and characterization of the Vibrio vulnificus phosphomannomutase gene Lee,J.H.;N.Y.Park;S.J.Park;S.H.Choi
  10. Ann. Oncol. v.13 In vitro comparative evaluation of trastuzumab (Herceptin) combined with paclitaxel (Taxol) or docetaxel (Taxotere) in HER2-expressing human breast cancer cell lines Merlin,J.L.:M.Barberi-Heyob;N.Bachmann https://doi.org/10.1093/annonc/mdf263
  11. Am. J. Physiol. Gastrointest. Liver Physiol. v.283 Helicobacter pylori induces apoptosis of rat gastric parietal cells Neu,B.;P.Randlkofer;M.Neuhofer;P.Voland;A.Mayerhofer;M.Gerhard;W.Schepp;C.Prinz https://doi.org/10.1152/ajpgi.00546.2001
  12. J. Immunol. Methods v.208 Optimization of the sulforhodamine B colorimetric assay Papazisis,K.T.;G.D.Geromichalos;K.A.Dimitriadis;A.H.Kortsaris https://doi.org/10.1016/S0022-1759(97)00137-3
  13. Eur. J. Cancer v.29 A comparison of clonogenic, microtetrazolium and sulforhodamine B assays for determination of cisplatin cytotoxicity in human ovarian carcinoma cell lines Perez,R.P.;A.K.Godwin;L.M.Handel;T.C.Hamilton https://doi.org/10.1016/0959-8049(93)90394-U
  14. Infect. Immun. v.70 Rickettsia-macrophage interactions: Host cell responses to Rickettsia akari and Rickettsia typhi Radulovic,S.;P.W.Price;M.S.Beier;J.Gaywee;J.A.Macaluso;A.Azad https://doi.org/10.1128/IAI.70.5.2576-2582.2002
  15. Infect. Immun. v.69 Differential interleukin-8 response of intestinal epithelial cell line to reactogenic and nonreactogenic candidate vaccine strains of Vibrio cholerae Rodriguez,B.L.;A.Rojas;J.Campos;T.Ledon;E.Valle;W.Toledo;R.Fando https://doi.org/10.1128/IAI.69.1.613-616.2001
  16. J. Immunol. Methods v.196 Improved protocol for colorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity Sepp,A.;R.M.Binns;R.I.Lechler https://doi.org/10.1016/0022-1759(96)00112-3
  17. J. Natl. Cancer Inst. v.82 Boyd. New colorimetric cytotoxicity assay for anticancer-drug screeing Skehan,P.;R.Storeng;D.Scudiero;A.Monks;J.McMahon;D.Vistica;J.T.Warren;H.Bokesch;S.Kenney;M.R.Boyd https://doi.org/10.1093/jnci/82.13.1107
  18. Microbes Infect. v.2 Epidemilolgy and pathogenesis of Vibrio vulnificus Strom,M.S.;R.N.Paranjpye https://doi.org/10.1016/S1286-4579(00)00270-7
  19. Infect. Immun. v.66 Differential early interactions between Salmonella enterica serovar typhi and two other pathogenic salmonella serovars with intestinal epithelial cells Weinstein,D.L.;B.L.O'Neill;D.M.Hone;E.S.Metcalf
  20. Am. J. Physiol. v.275 Enteroinvasive bacteria directly activate expression of iNOS and NO production in human colon epithelial cells Witthoft,T.;L.Eckmann;J.M.Kim;M.F.Kagnoff