Direct oxidation of 2′,7′-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production

Yunxia Q. O'Malley, Krzysztof J. Reszka, Bradley E Britigan

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2′,7′-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate. Incubation of DCFH 2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2′,7′-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.

Original languageEnglish (US)
Pages (from-to)90-100
Number of pages11
JournalFree Radical Biology and Medicine
Volume36
Issue number1
DOIs
StatePublished - Jan 1 2004

Fingerprint

Pyocyanine
Oxidation-Reduction
Reactive Oxygen Species
ametantrone
Oxidation
Methylphenazonium Methosulfate
Mitoxantrone
Streptonigrin
Spin Trapping
Vitamin K 3
Paraquat
Daunorubicin
Biological systems
Fluorescent Dyes
Superoxides
NAD
Catalase
Doxorubicin
Pseudomonas aeruginosa

Keywords

  • 2′,7′-Dichlorodihydrofluorescein
  • Ametantrone
  • Dihydrorhodamine
  • Free radical
  • Mitoxantrone
  • Phenazine methosulfate
  • Pyocyanin
  • Reactive oxygen species
  • Superoxide

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

Cite this

Direct oxidation of 2′,7′-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production. / O'Malley, Yunxia Q.; Reszka, Krzysztof J.; Britigan, Bradley E.

In: Free Radical Biology and Medicine, Vol. 36, No. 1, 01.01.2004, p. 90-100.

Research output: Contribution to journalArticle

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abstract = "Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2′,7′-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate. Incubation of DCFH 2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2′,7′-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.",
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AB - Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2′,7′-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate. Incubation of DCFH 2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2′,7′-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.

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