| Methylene blue(MB) is a widely used thiazine dye. Azures B(AZB), azure A(AZA), azure C(AZC) are demethylated metabolites of methylene blue. Methylene blue is often used to prevent saprolegniasis in aquaculture. However, foreign studies have indicated that they have reproductive toxicity, teratogenicity, mutagenicity and potential carcinogenicity. Accumulation of residues in aquatic animals may be harmful to non-target animals and humans. P-glycoprotein(P-gp), also known as transmembrane glycoprotein, can pump out drug. Drug can induce or inhibit the expression of Pglycoprotein. At the same time, expression of P- glycoprotein will affect toxicity of drug.The distribution and elimination of methylene blue in the aquatic animal have been reported rarely. Different animals have varied metabolism. Therefore, determination of methylene blue and its metabolites in allogynogenetic crucian carp by HPLC to inquiry the metabolism. Effects of methylene blue on P-gp expression have been studied by real-time fluorescence quantitative PCR and immunocytochemistry. These studies provide a theoretical basis for management and application of methylene blue.Researches as follows:(1) A kind of method has been established to detect methylene blue and its metabolites in allogynogenetic crucian carp. The method have good linear relationship within 0.010-1.0 mg/L, R2>0.9998. Linear equations of MB, AZB, AZA and AZC are y = 0.3738x- 0.4513, y = 0.0442x- 0.1672, y = 0.0455x- 1.1833, y = 0.1124x- 2.3458. Detection limit of method is 2.0μg/kg. Their variance coefficients are less than 10%.(2) The distribution and elimination rules of methylene blue and its metabolites in allogynogenetic crucian carp was investigated after bath treatment of 15 mg/L methylene blue. Allogynogenetic crucian carp were raised in aquariums. The concentrations of methylene blue and its metabolites in the main tissues were simultaneously detected by HPLC. The data were analyzed using pharmacokinetics software DAS3.0. The results showed that peak concentrations of methylene blue, azure B and azure A were 143.436, 110.770 and 8.444 μg/L at 0.083, 0.5 and 8 h, respectively. Four kinds of tissues concentration levels of Methylene blue, azrue B and azrue A from high to low were hepatopancreas, kidney, muscle and skin. Methylene blue, azrue B and azrue A were likely to accumulate in muscle and skin, which still can be detected until 64 d. Azrue C was not detected in tissues of allogynogenetic crucian carp.(3) Effect of MB on mRNA expression of P-glycoprotein in allogynogenetic crucian carp tissues were detected by real-time fluorescence quantitative PCR. Results indicated that P-glycoprotein relative expression was the highest in normal hepatopancreas and the lowest in normal kidney. There were significant differences in various tissues(P<0.01) by independent sample t-test. It could be inferred that the Pglycoprotein was widely existed in allogynogenetic crucian carp, but there were some differences in various tissues. The relative expression of P-glycoprotein was significant difference(P<0.01) between metabolism groups and contorl groups. The expression of P-glycoprotein in metabolism groups was lower than control groups in hepatopancreas. However, there was no similar pattern in kidney and skin.Effect of MB on protein expressions of P-glycoprotein in allogynogenetic crucian carp tissues were detected by immunohistochemistry. Results are as follows, the expression of P-glycoprotein was the highest in normal hepatopancreas, and normal skin was slightly higher than normal kidney. There were significant differences between normal hepatopancreas and skin, normal hepatopancreas and kidney(P<0.01) by independent sample t-test. However, difference was not significant between normal skin and kidney(P > 0.05). It could be concluded that the expression of P-glycoprotein was differences in various tissues, and was the highest in hepatopancreas. The metabolic process of methylene blue had a certain effect on the expression of P-glycoprotein in the hepatopancreas, kidney and skin.In summary, the method was established for simultaneous determination of methylene blue and its metabolites in aquatic products. Peak time of MB, AZA and AZB was diversity. Their residues could be detected until 64 d. However, AZC could not be detected in tissues. The expression of P-glycoprotein was differences in various tissues and was the highest in hepatopancreas. The metabolic process of methylene blue had a certain effect on the expression of P-glycoprotein in tissues. |
PDF Full Text Request