Study On The Cytotoxicity And Related Mechnism Of HepG-2 Cells Induced By Iodinated Haloacetamides

Iodinated haloacetamides(I-HAcAms)are emerging disinfection by-products(DBPs)and have received great concern due to their extremely high cytotoxicity.But up to now,little work was done about the toxicity of I-HAcAm except iodoacetamide(IAcAm).Therefore,the cytotoxicity,genotoxicity and other related research work of I-HAcAm should carried out in-depth,which is meaningful to the water utilities to better manage the drinking water,and give guidance for the DBPs control.Based on the information above,present study aimed to investigate the cytotoxicity and possible toxicity mechanisms of four I-HAcAms,i.e.diiodoacetamide(DIAcAm),iodoacetamide(IAcAm),bromoiodoacetamide(BIAcAm)and chloroiodoacetamide(CIAcAm),using HepG-2 cells.The results were as the following:1)All of I-HAcAms could cause the cytotoxicity on HepG-2 cells.The cytotoxicity followed a descending trend of DIAcAm>IAcAm>BIAcAm>CIAcAm.The cell growth rate for all treatments reduced in a time-and dose-manner.Moreover,the IC50 value of dihaloacetamides(di-HAcAms)was found to be significantly(p<0.05)related to the logarithm of the octanol-water partition coefficient(log P)which is used to measure the lipophilicity.The higher level of log P value,the higher capacity to cross cells membrane,the higher cytotoxicity was.Yet for IAcAm,the cytotoxicity cannot be explained by log p values.The much higher cytotoxicity of IAcAm than BIAcAm and CIAcAm may be attributed to fewer halogens per atom.IAcAm only has one halogen(i.e.iodine),which has a good leaving tendency due to the long bond length and low dissociation energy.2)The four I-HAcAms could stimulate the ROS generation on HepG-2 cells.The ROS intensity followed a descending order of DIAcAm>IAcAm>BIAcAm>CIAcAm and showed a dose dependent manner,i.e.the higher concentration on of I-HAcAm,the higher level of ROS was.These results were consistent with those obtained from cytotoxicity,demonstrating the close link between ROS generation and cytotoxicity of I-HAcAms on HepG-2 cells.Besides ROS,Nrf-2 and Gclc were also important index for oxidative stress.Nrf-2 is a central regulator of redox status in various biological processed.Nrf-2 can bind with antioxidant response element,controlling the transcription of a battery antioxidant enzymes,including glutamate-cysteine ligase catalytic subunit(Gclc).The mRNA expression of Nrf-2 and Gclc within HepG-2 cells generally increased with the increase of I-HAcAms concentration.The corresponding protein levels were also in line with mRNA expression trends.These results further confirmed the oxidative damage on HepG-2 cells induced by I-HAcAms.3)The four I-HAcAms could lead to apoptosis on HepG-2 cells.The apoptosis rate followed a descending trend of DIAcAm>IAcAm>BIAcAm>CIAcAm,and showed a positive linear relationship with the exposure time and exposure dose.Bax and Bcl-2 are pro-apoptotic and anti-apoptotic protein,respectively,serving as critical control points in the mitochondrial apoptosis pathway.The higher ratio of Bax/Bcl-2 indicated a promotion in apoptosis mediated by the mitochondrial pathway,while a lower ratio indicated an inhibition.In the present study,the ratio of Bax/Bcl-2 expression on HepG-2 cells generally increased with the increase of I-HAcAms concentration.The corresponding protein levels showed consistent trend as mRNA expression:Bax protein expression increased but Bcl-2 protein expression decreased with the increase of I-HAcAms dose.4)HepG-2 cells with NAC pretreatment recovered well from cytotoxicity induced by I-HAcAms or even grew better than the control group.Moreover,ROS generation for all I-HAcAms treatments was removed by addition of NAC.These results indicated that the NAC can protect cells from the cytotoxicity induced by I-HAcAms through ROS removal.Since the ROS can be scavenged by addition of NAC,the ROS-mediated apoptosis will also be eliminated by NAC.However,NAC treatment did not affect the apoptosis induced by BIAcAm and CIAcAm,and only partially reduced the apoptosis caused by DIAcAm and IAcAm.These results indicated that BIAcAm and CIAc Am induced a ROS-independent apoptosis,but for DIAcAm and IAcAm,the cell apoptosis was not only controlled by ROS,but also other factors.All of these results demonstrated that the mitochondrial apoptotic pathway played an important role in the apoptosis of all I-HAcAms treatment cells.