Evaluation and standardization of different purification procedures for fish bile and liver metallothionein quantification by spectrophotometry and SDS-PAGE analyses.
Fish bile metallothioneins (MT) have been recently reported as biomarkers for environmental metal contamination; however, no studies regarding standardizations for their purification are available. Therefore, different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, β-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Liver was also analyzed, since these two organs are intrinsically connected and show the same trend regarding MT expression. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated and a multivariate statistical analysis was then applied. A response surface methodology was also applied for bile samples, in order to further evaluate the responses for this matrix. Heat treatment effectively removes most undesired proteins from the samples, however results indicate that temperatures above 70 °C are not efficient since they also remove MTs from both bile and liver samples. Our results also indicate that the centrifugation times described in the literature can be decreased in order to analyze more samples in the same timeframe, of importance in environmental monitoring contexts where samples are usually numerous. In an environmental context, biliary MT was lower than liver MT, as expected, since liver accumulates MT with slower detoxification rates than bile, which is released from the gallbladder during feeding, and then diluted by water. Therefore, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix.
Pontifícia Universidade Católica - Rio de Janeiro (PUC-Rio), Chemistry Department, LABSPECTRO, Rua Marquês de São Vicente, 225, Gávea, CEP: 22453-900, Rio de Janeiro, RJ, Brazil. Electronic address: carolyrio@hotmail.com.
Carolina Lyrio
CL
Tenório-Daussat
Pontifícia Universidade Católica - Rio de Janeiro (PUC-Rio), Chemistry Department, LABSPECTRO, Rua Marquês de São Vicente, 225, Gávea, CEP: 22453-900, Rio de Janeiro, RJ, Brazil. Electronic address: marciamr@hotmail.com.
Marcia Carolina Martinho
MC
Resende
Universidade Federal do Estado do Rio de Janeiro - UNIRIO, Av. Pasteur, 458 - Urca, CEP: 22290-240, Rio de Janeiro, RJ, Brazil. Electronic address: robertaziolli@gmail.com.
Roberta L
RL
Ziolli
Pontifícia Universidade Católica - Rio de Janeiro (PUC-Rio), Chemistry Department, LABSPECTRO, Rua Marquês de São Vicente, 225, Gávea, CEP: 22453-900, Rio de Janeiro, RJ, Brazil. Electronic address: rachel.hauser.davis@gmail.com.
Rachel Ann
RA
Hauser-Davis
LCABIE UMR5254, Technopôle Hélioparc Pau Pyrénées, 2 avenue du Président Angot, Pau, 64053 Pau Cedex 09, France.
Dirk
D
Schaumloffel
Pontifícia Universidade Católica - Rio de Janeiro (PUC-Rio), Chemistry Department, LABSPECTRO, Rua Marquês de São Vicente, 225, Gávea, CEP: 22453-900, Rio de Janeiro, RJ, Brazil. Electronic address: tatispierre@puc-rio.br.
Tatiana D
TD
Saint'Pierre
D000818:Animals; D001646:Bile / Q000737:chemistry / Q000378:metabolism*; D023681:Cichlids / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods / Q000592:standards; D004784:Environmental Monitoring / Q000379:methods* / Q000592:standards; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D008668:Metallothionein / Q000302:isolation & purification* / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods / Q000592:standards; D014874:Water Pollutants, Chemical / Q000378:metabolism
D014874:Water Pollutants, Chemical; D008668:Metallothionein
Ecotoxicology; Fish bile; Metalloproteins; Metallothionein; Multivariate statistical analyses; Purification procedures