The Toxic Effects Of Cadmium And Mercury On L02Cells And The Interaction Between Cadmium And Serum Protein

Objective To study the effects of cadmium and mercury alone and in combination onDNA damage and apoptosis in human embryo hepatocytes （L02cells）; and then investigatethe interaction mechanism of cadmium chloride （CdCl₂） and bovine serum albumin （BSA）,and explore the the influence of CdCl₂on protein conformation.Methods L02cells were exposed to different concentrations （0.01～100μmol/L） ofcadmium chloride （CdCl₂）, mercury chloride （HgCl₂） and their mixtures for24hours. MTTmethod was used to measure the survival of L02cells, the Q value was measured by Finneylaw, and then the joint effects of these mixtures were judged depending on the Q value.Single cell gel electrophoresis （SCGE） and flow cytometry （FCM） were used to measurethe DNA damage and apoptosis of L02cells respectively. The fluorescence spectrum ofBSA and CdCl₂-BSA were measured at four different temperatures of298，302，306，310K, the Stern-Volmer, Arrhenius, Van ’t Hoff and small molecules and macromolecularligand equation were used to calculate the quenching constant, activation energy,thermodynamic parameters and binding constants and binding site; The distance betweenCdCl₂and BSA was calculated according to F rster fluorescence resonance energy transfertheory. At the same time, in order to investigate the influence of CdCl₂on BSAconformation, the UV-vis absorption spectrum, synchronous fluorescence spectrum, threedimensional fluorescence spectrum and circular dichroism of the systems were recorded at room temperature.Results0.01μmol/L of cadmium, mercury and their mixtures can stimulate the growth ofL02cells, but1μmol/L of cadmium, mercury and their mixtures can significantly inhibit thegrowth of L02cells. Q value exposed to cadmium and mercury was1.4, the joint effects ofcadmium and mercury are additional action. The rate of DNA damage and the percentage ofapoptosis were significantly higher than the control group after exposed to cadmium,mercury and their mixtures for24hours, and the rate of DNA damage and the percentage ofapoptosis show ascendant trend with the increase of individual dose. The quenchingconstant of BSA were27.854,28.527,32.223,33.664Lmol-1at four different temperaturesof298,302,306,310K, the binding constants of BSA were125.340,58.491,47.808,34.626M-1at four temperatures of298,302,306,310K, the activation energy was13.24, and thecorresponding thermodynamic parameters Δ S, Δ H and Δ G were223.364Jmol^-1K^-1,78.192KJmol^-1,(11.969,10.216,9.838, and-9.136KJmol^-1), respectively. The bindingdistance between CdCl₂and BSA was2.24nm. The UV-vis absorption spectrum displaythat with the addition of CdCl₂, the absorption spectrum intensity of BSA was not changedobviously, the synchronous fluorescence spectrum results showed that, with the increasingof CdCl₂, the fluorescence intensity of BSA decreased at△λ=15nm and△λ=60nm,and when△λ=60nm, the intensity decreased of BSA decreased more, and the largestemission wavelength had slight red shift; The three dimensional fluorescence spectrumshow that with the addition of CdCl₂, the fluorescence intensity of peak a was enhancedobviously （273.3²82.5）, the peak1was declined slightly （370.7³68.8）, the peak2wasdeclined significantly （563.4⁵40.5）; and the emission wavelength of peak1have slightred shift, the peak2have slight blue shift; The circular dichroism results show that with theincreasing of CdCl₂, there is a addition of α-helix structures （from51.93to52.31）, and thenthe content of α-helix reduced （from52.31to21.39）.Conclusions The joint effects of cadmium and mercury are additional action. Cadmium,mercury and their mixtures can cause DNA damage and cell apoptosis of L02cells, and they exist dose-effect relationship for certain. The main quenching mechanism offluorescence of BSA by CdCl₂is a dynamic quenching, and the CdCl₂could bind to BSAstrongly. The interaction between CdCl₂and BSA is mainly enthalpy-driven, and the mainforces between CdCl₂and BSA are hydrogen bonding and van der Waals force. The energytransfer between CdCl₂and BSA occurs with high probability, and then make the BSAoccur fluorescence quenching. At the same time, CdCl₂could affect the micro environmentand conformation of BSA.