The Protective Role Of Aldehyde Dehydrogenase 2 On Cyclophosphamide-induced Acute Toxicity

BackgroundCyclophosphamide(CY),a potent oxazaphosphorine nitrogen mustard alkylating drug,is widely used in the anti-neoplastic treatment of lung cancer,breast cancer,ovarian cancer,cervical cancer,multiple myeloma,and etc.Because of its immunosuppressive activity,it is also frequently used for treating autoimmune diseases,systemic lupus erythematosus,multiple sclerosis,and severe rheumatoid arthritis.However,these treatments of CY are associated with various side effects such as bone marrow suppression,hepatic injury,cardiotoxicity,hemorrhagic cystitis,et al.Acute cardiotoxicity,one of the most serious side effects,usually manifests as cardiomyopathy,hydropericardium,hemorrhagic myopericarditis and even fulminant congestive heart failure and sudden cardiac death.These adverse effects substantially hinder the implementation of the treatment plan and thus shorten the survival time of patients.European Society of Cardiology(ESC)guideline has been reported that the incidence of cardiac dysfunction and heart failure after high doses of CY accounts for up to 20%,and recommended early prevention and treatment of CY-induced cardiotoxicity.Nevertheless,the mechanism of CY cardiotoxicity remains unclear,and there is an urgent need to better understand the cardiac effects of CY in order to treat or minimize the cardiotoxicity.Previous studies have demonstrated that CY could generate substantial reactive oxygen species(ROS)and contribute to subsequent elevated levels of lipid peroxidation-derived aldehydes.Acrolein,one metabolite of CY,is a highly reactive ?,?-unsaturated aldehyde which could initiate and promote ROS production directly as well as be generated by lipid peroxidation,and thus forming a vicious circle resulting in cell death and tissue damage.Whether the acrolein-ROS-lipid peroxidation-induced aldehydes vicious circle leads to cardiomyocyte death and the composition of cell death(necrosis or apoptosis)remains elusive.Aldehyde dehydrogenase 2(ALDH2),is the main enzyme in the metabolism of ethanol.Besides its role in catalyzing the oxidation of acetaldehyde to acetic acid in ethanol metabolism,it is considered responsible for oxidation and detoxification of other reactive aldehydes such as 4-hydroxy-2-nonenal(4-HNE)and malonaldehyde(MDA).Recently,ALDH2 has been identified as an important endogenous enzyme protecting against various cardiovascular diseases including cardiac ischemia/reperfusion injury,diabetic cardiomyopathy,alcoholic cardiomyopathy,and drug-induced cardiomyopathy,et al.It has been revealed that the beneficial role of ALDH2 against cardiac injuries is mediated by alleviating toxic aldehydes,and regulating o mitochondrial injuries,autophagy,endoplasmic reticulum stress,apoptosis,et al.ALDH2*2 is a common variant of ALDH2,importantly,and more than 40%of the East Asians population carries the mutant allele,which results in a dramatic reduction in the enzymatic activity.ALDH2*2/*2 genotype has been proved as an independent genetic risk factor for coronary heart disease in Han population.And higher incidences of insensitivity to nitroglycerin treatment for angina,MI,hypertension,and other oxidative-related neurodegenerative diseases have also been associated with ALDH2*2 mutation in recent years.Since the affected world population of ALDH2*2 is huge,it is necessary that the risk for CY-induced cardiotoxicity should be re-evaluated in ALDH2*2 carriers.Furthermore,based on the previous studies,we make the hypothesis that the ALDH2 plays a protective role against CY-induced cardiotoxicity.However,the related mechanisms have not been investigated.ObjectivesThe main objectives of this experiment are to investigate:1.Whether ALDH2 deficiency mice are more sensitive to CY-induced cardiotoxicity.2.The regulation and mechanism of ALDH2 to CY-induced cardiotoxicity.3.Whether the activation of ALDH2 enzymatic activity influence the anti-tumor effect of CY.Methods(I)For vivo study1.Animal model:C57BL/6J mice(WT)and ALDH2-/-mice(KO),8w old,were divided into three groups:WT group,WT+CY group,and KO+CY group.One dose of CY(200 mg/kg)was used to induce cardiotoxicity by intraperitoneally.2.Then C57BL/6J mice,8w old,were divided into two groups:WT+DMSO+CY group and WT+Alda-1+CY group.An ALDH2 agonist,Alda-1 inj ection(20 mg/kg,i.p.)daily or an equivalent volume of dimethyl sulfoxide(DMSO)was started before the day of CY administration and continued for 3 days.3.Echocardiographic assessment was performed to measure cardiac function.In M-mode images,we measured and calculated left ventricular end-diastolic dimension(LVEDD)and left ventricular end-systolic dimension(LVESD),left ventricular ej ection fraction(EF)and fraction shortening(FS).4.Immunofluorescence staining of EBD-CaV3 was performed to show necrosis in the myocardium of mice induced by CY administration.5.Terminal deoxynucleotide nick-end labeling(TUNEL)assay was performed to show apoptosis in the myocardium of mice induced by CY administration.Western blotting was performed to measure the protein expression of 4-HNE,Bcl2,Bax,Caspase3,and ?-actin in the myocardium of mice after CY administration.6.Immunohistochemical staining was performed to show acrolein accumulation in the myocardium of mice induced by CY administration.7.ROS fluorescent probe-dihydroethidium(DHE)was performed to detect the level of reactive oxygen species(ROS)in the myocardium of mice induced by CY administration.8.RT-qPCR and western blotting were performed to measure the mRNA and protein expression of ALDH2.9.ALDH2 enzymatic activity measurement:Isolate the mitochondria from myocardial tissue and measure the conversion of NAD+ to NADH to determined the enzymatic activity of ALDH2.10.Statistical analysis:Data were expressed as Mean ± SEM.SPSS was used for statistical analysis.P<0.05 was considered statistically significant.(?)For vitro study1.Acrolein-induced cell injury model:Rat cardiomyoblasts cell line(H9C2)was cultured in DMEM with 10%of FBS and 1%of penicillin/streptomycin.H9C2 cells were treated with acrolein for 6 hours in the absence or presence of Alda-1(20?M).Pan-caspase inhibitor zVAD was used to inhibit cell apoptosis.2.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to determine cell viability after acrolein treatmentResults1.ALDH2 deficiency exacerbates CY-induced cardiotoxicityCompared with the Con group, cardiotoxicity was induced by CY,showed as decreased EF and FS,increased levels of serum LDH and CKMB.Compared with WT+CY mice,ALDH2 deficiency exacerbated CY-induced cardiac injury,as presented by significantly reduced EF and FS,increased serum LDH and CK-MB.2.ALDH2 deficiency accentuates CY-induced cardiac cell deathAs evidenced by EBD-positive staining,CY led to the loss of cardiomyocyte membrane integrity(an indicator of necrosis)in WT mice.ALDH2 deficiency markedly increased the CY-induced cardiac cell necrosis compared with WT+CY group.Except for necrosis,apoptosis percentage evaluated by TUNEL staining was markedly increased in WT+CY,and ALDH2 deficiency significantly increased the CY-induced cardiac cell apoptosis.The key proteins related to cellular apoptosis were also assessed.Compared with the WT group,the expression levels of Bax and Caspase-3 were significantly increased whereas the expression levels of Bcl-2 were decreased in WT+CY group.Furthermore,the expression levels of Bax and Caspase-3 were increased whereas the levels of Bcl-2 were decreased in KO+CY group compared with WT+CY group.3,ALDH2 deficiency accentuates CY-induced accumulation of toxic aldehydesCompared with the WT group,the levels of protein-acrolein adducts were remarkably elevated in WT+CY group which were further elevated in ALDH2 KO+CY group.Similarly,CY resulted in more accumulation of protein-4-HNE adducts compared with the WT group.The levels of protein-4-HNE adducts were further elevated in KO+CY compared with WT+CY groupBesides,levels of ROS were higher after CY administration and ALDH2 deficiency ameliorated the CY-elevated ROS levels.4.Cardiac ALDH2 activity was inhibited in CY-induced cardiotoxicityThe mRNA and protein expression levels of ALDH2 had no obvious changes,whereas ALDH2 activity was reduced in WT+CY group compared with the WT group5.ALDH2 activation attenuates CY-induced cardiotoxicityPretreatment with Alda-1 markedly ameliorated CY-induced LV contractile dysfunction,together with the reduction of serum CK-MB and LDH compared with WT+DMSO+CY group.6.ALDH2 activation attenuates CY-induced cardiac cell deathCompared with WT+DMSO+CY group,WT+Alda-1+CY group showed decreased CY-induced cardiomyocyte necrosis.In line with these findings,Alda-1 decreased the percentage of apoptotic myocyte compared with WT+ DMSO+CY group.Compared with WT+DMSO+CY group,the expression levels of Caspase-3 and Bax were significantly decreased in WT+Alda-1+CY group,and Bcl-2 was markedly increased in WT+Alda-1+CY group.7.ALDH2 activation attenuates CY-induced toxic aldehydes accumulationALDH2 activation rescued the CY-induced toxic aldehydes accumulation.Compared with WT+DMSO+CY group,the accumulation of both protein-acrolein adducts and protein-4-HNE adducts were reduced WT+Alda-1+CY group.Alda-1 also suppressed the CY-elevated ROS levels by 45.05%compared with WT+DMSO+CY group.8.ALDH2 activation attenuates acrolein-induced cardiomyocyte necrosis and apoptosisApproximately 78.28%of acrolein-induced cell death could not be blocked by zVAD,suggesting that acrolein-induced cardiomyocyte death was mediated primarily by necrosis.The protective effect of Alda-1 could not be abolished by zVAD.Approximately 49.3 1%of acrolein-induced necrosis was blocked by Alda-1.9.ALDH2 activation do not antagonize the anti-tumor efficacy of CYGiven the systemic effects of Alda-1,we further investigated whether Alda-1 pretreatment could antagonize the specific anti-tumor efficacy of CY by using theBALB/c mouse model of breast cancer(4T1).There were no significant differences in growth rate of tumor tissue among DMSO,DMSO+CY and Alda-1+CY group.The expression levels of Bax and Bcl-2 in tumor tissue were also not different among DMSO,DMSO+CY and Alda-1+CY group.The tumor weight of DMSO+CY and Alda-1+CY group were respectively smaller than the DMSO group.Conclusion1.Our findings demonstrate that ALDH2 deficiency mice are more sensitive to CY-induced cardiotoxicity as reflected by increased cardiac dysfunction and serum myocardial markers.2.ALDH2 activation protects against CY-induced cardiotoxicity mainly by removing toxic aldehydes accumulation and ROS production,breaking the toxic aldehydes-ROS-toxic aldehydes cycle,and thus alleviating cardiac cell death.3.ALDH2 activation do not antagonize the anti-tumor efficacy of CY in the mouse model of breast cancer.IntroductionCyclophosphamide(CY)is a widely used chemotherapeutic agent that is associated with several side effects.Several antineoplastic protocols required CY are associated with several side effects,such as urotoxicity,immunotoxicity,hepatotoxicity,cardiotoxicity,and nephrotoxicity.Hepatotoxicity is theoretically considered as one of the most severe adverse effects among them.Because the liver is the key organ which is responsible for the metabolism of CY and its reactive metabolites.However,the extent,mechanisms and potential prevention and treatment strategies of CY-induced hepatotoxicity are largely unknown.Previous studies have revealed that CY could generate reactive oxygen species(ROS)and contribute to succeeding elevated levels of highly cytotoxic oxidative stress-derived lipid peroxidation aldehydes,such as 4-hydroxynonenol(4-HNE)and malondialdehyde(MDA).Besides,acrolein(ACR),one of CY metabolites,could directly initiate oxidative stress and be generated by lipid peroxidation.However,whether the vicious circle plays a key role hepatotoxicity induced by CY need to be explored.Aldehyde dehydrogenase 2(ALDH2)has recently been identified as an important endogenous protective enzyme in many hepatic diseases,such as alcoholic-induced hepatic steatosis,hepatic ischemia/reperfusion injury,hepatic fibrosis,and hepatocellular carcinoma.ALDH2 is the main enzyme to metabolize a variety of toxic aldehydes in physiological and pathological processes.Previous evidence showed that ALDH2 plays its protective roles in many organs by reducing oxidative stress and toxic aldehyde,such as heart,brain,liver,and kidney.However,whether ALDH2 plays a key role in hepatotoxicity induced by CY remains elusive.ObjectivesIn this study,we determined to demonstrated the protective effect and related mechanisms of ALDH2 on hepatotoxicity induced by CY.MethodsOne dose of CY(150 mg/kg)was used to induce hepatotoxicity by intraperitoneally(i.p.).And Alda-1 injection(20 mg/kg,i.p.)daily was used to activate ALDH2 enzymatic activity before the day of CY treatment and continued for 3 days.And then the liver function indicators and hepatic micromorphology were determined.1.Adult male ALDH2 wild type(WT)and ALDH2-/-(KO)mice were divided into four groups:WT,WT + CY,KO + CY and WT + CY +Alda-1.2.Biochemical parameters were determined to evaluate the hepatic and renal function.3.Hematoxylin and Eosin(H&E)staining was used to detect the hepatic micromorphology after CY administration.4.The level of ROS was evaluated by using dihydroethidium(DHE)method.5.Acrolein and 4-HNE accumulation levels in liver were determined.Thiobarbituric acid reactive substances(TBARS)was used to detect the level of MDA in liver after CY administration.Results1.ALDH2 deficiency exacerbated the hepatotoxicity induced by CYCompared with the WT group,the level of plasma ALT was increased by 37.7%in WT+ CY group.And the levels of plasma ALT were increased by 35.8%in KO +CY group compared with WT + CY group.In WT+CY group,we found obvious hepatic hydropic degeneration and vacuolization.Furthermore,in KO+CY group,the damage was further aggravated,showed as severe hydropic degeneration,vacuolization,and nuclear fragmentation.2.Alda-1 attenuated CY-induced hepatotoxicityCompared with WT + CY group,plasma ALT was decreased by 21.1%,and the hepatic structure was almost normal in WT + CY + Alda-1 group.3.Alda-1 attenuated CY-induced oxidative stressCompared with WT + CY group,level of reactive oxygen species(ROS)induced by CY administration was increased significantly in KO + CY group and decreased significantly in WT + CY +Alda-1 group(P<0.05).4.Alda-1 attenuated CY-induced reactive aldehydesCompared with WT + CY group,levels of toxic aldehydes(acrolein,4-HNE,MDA)accumulation induced by CY administration were increased significantly in KO+ CY group and decreased significantly in WT + CY + Alda-1 group(P<0.05).ConclusionOur findings demonstrate that ALDH2 deficiency mice are more sensitive to CY-induced hepatotoxicity.Furthermore,ALDH2 plays a protective role against CY-induced hepatotoxicity through attenuating oxidative stress and detoxifying toxic aldehydes.