Studies On Absorption And Distribution Of Enrofloxacin In Earthworms Body And Its Effects On Cytochrome P450

Environmental protection and food safety have gradually become one of the public foci.With the increase of environmental protection consciousness,the environmental impact of a new type of pollutants-personal care products（PPCPs）has been paid more and more attention.The influence of drug residues on the ecological environment has gradually become a new focused research area.Enrofloxacin as one of the most widely used veterinary antimicrobial drugs has been proven effective against both gram-negative and gram-positive microorganisms and used widely in aquatic and livestock breeding to help maintain the health condition of domestic animals and ensure their well-being.However,its excessive use has caused substantial amounts of drugs and their metabolites being released to the environment through the excreta of animals.These drug residues and their metabolites may accumulate in soil and disturb the balance of the soil ecosystem.As a widely distributed group of large soil animals,the earthworms play important functions in ecosystem.They are one kind of good bioindicators of soil environment,they can be used as key indicators to predict the effects of chemicals on the soil environment and other soil invertebrates.This paper mainly studied a detection method for enrofloxacin residues in earthworm body,the uptake and distribution of enrofloxacin in earthworms,and the effects to protein expression of two kinds of typical cytochrome P450 subunit.An assay method using high performance liquid chromatography（HPLC）with fluorescence detection was studied and established to quantify residues of enrofloxacin in earthworms firstly.Sample pretreatment method was just as follows:The extraction solution comprised 0.1 mol·L^-1 phosphoric acid（pH12）+acetonitrile（1+3v/v）.The initial extract was defatted,evaporated,and re-dissolved in the mobile phase comprising 0.025 mol·L^-1 phosphoric acid/triethylamine buffer（pH3.0）+acetonitrile（83+17 v/v）.The fluorescence detector emission wavelength was 450 nm,and the excitation wavelength 280 nm.The linear range of the standard curve was 5⁵00 ng·mL^-1.The recovery rates of 3concentration groups were between 81%and 88%with an average recovery of 84.5%and coefficient of variation<10%.The retention time was from 11 to 12 minute,and the lowest detection limit of enrofloxacin was 2 ng·g^-1.Then studied the absorption of enrofloxacin by earthworms through the contact filter paper method and natural soil test method.In the contact filter paper test,Earthworms were exposed to filter papers soaked in enrofloxacin with concentrations of 0,0.16,1.6,16,and 32μg·cm^-2.After48 hours exposure,the average enrofloxacin concentration in earthworms from the respective groups was 0,1.53,16.81,113.32,and 120.83μg·g^-1Natural soil was added with EF at 0,1,10,100,and 500 mg·kg^-1concentrations.Then breed earthworms of Eisenia fetida into the natural soil.After these samples would be taken at days 1,3,7,14,21and 28 of exposure for determination of the content of Enrofloxacin in earthworms.The results showed that the content of EF in earthworm was increasing in1⁷ days,and basically reached peak after 7 days.The peak enrofloxacin concentration in earthworms from the 1,10,100,500 mg·kg^-1concentration groups was 0.247,0.591,2.798 and 16.098 mg·kg^-1.When the absorption reached the peak,curve would show a certain decline in short period.But the content of EF in earthworm respectively reached basic balance in the time of 21²8d,14²8d,21²8d,and 14²8d.At this time,the average content of EF in each group’earthworms was 0.220,0.577,3.486,13.971 mg·kg^-1.From these we can see that earthworm through surface and intake has a good absorption of enrofloxacin.The EF content in earthworm would increase with the extension of time and elevated outside enrofloxacin concentration.But it would eventually tend to dynamic equilibrium state.And the concentration of EF in different groups’earthworms was much lower than that in the soil,and the enrichment coefficient were 0.220,0.058,0.025,0.028.Obviously,the absorption and accumulation effects of EF in earthworms is not very significant.This paper also carried out research on enrofloxacin in earthworm distribution.The results showed that,When the earthworm uptake of EF reached equilibrium,the content of EF in various tissues and organs were sorted from high to low as gizzard>bowel>spermatheca>seminal vesical>body wall.Enrofloxacin is mainly distributed in the digestive organs such as gizzard and bowel,least in body wall.In the absorption process,when the content reaches the peak,it would decrease.These results conformed to the change trend in natural soil test.Cytochrome P450（CYP450）is one of the most important hemoglobin enzymatic family which involves in the biotransformation of many endogenous and exogemous compounds in vivo.It plays an important role in the metabolism of drugs and has extremely wide metabolic substrates.A lot of drugs can affect the CYP450,play a role in inducing or inhibiting,thereby affecting their own or other similar drugs.This paper takes CYP2B6 and CYP2C9 as the target protein,studied the effects of enrofloxacin on earthworm cytochrome P450.The results showed that CYP2B6/β-actin in the four concentration groups of 0,10,100,500mg·kg^-11 were 1.930,3.120,2.953,2.406;CYP2C9/β-actin were 2.473,3.891,3.747,3.141.It’s not difficult to see that within the concentration range setted in the test,enrofloxacin mainly played a role of inducing in earthworm cytochrome P450.And the induction first enhanced and then weakened with the increasing concentration of enrofloxacin.The results of this study got some basic data for further research on the toxic effects of enrofloxacin and its metabolites to ecological environment,and also provided reference for the environmental impact assessment and pollution control of veterinary drug residues.