Verification Of HLA-B~*1301as A Biomarker Of Trichloroethylene Susceptibility And The Mechanism Of Trichloroethylene-Induced Immunity

As one of the most powerful chlorinated hydrocarbon solvents, trichloroethylene (TCE) is widely used in various occasions. TCE exposure can cause health issues in occupational exposed population. It was reported that there were94positioning incidents occurred during the years1993-2008in Longgang District, ShenzhenCity. There were ten deaths among430poisoning patients. The direct economic loss was40million Yuan. The TCEhad highest correlation with the disease upon an exposure. Analysis of the registedoccupational diseases data showed that there were232patients reported with occupational diseases in10years (1993-2000) in Baoan District, Shenzhen.Over90%patients suffered from occupational poisoning by organic solvents, especially by TCE. From1990to2005, there were896cases of occupational diseases were reported in Shenzhen, forty of which were dead. Most of the deaths cases(50.0%)were caused by dermatitis medicamentosa-like of TCE(DMLT). Given the findings from Baoan District, Shenzhen, in2006, the economic cost of treating one DMLT patient was about80,000Yuan, and the economic burden was280thousandYuan for dealing with each related death. To avoid the more extra economic loss, the primary and secondary prevention in occupational health play an increasingly important role.As amilestone, biomarkers can be used for screening susceptible population, to protect high-risk populations and to economize social resources. It has great significance to further investigate biomarkers in the TCE-exposed population, evaluate the TCE exposure risk, to understand the TCE pathogenesis mechanism, and to protectthe susceptible people.The aim of present study including follow two aspects.Firstly,to verify the effectiveness ofHLA-B*1301allele as the biomarker of DMLT through a cohort study.Secondly,to establish an animal model for future investigating the mechanism of DMLT.1. Verification of HLA-B*1301allele as the biomarker of DMLTTotally,1764occupational TCE exposed workers were recruited in theretrospective cohort study in Shenzhen City.The surveys were done with204factories. Among workers1651were met the studycriteria and finished the follow up. The workers were composed of948males (57.4%) and703females (42.6%). The age rangewasl7to58years(average27.03years). Before the end of study, the telephone follow-up was done for90days.The genotyping analysis of the1651workers was done in the laboratory. Among them,158(9.57%) workers were found to carry HLA-B*1301, and12cases(7.59%) were suffered from DMLT during the study. The other1493(90.43%) workers did not carry HLA-B*1301, and4works suffered from DMLT. The HLA-B*1301(+) workers had a significantly higher DMLT incidence than the HLA-B*1301(-) workers (RR:28.35;95%CI:9.25-86.85). Attributable risk percentage was96.44%, suggesting that the HLA-B*1301allele was a key factor of DMLT. The result verified the results of our previous case-control study. After adjustment of the confounding factors, multivariate logistic regression analysis showed that the type of exposure (OR:8.88;95%CI:2.21-35.63) and HLA-B*1301genotype (OR:20.17;95%CI:5.74-70.58) were the major factors impacting DMLTincidence. Therefore, HLA-B*1301allele screening of pre-employment medical examination is an effective measure to reduce the incidence of DMLT. ROC analysis showed anegative predictive value of99.7%, supporting that the HLA-B*1301allele detection is particularly suitable for use inpre-employment screening. We assume that the HLA-B*1301genetic screening of all workers in the pre-employment, one case of DML Tcould be avoided by screening every143candidates. So HLA-B*1301gene is a very effective susceptibility marker even in the general population screening. The direct cost of screening143workers is7,150Yuan, which is far below the treatment and care costs (32,043to64,540yuan) of one occupational DML Tpatient. The results provide reference information on the selection of early prevention measures.2. Establishment of DMLT animal model 1)Sensitization study of TCE and dichloroacetyl chlorideGuinea pigs, BALB/cmice and MRL/lprmicewere used as the experimental subjects. Based on the OECD (406) skin allergy test method, three induction and one inspiration were applied to the animals. Serum was collected at24h post the last inspiration. The right lobe of the liver was fixed by10%formaldehyde. Approximately3x3mm liver tissue was frozen in liquid nitrogen for RNA preparation later. The spleen and residue liver tissues were homogenized individually. Cytokines were measured in liver and spleen supernatants. The results showed that dichloroacetyl chloride (DCAC) can cause skin erythema reaction among72.7%of the guinea pigs. According to Magnusson sensitization scoring system. DCAC was classified asa strong allergen. In the sensitization experiments of BALB/c and MRL/lpr mice, the animal showed a transient anesthesia performance, the weakening of activity, even the sleep state at about20min post the administration of TCE. The animals of the DCAC and control groups were active,and no abnormal reactions were observed. Similar result was observed in a repeat experiment. In the induction experiment, same concentration of TCE and DCAC were also painted on the animal ear skins. The obvious skin erythema and edema were observed on guinea pig. The histological examination revealed that there were significant skin inflammatory cell infiltration in the dermis telangiectasia, edema and allergicreaction in TCE and DCAC groups. No abnormal reactions were observed with BALB/c and MRL/lpr mice. Aminotransferase test results showed that the aminotransferase levels were comparable among guinea pig, BALB/c mouse and MRL/lpr mouse groups after TCE treatment. The guinea pig and BALB/c mouse showed slightly increased aminotransferase after DCAC treatment. The P values were0.053and0.079. Liver pathology results showed that none abnormal was found with the experimental groups. The eight cytokines IL-2, IL-4, IL-5, IL-10, IL-12, IFN-γ, GM-CSFand TNF-α were measured in the sera,liver homogenates and spleen homogenates. The results showed that the TCE group had significantly lower levels of IL-2and IFN-ythan thecontrolgroup (P<0.05). The other six cytokines had no significant difference among the control, TCE and DCAC groups. There were significantly higher levels of IL-4and GM-CSF in sera than in liver and spleen homogenates (P<0.05). The liver homogenates had higher levels of IL-2, IL-12, IFN-γ,TNF-α than the serum and spleen homogenate (P<0.05). The liver had higher concentration of GM-CSF than spleen (P<0.05) but lower than serum (P<0.05). The spleen homogenate had higher IL-5and IL-10than the liver homogenate and serum (P<0.05). The data demonstrated that it was unsuccessful in establishing the animal model for DMLT. However, the experimental results identified DCAC as a strong allergen, may lead to liver injury of the guinea pig and BALB/c mouse. It also indicated that TCE and DCAC dissolved in olive oil did not cause allergic reaction in BALB/c and MRL/lpr mice. The results of these experiments have a certain reference value for future research..TCE has a complicated metabolic processe in the body. After getting into the body, TCE and its metabolites may conjugate to protein carriers to form complete antigen. Immune response was induced after recognition of the complete antigen by antigen presenting cells. Alternatively, the TCE and its metabolites bind to endogenous proteins (such as heat shock proteins, etc.) to form a super antigen. At the same time of exposure to TCE, the presence of the viral infection or Corynebacterium parvuminfection provided in vivo biological adjuvant molecules, causing the body to produce a strong immune response. To test this adoptive immunity based hypothesis, immunogens were prepared from TCE and DCAC induced animal. Then, the immunogens (animal tissue or serum) were administrated to animals to observe allergic reaction.1) Establishment of DMLT animal model based on adoptive immunityThe SPF level BALB/c mice were divided into two groups. The mice in experimental group (+) were induced with TCE every three days. The other group served as control (-). Induction was performed for three times. At14h post last induction, serum was collected. Mice were euthanized for preparation liver, spleen and kidney homogenates. Meanwhile, human epidermal cells were expanded. One aliquot of the cells was fixed with formalin, followed by three washes with sterile saline solution. The cells were adjusted to1.67×105cells/L. The other aliquot of cells were incubated with10mM TCE for24h, followed by formalin treatment and sterile saline washes. The cells were adjusted to1.67×105cells/L. The sera (+/-), liver homogenates (+/-), spleen homogenates (+/-), renal homogenates (+/-) andhuman epidermal cells (+/-), serving as adoptive immunity test substance, were administrated to the recipient mice. Animal reaction was observed.At24hours post injection of sera, homogenates and cells, mice were examined. The injection of cells and some tissue homogenates (sera, kidney and spleen) resulted in no abnormal reaction as observed with the control group. All mice had smooth hair and were active. Few mice had non-completely absorbed papule. In contrast, most mice injected with the liver homogenate (TCE-/+) showed fluffy hair, the weakened activity and increased respiratory rate as the disease state; most abdomen papules were unabsorbed. At96h post injection, the mice of all groups had smooth hair, were active and behaved normally. Few kidney homogenate group mice showed tiny papule on the abdomen injection sites, while the liver homogenate group mice had obvious papule on side abdomen, which turned out to be granuloma-like nodules caused by non-completely absorbed homogenates. Painting testing cells and homogenates on the mouse ear skin resulted in no obvious reaction. The obvious paw edema was observed. The multivariate analysis of variance revealed that each set of data had statistically significant difference at Oh,24h,48h,72h and96h five-time-points. Further analysis showed that the positive control group (DNCB) was significantly different with the rest groups (P<0.01). The negative control group (NS) had no significant difference with liver homogenate groups, spleen homogenate groups, kidney homogenate groups, serum groups, and cell groups (P>0.05). No significant difference was between TCE (-) and TCE (+) in each group (P<0.05). The data demonstrated that TCE treatment of tissue homogenates and epidermal cells before injection into mice did not cause significantly differentpaw edema.For the establishment of animal model, the target phenotype was affected by many factors including animal genetic characteristics, route of administration, the immune status, the testing substance and it smetabolites, and complex formation. Some research found that the species, strain and gender have significant impacton the absorption, distribution and metabolic of TCE, thus affecting the toxic effects of TCE. Therefore, in the future model building research, a new breakthrough may be achieved by using of different solvent, building composite antigen of TCE and its metabolites or choosing animals with aslow metabolic rate of TCE.In summary, the cohort study futher verified the results of previous case-control study. The preliminary cost-effectiveness analysis was done with HLA-B*1301genotype as a DMLT susceptibility biomarker. This study provided valuable information for HLA-B*1301allele as a susceptibility biological marker used inpre-employment genetic screening. To explore the mechanism of DMLT, BALB/c and MRL/lpr mice were used in animal model establishment. Overall the animal model building was unsuccessful, however, the result still provided helpful information for future DMLT animal model research.