Determination of total antioxidant capacity, instead of the measurements of limited
number of antioxidants, is very important for the understanding of how antioxidants
interact with reactive oxygen species (ROS). Several techniques already exist with
this propose, although some of them are extremely time-consuming. A new methodology
is proposed, based on the detection of ROS by fluorometry (ex/em: 485/520 nm) employing
2',7'-dichlorofluorescein diacetate (H(2)DCF-DA) as substrate. Supernatant of homogenized
samples from different organs (gill, muscle, liver, and brain) of the teleost fish
Jenynsia multidentata (Anaplebidae) were exposed to peroxyl radicals generated by
thermal (35 degrees C) decomposition of 2,2'-azobis (2 methylpropionamidine) dihydrochloride
(ABAP, 4 mM). Different protein concentrations (0.5, 1, 2 and 8 mg/ml) were assayed
to get the best signal and curve fitting of fluorescence data over time (30 min).
Total antioxidant capacity against peroxyl radicals was estimated as the difference
in ROS area with and without ABAP, relative to the fluorescence registered without
ABAP. For application of this methodology, J. multidentata specimens were exposed
for 24 h to microcystins, cyanotoxins known to induce oxidative stress. Almost all
organs showed a lower antioxidant capacity (p<0.05) in samples with 8 mg proteins/ml,
when compared to protein content of 1-2 mg/ml. In liver samples, higher (p<0.05) free
iron content was determined in samples with 8 mg proteins/ml. Sensitivity test employing
GSH spiked in homogenized samples showed the protocol efficiency in detecting total
antioxidant capacity. In the test with microcystins a dose-dependent decrease (p<0.05)
of antioxidant competence in gills and brain and an inverse result with liver samples
were observed. The use of antioxidant defenses was efficient in avoiding oxidative
damage, as the content of oxidized proteins was not altered. Data obtained show the
potential of this new methodology to be used in ecotoxicological studies.