A Study On Biomarkers And The DNA Adduct Characteristics Of Formaldehyde And Styrene.

Formaldehyde, a familier and important chemistry material, has been widely used in architecture, decorated material, household-use chemicals. It is a colorless and stimulative gas and easily to dissolve in water at ordinary temperature. Its aqueous solution (35%-40%) is called formalin which often act as the antiseptic to immerse specimen. Long-term exposure to low concentration of Formaldehyde can impair human immune system, respiratory apparatus, nervous system ,skin and liver, etc. High concentration of Formaldehyde has genetic toxicity. Formaldehyde has been listed as suspicious mankind's carcinogen (2A group) in the American occupational health threshold limit value. Formaldehyde is mocromolecule aldehyde chemical compound that has electropositivity and vivid chemical property. It can attack nucleophilic group and result in the damage of giant molecular substance such as protein, nucleic acid.Styrene, a kind of suspect carcinogen (2B group) with wide use, is the main chemical material of synthetic resin, synthesizes rubber, plastics, coating, drug , perfume and so on. Styrene is absorbed mainly through respiratory tract (account 89% of total absorption). The long-term exposure to low concentration of Styrene , has the chronic toxic action to the nervous system and liver. It is reported that Styrene, belonging to mid-class toxic chemical compound, also has the harmful fuction to the blood, kidney, heart and genital system.It is clear that the metabolic way of Formaldehyde and Styrene in domestic and international research. However, the metabolic mechanism of them is still on going research. It is also lacks the systematic study on biological monitoring and harm to organism, and it is difficult to quota eliminate the smoking influence to the biological monitoring.Along with the rapid development of theory and technology of molecular biology, the study of biomarker, a brand-new field, has gradually draw attention in the field of Preventive medicine throughout the world. What biomarker reflecting is the molecular changes of physiology, biochemical, immunity and heredity, etc. These changes are evoked by the interaction of biosystem and environmental factors-chemical, physical and biological factors. Biomaker is an important milestone that manifest environmental medicine has developed at molecular level. The research about it has vital value in the field of molecular epidemiology, molecular toxicology, labour hygiene, environmental medicine, etc.DNA adducts, a biomarker of biological effective dose (target dosage), is formed when toxic compound enter a human body and interacte with DNA. DNA adducts may become the minimum factor to lead to carcinogenicity, malformation and mutation once it escapes the procedure of DNA repair.Chemical metabolites-the product of toxic compound oxidize and degradatiohn in the human body, is the biomarker of internal dose. It reflects the state of absorption, metabolism and excretion of toxic compound in body.Mercapturie acid is another biomarker of internal dose. Electrophilic compound and glutathion can form mercapturie acid metabolites under the effect of glutathione transferas in vivo. There are high dose-response relations between the concentration of DNA and proteinum adducts and mercapturie acid.Genetic polymorphism is an important biomarker of susceptibility. Different morphous of biological metabolic enzyme have different effect in the procedure of exogenous compound has toxic effect on organism, and determine the affectability of organism to chemical poison. As a result, it can be taken as an important screening index to occupational contraindication.The detection of biomarkers consides not only the variation of concentration of noxious substance in the air but also the ambulation of contact persons between different workplaces. Moreover, various absorptive path ( respiratory passage, skin, alimentary canal) , vocational factor , various physical and chemical factors such as workload (effect absorption), the ability to metabolitic noxious substance (absorption, distribution, bioconversion), individual affectability also be taken into account. For these reasons,the detection of biomarker represents exposure level more efficaciously than aerial detection. Consequently, it is an ideal means to estimate synthetic contact with noxious substance[Objective]With biomarkers of biological effective doses( target dosage)-DNA adduct, biomarkers of internal doses-chemical metabolites, metabolites of mercapturie acid, biomarkers of susceptibility-genetic polymorphism for the breakthrough, we approached to the chemical damage mechanism of Formaldehyde, Styrene to DNA, studied the applied value of biomarker as a factor for exposure monitoring of both of them, and offered scientific theoretical basis for biological monitoring,precaution,and evaluation of the risk of population group exposed to Formaldehyde and Styrene.[Methods]1. To moitor Formaldehyde and Styrene in work place air and their metabolites in urine by HPLCAccording to "The development standard on the method of the moitoring intoxicant in work place air" and "The trituration standard of the method of creature material analysis (wet kind and blood kind)", to develop the monitoring method for Formaldehyde in the work place air(including the method of collecting sample in spot and the analytic method with HPLC), the determinate method of the metabolite (formic acid ) in urine exposed to Formaldehyde and of the metabolites (MA, PGA and UMA) in Urine exposed to Styrene by HPLC, and applied a standardizational method for the measurement of Formaldehyde in the work place air and the biological monitoring of occupational exposed to Formaldehyde and Styrene.2.To study the DNA adduct characteristics of Formaldehyde and Styrene by culturing in vitro and in vivo, migration method of ultraviolet absorption spectrometry and HPLC methods. Takes the calf thymus DNA(20.0mg/ml), drops in the different density of Formaldehyde, Styrene and their metabolites, maintains 24h in the 37℃water bath, screens compounds which occur chemical reaction with DNA by migration method of ultraviolet absorption spectrometryTakes four kind of single deaeration nucleotides in the phosphate buffer solution (PH 7.2), carries on reaction with screened compounds separately, fills N₂ and seals tubes, maintains 18h in the 37℃water bath, Analysis intermixture by HPLC and determined the biding site. Simultaneously uses this reacting system to study the chemical bond type, chemical reaction progression of DNA adducts and so on.Separates the lymphocyte from the healthy adult circumference blood using the ficoll density gradient gentrifugalism technology, then configurates the cell suspending liquid with the nutrient fluid. Drops in the different density screened compounds to contaminate and fosters. Withdraws DNA form contaminated lymphocyte with the reagent box, and then conformed that screened compounds can induce DNA addiction reaction in human lymphocyte by using of migration method of ultraviolet absorption spectrometry, which is harmful to mammalian cells in a certain degree.3.To study on biological marker exposed to Formaldehyde- formic acid and biomarkers of susceptibility-ALDH2, CYP2E1 by HPLC and PCR-RFLP respectively. To discusse the possibility that formic acid took the monitoring index of Formaldehyde, and seek genes of susceptibility to use in the Formaldehyde high-risk group screening.To Sample Formaldehyde in work place air by silica gel tube soaked in 2,4-dinitrobenzene and determine it's concentrations by HPLC.To Sample urine in the morning and the end of work shift from workers exposed to Formaldehyde with the polyethylene plastic bottle. Drops in the phosphate buffer solution (PH7.6) and the derivating agent 1-brominemethyl -5-fluorine benzene, maintains 1h in the 60℃water bath and extracting with n-hexane. determines formic acid by HPLC.To determine the nicotine and formic acid in urine from smokers using the HPLC, and to discusse the correlational relationship between nicotine and formic acid in urine, thus quota elimination influence that smoking factor to formic acid determination.To gather the venous blood samples from persons exposed to Formaldehyde, loads in the EDTA Anti-coagulate tube, mixes and Preserves in refrigerate. Separates the lymphocyte from the blood samples using the ficoll density gradient gentrifugalism technology, and extractes DNA in the whole blood by the saturated phenol/chloroform, Then analysis the polymorphism of ALDH2 and CYP2E1.4. To study on biological marker exposed to Styrene- UMA, MA and PGA and biomarkers of susceptibility- CYP2E1, EPHX1, GSTT1 and GSTM1 by HPLC and PCR-RFLP respectively. To discusse the possibility that UMA, MA and PGA took the monitoring index of Styrene, and seek genes of susceptibility to use in the Styrene high-risk group screening.To Sample Styrene in work place air by activated charcoal tube and determine it's concentrations by HPLC.To Sample urine in the morning and the end of work shift from workers exposed to Styrene with the polyethylene plastic bottle, extracte with n-hexane, determine UMA, MA and PGA by HPLC.To gather the venous blood samples from persons exposed to Styrene, loads in the EDTA Anti-coagulate tube, mixes and preserves in refrigerate. Separates the lymphocyte from the blood samples using the ficoll density gradient gentrifugalism technology, and extractes DNA in the whole blood by the saturated phenol/chloroform, Then analysis the polymorphism of CYP2E1, EPHX1, GSTT1 and GSTM1.[Results]1. The method of measuring air Formaldehyde in workplace is estabolished in the study, samples are collected with silica gel dipping in DNPH to keep the samples stable during storage. The recovery rate of Formaldehyde is > 95% , and RSD <0.88%, and detecting limit is 0.0053μg/ml, and sampling efficiency >95%. The samples of air Formaldehyde is stable preserved in room temperature at lest 7 days. In the study on the method of measuring urinary metabolites of subjects exposured to Formaldehyde, the recovery rate of formic acid >90%, and CV<5.7%, and detecting limit is 0.15μg/ml. Preserving the urine samples in 4℃, RSD<8.93% in two weeks. In the study on the method of measuring urinary metabolites of subjects exposured to Styrene, the recovery rate of UMA, MA, PGA >90% and CV <6.3%. Preserving the urine samples in 4°C, RSD <8.3% in one week, and the detecting limits of UMA, MA and PGA are 0.02 0.3 and 0.1 mg/g creat.2. In vitro model system, one molecular Formaldehyde and one molecular dAMP form stable adduct through covalent bond. SO—the intermediate metabolite of Styrene—binds with dGMP and dAMP of four dNTPs. One molecular SO forms stable adducts binding with one dGMP through covalent bond,but the adduct is instable and weak formed by SO and dAMP.3. Health people peripheral lymphocytes contaminated by single Formaldehyde, Styrene or 7'8-SO indicate that active CHO- contained in Formaldehyde made them directly attack nucleate base. When 40μmol / L, Formaldehyde can weakly bind with human lymphocytes DNA. When >40μmol / L, Formaldehyde easily bind with human lymphocytes DNA, in accordance with the report that Formaldehyde bind with calf thymus DNA in vitro testing, which manifests that Formaldehyde is one genetoxin. In high concentration, Styrene can weakly bind with lymphocytes DNA in vitro cell culture, different from the response with calf thymus DNA. The intermediate metabolite -SO- stably binds with lymphocytes DNA because of its nucleophilicity, the same response with calf thymus DNA in vitro., which disclose Styrene and SO are both genetoxicant, and both covalent bind with DNA, but the binding reaction formed by SO is stronger than Styrene itself.4. Detecting the level of formic acid - one metabolite of Formaldehyde in vivo, in morning urine and the end of work shift and the concentration of air Formaldehyde in three different workshops, we may reguard the level of the increment of formic acid in morning urine as biomarker that subjects exposed to Formaldehyde through statistical analysis. Meanwhile, according to the experimental result, BTLV (biological threshold limit value) of the level of the increment of formic acid in morning urine for laborers exposed to Formaldehyde was recommended matching with current MAC of Formaldehyde as: 19.7 mg/g creat.5. Selects 50 smokers who smoke the diffirent quantity cigerattes and periode of smoking is over 1 year, and 20 non-smokers, gathers the smoker urine, determines nicotine and formic acid by HPLC. From the divariant quantity correlation analysis, we find that it has the significance correlational between the quantity cigerattes smoked and the increase of formic acid (R2 = 0.9303, P<0.05). So smoking can affect the Formaldehyde metabolite in the biological monitor.6. Detecting the genotype of ALDH2 and CYP2E1 of 107 workers exposed to Formaldehyde through collecting peripheral blood lymphocytes, we can find the level of urine formic acid increment was influenced by genotypes of ALDH2. The metabolism of ALDH2 *1 homozygotic genotype to Formaldehyde is more active than ALDH2 *2 homozygotic genotype (the difference of the two mean rank is 11.38), but the polymorphism of Rsa I/Pst I site of CYP2E1 5'-franking region Rsa I/Pst I site does not influence the level of urine formic acid.7. Detecting the level of the metabolites of Styrene, UMA, MA and PGA, in urine collected in morning and the end of work shift and detecting individual exposure dose of workers exposed to Styrene, through statistical analysis, we may reguard that UMA, MA, PGA and (MA + PGA) in morning urine and urinary UMA in the end of work shift as biomarkers of workers exposed to Styrene. Meanwhile, according to the experimental result, the recommended values of BTLV matching with current TWA of the UMA, MA, PGA, (MA + PGA) in morning urine of laborers exposed to Styrene are 11 mg/g creat for UMA, 607 mg/g creat for MA,386 mg/g creat for PGA, 992 mg/g creat for (MA + PGA), 29 mg/g creat for UMA in the end of work shift.8. Detecting the genotype of CYP2E1, EPHX1, GSTT1 and GSTM1 of 100 workers exposed to Styrene through collecting peripheral blood lymphocytes, we find activity CYP2E1PstI/ RsaIC2C2 ,no-activity GSTM1(-) and EHPX1(-) can be taken as Styrene's susceptible biomarker, can be used to screen Styrene occupational contraindication. That it has remarkable correlation between CYP2ElPstI/the RsaIC2C2 genotype and of Styrene metabolism in the increase poisonous process,and it has also remarkable correlation between GSTMI(+) genotype, the EPHXI high-activity group genotype and Styrene metabolism reduce the poisonous process. It is not discovered that it has the remarkable statistics significance between the GSTTI genotype and UMA in urine.[Conclusions]1. The method of measuring air Formaldehyde in workplace, the method of measuring urinary metabolites of subjects exposured to Formaldehyde, and the method of measuring urinary metabolites of subjects exposured to Styrene, is estabolished in the study. These methods fit the demands of "The development standard on the method of the moitoring intoxicant in work place air" and "The trituration standard of the method of creature material analysis (wet kind and blood kind)". They are available in determination of air Formaldehyde in workplace and measuring urinary metabolites of subjects exposured to Formaldehyde and Styrene.2. The lively aldehyde group in the Formaldehyde enable to attack the basic group in nucleic acid. One molecular Formaldehyde and one molecular dAMP form stable adduct through covalent bond. They have the same result that contaminated test with human lymphocytes DNA and chemical reaction test with calf thymus DNA in vitro, which manifests that Formaldehyde has the heredity poisonous effect.The Styrene,t hrough its active intermediate SO, attacks basic group in nucleic acid. One molecular SO forms stable adducts binding with one dGMP through covalent bond. They have the same result that contaminated test with human lymphocytes DNA and chemical reaction test with calf thymus DNA in vitro, which manifests that Styrene has also the heredity poisonous effect.3. The level of the increment of formic acid in morning urine enable to be biomarker that subjects exposed to Formaldehyde, BTLV (biological threshold limit value) of the level of the increment of formic acid in morning urine for laborers exposed to Formaldehyde was recommended matching with current MAC of Formaldehyde as: 19.7 mg/g creat.The increases of the formic acid caused by smoking may figure out from nicotine in urine. It is eliminated which smoking to the formic acid biology monitor influence from the urinary formic acid of smoking worker exposed to Formaldehyde subtract the increases of urinary formic acid caused by smoking.CYP2E1 does not affect the Formaldehyde metabolism activity. ALDH2 may take as the biomarker of susceptibility for Formaldehyde, using in the Formaldehyde high-risk group screening.4. UMA, MA, PGA and (MA + PGA) in morning urine and urinary UMA in the end of work shift may take as biomarkers of workers exposed to Styrene, the recommended values of BTLV matching with current TWA of the UMA, MA, PGA, (MA + PGA) in morning urine of laborers exposed to Styrene are 11 mg/g creat for UMA, 607 mg/g creat for MA,386 mg/g creat for PGA, 992 mg/g creat for (MA + PGA), 29 mg/g creat for UMA in the end of work shift.GSTTI does not affect the Styrene metabolism activity. CYP2E1, GSTM1 and EPHX1 may take as the biomarkers of susceptibility for Styrene, using in the Styrene high-risk group screening.[Characteristics and significance]Choosing biomarkers as breakthrough point, in technology we developed determination methods of biomarkers and provided standard methods for biological monitoring of Formaldehyde and Styrene. In mechanism of action we studied the formation, conjoint position, type of conjoint chemical bond and reaction order of DNA adducts and gave the theoretical foundation for the mechanism of chemical impairment of Formaldehyde and Styrene. In biological effect we have discussed the characteristics of addition reaction between Formaldehyde, Styrene and DNA in human lymphocyte, and primarily certified both of them can induce the additive effect in human lymphocyte, consequently, it can impair mammalian cells. In the evaluation of biological monitoring, we studied the five biomarkers separately and judge the possibility of application of biomarkers, such as metabolites as exposure monitoring factors. We also promoted the biological threshold limit value, and provided the foundation for the quality evaluation of biological monitor. In screening for biomarkers of susceptibility, we studied the genetic polymorphism of five metabolic enzyme for Formaldehyde and Styrene, and find the susceptibilite gene. The findings provided the theoretical foundation to the high-risk group's screening and primary prevention of occupational impairment for Formaldehyde and Styrene.