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However, in Brazilian fish species, these biomarkers standardization need to be carried out for subsequent utilization as possible biomarkers in each indicator species. It is particularly necessary to use the biological indicators in the aquatic environment, especially because it is the fate for a variety of pollutants from focal or non-point sources. Fish species are useful as bioindicators, since they are promptly exposed to diverse anthropogenic water contaminants Sturm et al, The mixed function oxygenases MFO system plays an essential role in the metabolism of xenobiotics thanks to the function of cytochromes P CYP that mediate the NADPH-dependent oxidation of endogenous compounds and xenobiotics.

This complex associates to the Aril receptor nuclear translocase ARNT , which bind to the promoter region of CYP1A gene, activating the transcription and subsequently, protein synthesis Hahn and Chandran, The CYP system in the fish is structurally similar to that in mammals, but some differences are found; for example, the enzyme activity of fish liver microsomes is lower than in mammals, and the system could be regulated by exogenous factors Klotz et al , Stegeman and Hahn, In recent years, research in fish CYP1A has increased with the attempt to provide data to biomonitoring and pollutant metabolism Da Silva et al, Ancistrus multispinis Regan, cascudo is a Brazilian tropical fish belonging to the Loricariidae family that can be found in many Brazilian rivers.

It has been chosen due to its detritivorous habit, meaning that the fish is in contact with xenobiotics that interact with algae from stone or sediment, tolerance to hypoxia and facility to maintain in laboratory. This species could be considered a model for ecotoxicological studies since it could be in contact with xenobiotics indifferent ways. To cope with this, the spectrophotometric method was adapted for CYP analysis in Ancistrus multispinis.

Additionally, the western blotting method for CYP1A analysis was also tested for this species, improving even more the interpretation of results. Male and female fishes weighing approximately Fishes were randomly taken, measured, weighed and grouped in 30 liters test aquaria 12 fishes in each aquarium. Control group received an IC injection of sunflower oil in the same volume as the induced group. Both the groups were kept under the same experimental conditions for 96 h before liver collection. Liver was removed, washed with saline solution and immediately frozen in liquid nitrogen before microsomes isolation.

Microsomes were obtained from liver following Stegemann et al, , and Silva de Assis Livers were thawed and homogenized approximately 1g liver tissue in for volumes of homogenization buffer 0,08M Na 2 HPO 4 ; 0.


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Homogenate was centrifuged at The supernatant was ultracentrifuged at All the spectroscopic CYP analyses were carried out with this fraction. Protein concentration was determined using bovine serum albumin BSA as a standard Bradford, Total cytochrome P CYP. Analysis of CYP was based on the methods described before Omura and Sato, , Johannesen and Depierre, using differential visible spectroscopy with some adaptations. This protocol compares an oxidized aliquot of microsomal fraction with another reduced aliquot, which is bubbled with carbon monoxide. Liver perfusion was performed with an isotonic saline solution 0.

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Because liver samples of Ancistrus multispinis were very small, the perfusion was carried out by injecting the isotonic solution into each fragment of tissue until it showed a yellowish color. Blood of A. The hemoglobin was treated exactly as the microsome samples and UV-vis spectra were recorded. Analysis was carried out in a Beckman DU spectrophotometer. TRIS; 0. The primary antibodies used were the antibody donated by John J. The secondary antibodies used were peroxidase conjugated goat anti-rabbit IgG.

Protein bands were detected according to the. Amersham Pharmacia Kit for chemiluminescence detection of Western blots and the protein bands were visualized by autoradiography on Fuji Medical X-ray film. Autoradiograms were scanned and densitometrically analyzed using NIH image.

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Statistical analysis. Statistical analysis was carried out by t -test followed by Welch test. These spectra were composed of the expected nm peak, which resulted from the combination of the CYP with carbon monoxide CO , and of an additional peak near nm that could be caused by the degradation of the CYP into CYP or by contamination of the microssomal sample with hemoglobin Fig.

Spectra obtained from microsomes prepared after the liver perfusion showed a peak at nm and a peak at nm Fig.

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The effect of hemoglobin from blood sample from A. There was a coincidence between the spectra valleys around nm and also between the peaks around nm. Comparison of these spectra led to conclude that the nm peak could have been caused by the hemoglobin contamination. Results of the western blotting method are shown in Figure 5. Evidently, the 3-MC effectively induced A. The value obtained for the 3-MC group 3.


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  • There are generally marked differences between the species in P enzyme content, activity, and susceptibility. It could explain the different physiology, habitats, behavior, and feeding habitats, to uptake, accumulate, distribute, and metabolize contaminants. There are only few studies in the literature about P of Brazilian fishes and because of this, it is important to adapt the methods for each species.


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    Unusual spectra were obtained following the classical method for detection by Omura and Sato Studies on Hypostomus punctatus , a fish that belonged to the same Order as A. Assays with rat lung microsomes showed similar results and attributed them to the residues of hemoglobin since tissues could not be adequately perfused. Other study with a Neotropical freshwater fish species also described similar uncommon spectra and adopted tissue perfusion to decrease influence of hemoglobin contamination.

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