Uma abordagem proteomica na identificação do citocromo P450 em Prochilodus Scrofa : uma nova ferramenta em ensaios ecotoxicologicos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Cytochromes P450 constitute a superfamily of the phase I enzymes whose primary task is the detoxification of both endogenous and xenobiotic compounds. This activity is only possible through the association of CYP with cytochrome b5 and the enzyme NADPH cytochrome P450 reductase. This association is possible because of membrane environment. Any alteration in this environmet leads to altereation on the functionality of CYP system. Based on this information and in the fact that various pollutants have its solubility increased by the surfactant addition, we had done in vitro tests using hepatic microsomes of Curimbatá - a Brazilian fish belong the family Prochilodontidae ? and observed the effects of Triton X-100 and Tween 80 in CYP system. Both surfactants are commonly used in pesticides solubilization. Total CYP and EROD were strongly inhibited by Triton X-100 and Tween 80 in a concentration-dependent way; the content of CYP was reduced until zero while EROD activity was completely inhibited in the presence of Triton X-100 and more than 40% inhibited in the presence of Tween 80. Each surfactant causes a different effect on each antioxidant enzyme. No effect was detected in SOD activity in the presence of even Triton X-100 or Tween 80. Triton X-100 increase catalase activity, while Tween 80 decreases this enzyme activity. The molecular structure of the surfactants causes the alteration of this system, since they are able to interact with the microsomal protein, especially with monooxigenase?s components, altering their conformation and, consequently destroying their function. Our results in this first part suggest that surfactants can interact with components of the microsomal system leading to inhibition of CYP. Therefore, CYP activity, which has been used as a biomarker of xenobiotic exposure, should be used as a marker in association with other enzymes. With those results we suggest the hypothesis that surfactants that are commonly used in the purification process could alter the CYP activity. Since that CYP is a membrane protein, the solubilization process is necessary before the purification process to separated membrane lipids of proteins. The use of surfactants in the process could mask the real concentration of CYP obtained. Then, in the second part of this project we develop a new method to of purification that excludes the necessity of surfactants. The purification of CYP1A was done by Reverse Phase HPLC on a C18 column. Purified CYP1A was characterized with respect to electrophoretic, immunochemical and biocatalyst properties. CYP1A fractions produced a single uniform band on SDS-PAGE with an apparent molecular mass of 58 kDa. Purified CYP1A of P. scrofa showed strong cross-reactivity with antibodies directed against CYP1A from trout. The fraction was also encapsulated in two different reconstituted systems; one composed of neutral lipids and another of negatively charged lipids. In both of them, we could detect EROD activity but not PROD activity, which confirms that the CYP1A was purified with all its enzyme activity. There was an increase of activity when CYP1A and NADPH cytochrome P450 (CYP) reductase were encapsulated in negatively charged lipids, which confirms that the charge of lipid is essential to CYP1A activity. All these characteristics strongly suggest that this new procedure is efficient for purifying hepatic CYP1A from P. scrofa, showing that the CYP1A isoform of this fish has a highly conserved protein region. However the process was not efficient to separate the isoforms of CYP1A. In the third part of project we aimed the identification of CYP1A1 and CYP1A3 isoforms through bidimensional electrophoresis and MALDI ? TOF mass spectrometry. Three major spots were detected. These spots had the same molecular weight, but presented pI in a range of 5.5 to 6.0, suggesting the presence of 3 isoforms of CYP1. Spots were collected, and treated with Trypsin and the resultant peptides were measured by MALDI-TOF Mass Spectrometry. Tryptic peptide mass fingerprint of CYP1A showed the presence of 10 masses that matched the expected tryptic peptides obtained through theory digestion of the database sequence. These values corresponded to 31% of the translated amino acids of CYP1A family of other fishes and mammals. All spots had the same profile of fragmentation, which confirms that they are isoforms. Among these peptides, two demonstrated major importance T3, which is a conservative domain of CYP1A and T2, which could represent a more conservative region that occurs only in fishes. All these characteristics strongly suggest that this new procedure efficiently identifies, simultaneously, different isoforms of hepatic CYP1A from P. scrofa and its conservative region of protein. Due to the sensibility of the technique, in this last part of the project we try to identify the CYP isoenzymes that are expressed in hepatic microsomes of Curimbatá using one ? dimensional (1 ? DE) and two dimensional (2 ? DE) gel electrophoresis followed by tryptic peptide mapping (PMF). Both 1 ?DE and 2 ? DE showed that 3 ? MC induces not only CYP1A1 and CYP1A3, but other biotransformation enzymes presents in endoplasmic reticulum like cytochrome b5 (~15kDa), NADPH cytochrome P450 reductase (~75kDa) and microsomal Glutathione ? S ? Tranferase (~20kDa). There were 25 proteins that appeared only in 3 ? MC treated group and a matching of 38% between control and 3 ? MC gel. The results demonstrated here show that CYP system associated to the proteomic approach have great potential to be utilized in the identification of proteins differentially expressed according to environmental situation

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biomarcadores peixe

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