Cell Cycle Control Genes And Susceptibility To Chronic Benzene Poisoning

Ph.D Candidate: Mr. Pin SunSupervisor: Prof. Zhao-lin XiaBenzene is a prototype hematotoxic and genotoxic carcinogen. Chronic benzeneexposure results in progressive depression of bone marrow function, but people varyin their response to this toxic agent. One of the important mechanisms of benzenepoisoning (BP) is DNA damage caused by its reactive metabolites. The P53 proteinactivated by these damaged DNA can lead to increased expression levels of p21 andGadd45a genes, resulting in the cell cycle being interruptedion and preventingreplication of damaged DNA, which is important for genomic stability, while theexpression level of P53 is also regulated negatively by MDM2 and P14^ARF. A numberof genetic polymorphisms have been identified in these genes, some of which arerelatively common and can result in change of enzyme activity or the configuration,mRNA expression as well, which might be responsible for human susceptibility to BP.So far there has been limited data on association between cell cycle control genes andsusceptibility to BP.A rapid, reliable and simple method detecting hydroquinone (HQ) and catechol (CAT)in urine by high performance liquid chromatography with electrochemical detectorwas developed. By this method, we detected the concentration of HQ and CAT inurine of 43 BP patients and 43 workers without the poisoning manifestations butoccupationally exposed to benzene. The results indicated that there was no significantdifference in the concentration of HQ and CAT between case group and control group.The results also indicated that the concentration of HQ in female-workers group washigher than in male-workers group, so female-workers should be paid more attentionin occupational health surveillance. Meanwhile, in the case and control groups themRNA expression of p53 and p21 gene in peripheral blood lymphocytes was detectedby Real-time PCR. The results indicated that the expression of p21 was dependent onthe expression of p53. However, no relationship was found between sex, exposure duration, exposure level and the expression levels of p53 and p21 in peripheral bloodlymphocytes.In order to elucidate roles of cell cycle control genes in human susceptibility to BPand effects of benzene exposure duration and life styles, a case-control study wasconducted. 311 BP patients come from Shanghai, Guangzhou, Hangzhou, Maanshan,Wuhu and Cixi and 311 workers without poisoning manifestations but occupationallyexposed to benzene were investigated. The cumulative exposure level was estimatedwith method described by Dosmeci, and the lifestyles such as cigarette smoking andalcohol consumption were also explored. The results of occupational epidemiologicalsurvey showed that there was no statistic difference between distribution of sex, race,age, work-age and cumulative exposure level in case and control groups, whichindicated that it was comparable with the case and control groups.With method of polymerase chain reaction-restriction fragments length polymorphism(PCR-RFLP) and created restriction site combined with restriction fragment lengthpolymorphism (CRS-RFLP), genetic polymorphisms in p53, p21, mdm2, Gadd45aand p14^ARF were genotyped. The associations between these genetic polymorphismsand risk of developing BP, and the potential modifying effects of intensity of benzeneexposure and life styles were also investigated. The results were as followings:(1) No variant allele was detected for polymorphism of Gadd45a c.25 G＞A. Thefrequency of the variant allele for polymorphism of mdm2 Dell518 was notconsisted with those studies which were reported in Chinese population.(2) The proportion of p21 Ex2+98 CA+AA, p21 Ex3+70 CT+TT and p14^ARF g. 22008GA+AA genotypes in the case group was lower than that of the control group(67.00% vs. 77.51%, 78.07% vs. 84.59%, and 45.27% vs. 55.94%, respectively).There was a 0.54-fold reduced risk of BP for individuals carrying p21 Ex2+98CA+AA genotypes (OR_adj=0.54; 95%CI: 0.33-0.87; P=0.01) compared with thesubjects carrying CC genotype. Compared with the subjects carrying p21 Ex3+70CC genotype, the risk of BP for the individuals carrying p21 Ex3+70 CT+TTdecreased (OR_adj=0.55; 95%CI: 0.31-0.98; P=0.04). Compared with the subjectscarrying p14^ARF g. 22008 GG genotype, there was a 0.53-fold decreased risk of BPfor individuals carrying p14^ARF g. 22008 GA+AA genotypes (OR_adj=0.53; 95%CI:0.53-0.82; P＜0.01).(3) Potential interactions were found among intensity of benzene exposure and polymorphism of p14^ARF g.22008 G＞A (χH²=8.88, P=0.012), sex andpolymorphism of mdm2 Dell518 (χH²=11.402, P＜0.01). In the moderate or highbenzene exposure group, compared with individuals with p14^ARF g.22008 GGgenotype, the risk of BP for those carrying p14^ARF g.22008 GA+AA genotypesfurther decreased (OR_adj=0.23; 95%CI: 0.08-0.64; P＜0.05 ; OR_adj=0.31; 95%CI:0.13-0.76; P＜0.01; respectively). Among the male, compared with those withmdm2 Dell518 WW genotype, there was a 0.36-fold decreased risk of BP forthose carrying mdm2 Dell518 WM+MM genotypes (OR=0.36; 95%C: 0.22-0.61;P＜0.01).(4) Stratified analysis of different genetic polymorphisms suggested that there arepotential interactions among different genetic polymorphisms (p53 intron3 and p53Ex4+119 C＞G, p21 Ex2+98 A＞C and mdm2 IVS1+309 T＞G, mdm2 Dell518 andp14^ARE g.22008 G＞A, Gadd45a g.-907 G＞C and Gadd45a g.1260 T＞C,respectively). The risk of BP may decrease for the individuals carrying genotypesof mdm2 Dell518 WW and p14^ARF g.22008 GA+AA (OR=0.21; 95%CI: 0.10-0.44;P＜0.01), mdm2 Dell518 WM+MM and p14^ARF g.22008 GG (OR=0.28; 95%CI:0.15-0.52; P＜0.01),mdm2 Dell518 WM+MM and p14^ARF g.22008 GA+AA (OR=0.26; 95%CI: 0.14-0.47; P＜0.01), Gadd45a g.-907GG and Gadd45a g.1260TC+CC (OR=0.28, 95%CI:0.15-0.52, P＜0.01), respectively.(5) Diplotypes analysis of polymorphisms in p21, mdm2, and Gadd45a genesshowed that the risk of BP decreased in the subjects with 1/2 diplotype (CC/AT;OR=0.51; 95%CI, 0.30～0.85; P=0.01) or 2/2 diplotype (AT/AT; OR=0.50;95%CI, 0.28～0.90; P=0.02), compared with those with 1/1 diplotype (CC/CC)of p21 gene; the subjects with 2/4 diplotype (MG/MT; OR=0.20; 95%CI, 0.05～0.81; P=0.02), compared to those with 1/1 diplotype (WT/WT) of mdm2 gene;the subjects with 1/2 diplotype (GT/CC; OR=0.39; 95%CI, 0.17～0.90; P=0.02)or 1/3 diplotype (GT/GC; OR=0.40; 95%CI, 0.18～0.87; P=0.02), comparedwith those with 1/1 diplotype (GT/GT) of Gadd45a gene, respectively.Diplotypes analysis of polymorphisms in p14^ARF gene showed that the risk of BPdecreased in subjects with 1/6 diplotype (TGG/TAA) (P＜0.01), whereasincreased in those with 2/4 diplotype (TGA/TAG), compared with those with 1/1diplotype (TGG/TGG).In conclusion, we first found that the risk of BP is associated with specificpolymorphisms of p21 or p14^ARF genes. The altered risk of BP is also regulated by intensity of benzene exposure and modified by factors such as sex and potentialinteractions among different genotypes of these polymorphisms. Polymorphisms inthese cell cycle control genes may contribute to individuals' genetic susceptibility toBP, which partially throws light on different endpoint for workers with similarbenzene exposure history. Results in our study will provide theoretical evidence forhealth surveillance and screening effective biomarkers of susceptibility.