A Study On The Associations Between Drug-metabolizing Enzymes And Antituberculosis Drug-induced Liver Injury

Background and ObjectiveAccording to the World Health Organization(WHO)2016 data,mycobacterium tuberculosis continues to infect one-third of the world`s population,of which 1.8 million died of it.In China,the number of new tuberculosis patients each year is up to 0.9 million.At present,the treatment of antituberculosis is that four medications(isoniazide,rifampicin,pyrazinamide and ethambutol)are used in combination.However,in the antituberculosis treatment process,the most frequent adverse effects are liver injury,skin reactions,gastrointestinal and neurological disorders,in which liver injury decreases treatment effectiveness,ultimately contributing to relapse,treatment failure,or the emergence of drug-resistance.Multiple mechanisms can culminate in antituberculosis drug-induced liver injury,but metabolism and genetics play distinct roles in this process.The accumulation of toxic metabolites(hydrazine and acetyl hydrazine)in hepatocytes induces the excessive production of ROS and reactive nitrogen species,contributing to antituberculosis drug-induced liver injury;On the other hand,the NAT2,GST,CYP2E1,HLA genotypes might play a role in determining the risk for the antituberculosis drug-induced liver injury.However,in recent years,liver injury could be a hypersensitivity reaction,because it has characteristics such as delay in onset and no relationship between the dose of the drugs and the risk of liver injury.In addition,there were cases with a fast onset upon rechallenge and cases associated with fever,rash.So the immune-mediated reactions can also lead to the occurrence of liver injury.The aim of this study is to analyze the symptoms related to liver injury induced by RIF and INH co-therapy,and to evaluate the risk of RIF and INH co-therapy-induced moderate and severe liver injury in patients with these symptoms.At the same time,We investigate the relationship between genetic polymorphisms of the two important drug-metabolizing enzymes NAT2(N-acetyltransferase 2)and CYP2E1(cytochrome P450 2E1)and antituberculosis drug-induced liver injury in Henan province;also first examine whether liver injury is associated with evidence of an immune-mediated reaction.Methods1.Subjects: The blood samples used in this study were taken from patients in Henan Province Infectious Diseases Hospital.All patients signed informed consent.The program was approved the Zhengzhou University Medical Ethics Committee,Henan Province Infectious Diseases Hospital Medical Ethics Committee before implemented.2.Clinical cases analysis: An analysis was conducted in 65 tuberculosis patients(during treatment with RIF and INH)between 2016 and 2017 in Henan Province Infectious Disease Hospital.The clinical characteristics and the risk of RIF and INH co-therapy-induced liver injury were recorded.3.Genomic DNA extraction and genotype analysis: A total number of 64 patients who received RIF and INH co-therapy-induced liver injury are case group,of which 42 cases were mild patients,18 cases were moderate patients,4 cases were severe patients.CYP2E1 and NAT2 genotypes were determined using a polymerase chain reaction and direct sequencing method to study the susceptibility between the gene polymorphisim and liver injury.4.Determination of anti-CYP3A4 and CYP2E1 antibodies in serum of patients with liver injury: A total number of 26 patients who received RIF and INH co-therapy-induced liver injury are case group,of which 16 cases were mild patients,8 cases were moderate patients,2 cases were severe patients.Western blot was used to study whether the anti-CYP3A4 and anti-CYP2E1 antibodies were detected in each blood sample.The relative expression of anti-CYP3A4 and anti-CYP2E1 antibodies in each blood sample was detected by enzyme-linked immunosorbent assay(ELISA).Results1.Relationship between liver injury-related clinical characteristics and liver injury in tuberculosis patientsIn this group of data,there was a total of 65 cases of RIF and INH co-therapy-induced liver injury in tuberculosis patients,men accounted for 67.69%,far higher than the incidence of women.Liver injury occurred in the treatment after 2 weeks,drinking and smoking would not increase the risk of moderate or severe liver injury.In 65 cases,36 patients(55.38%)had clinical characteristics related to liver injury during treatment,the highest incidence was loss of appetite,followed by weakness,nausea,fever,abdominal distention,anorexia,skin rash and skin itching.Patients with weakness,fever,nausea,loss of appetite have a higher risk of moderate and severe liver injury.Weakness,fever,nausea,loss of appetite may be the indicative symptom of moderate and severe liver injury.2.The relationship between genetic polymorphisms of the two important drug-metabolizing enzymes NAT2 and CYP2E1 and liver injury in tuberculosis patientsCompared two genotypes of CYP2E1 gene(C1/C1,C1/C2+C2/C2),C1/C1 genotype didn't have a higher risk of liver injury in tuberculosis patients(OR=1.478,p>0.05).There was no difference between the patient and the control group about the severity of liver injury(OR=0.961,p>0.05).Compared fast acetylator genotypes of the NAT2 gene,the media and slow acetylator genotypes didn't have a higher risk of liver injury in tuberculosis patients(p>0.05).The genotype of NAT2*5/7 with a higher OR than other genotypes(OR=5.304,p>0.05)is more likely to liver injury.The genotype of NAT2*4/7 ? NAT2*5/7 with a higher OR than other genotypes,2.235(p>0.05)?2.050(p>0.05)respectively are more likely to moderate and severe liver injury.3.Correlation between the expression of anti-CYP3A4 and CYP2E1 antibodies and the severity of liver injuryWestern Blot experiments showed that anti-CYP3A4 and CYP2E1 antibodies were present in the serum of patients with moderate and severe liver injury;ELISA showed that in moderate and severe liver injury,anti-CYP3A4 and CYP2E1 antibodies were significantly different from those with mild liver injury patients(p<0.001).Conclusions1.55.38% of tuberculosis patients experience clinical characteristics related with liver injury during treatment.patients with fever,fatigue of RIF and INH co-therapy-induced liver injury have a higher risk of moderate and severe liver injury.2.CYP2E1 and NAT2 genotypes have no association with susceptibility to the risk and the severity of RIF and INH co-therapy-induced liver injury.3.Immune mechanisms cause RIF and INH co-therapy-induced liver injury.