CYP1A1 RS4646903 polymorphisms and association with dioxin concentration in blood of Vietnamese victims exposed to chemical warfare/dioxin

Journal of military pharmaco-medicine n 0 1-2020 226 CYP1A1 RS4646903 POLYMORPHISMS AND ASSOCIATION WITH DIOXIN CONCENTRATION IN BLOOD OF VIETNAMESE VICTIMS EXPOSED TO CHEMICAL WARFARE/DIOXIN Dao Hong Duong1; Nguyen Hoang Thanh2; Nguyen Ba Vuong2 SUMMARY Objectives: To study CYP1A1 rs4646903 polymorphisms and its association with dioxin concentration in blood of Vietnamese victims exposed to chemical warfare/dioxin. Subjects and methods: A cross-sectional analysis was implemented on 100 Vietnamese victims exposed to chemical warfare/dioxin in 103 Military Hospital and 17 Military Hospital from January 2014 to December 2016. These victims had been measured the concentrations of 17 dioxin and dioxin-like compounds in blood by using High-Resolution Gas Chromatography/High-Resolution Mass Spectrometry - HRGC/HRMS, identified CYP1A1 rs4646903 polymorphisms by Sanger sequencing. Results and conclusion: The proportion of patients with A/A, A/G, G/G, T/G genotypes were 29%, 45%, 24%, 2%, respectively; the percentage of patients carrying allele A was 51.5%, allele G 47.5%, and allele T 1%. There were significant differences in 2,3,7,8-tetrachlorodibenzo-p- dioxin concentrations, total toxic equivalency of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in the CYP1A1 rs4646903 genotype model, dominant model, and recessive model. The dioxin concentrations and toxic equivalents were higher in those with dominant allele genotype (A) than those with recessive alleles only (G, T). There were significant correlations between the genotypic distribution and dioxin concentration. 2,3,7,8-tetrachlorodibenzofuran concentration had the highest sensitivity of defining allele A with the area under the curve = 0.833; cut-off point at position 1.17 ppt had the highest Youden J index (sensitivity 89.2%; specificity 76.9%). * Keywords: CYP1A1; Dioxin. INTRODUCTION Dioxin is the most toxic chemical group known to human being. In recent years, with the remarkable progress of molecular biology, scientists have shed some light on the mechanism of molecular effects of dioxin on the structure and function of genes relating to dioxin metabolism like CYP1A1 [5, 6, 7]. CYP1A1 is the gene that encodes CYP1A1, the enzyme that plays an important role in the metabolism of aromatic hydrocarbons, including dioxin. Changes in the structure of the CYP1A1 gene affect the structure and function of the enzyme, thereby disrupting the metabolism of ligands like dioxin [9]. Analysis of polymorphisms of the CYP1A1 gene on subjects exposed to dioxin showed a clear difference in dioxin concentration as well as the total toxic equivalency (TEQ) between the gene variants of CYP1A1 [6, 10]. 1. Vietnam Military Medical University 2. 103 Military Hospital Corresponding author: Dao Hong Duong (dr.duongj9@gmail.com) Date received: 7/01/2020 Date accepted: 10/02/2020 Journal of military pharmaco-medicine n 0 1-2020 227 In Vietnam, the largest amount of dioxin in the world and impure substances in defoliants, mainly agent orange, was sprayed by the US Air Force to the southern battlefields during the period from 1961 to 1972. The serious impact of dioxin on the health of exposed victims over many generations has been studied at various levels by domestic and foreign scientists [1]. Currently, there are not many studies on dioxin metabolic genes such as CYP1A1 in the population of victims exposed to agent orange/dioxin. Therefore, we conducted the study: To research on CYP1A1 rs4646903 polymorphisms and its association with dioxin concentration in blood of Vietnamese victims exposed to chemical warfare/dioxin. SUBJECTS AND METHODS 1. Subjects. 100 people exposed to agent orange/dioxin, living around Da Nang and Bien Hoa airbases were admitted to 103 Military Hospital and 17 Military Hospital from January 2014 to December 2016. * Inclusion criteria: - Living in hot spots of dioxin contamination around Da Nang and Bien Hoa airbases. - Duration of residence in the area ≥ 5 years. - Testing for concentration of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) ≥ 2 ppt. 2. Methods. - Study design: A cross-sectional analysis. - Dioxin analysis: a 40 mL blood sample was taken from the patient’s peripheral vein during hospitalization. All samples were stored at -80°C until analysis. The concentration in blood of 17 polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) congeners were measured by using high- resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) at German Eurofins Center based on US EPA method 1613 [4]. The TEQ values were calculated by multiplying the concentrations of each congener by its toxicity equivalency factor (TEF) value based on the WHO standards (2005). Sample values below the detection limit were assigned a value of one of half the detection limit to estimate the total dioxin concentration [8]. - CYP1A1 rs4646903 polymorphic analysis: Genomic DNA was extracted from peripheral blood by using DNA blood mini kit. DNA amplifications were performed in batches by using validated TagMan probes for rs4646903 SNP on a 7500 Real-time PCR System (Applied Biosystems, USA). Thermal cycle: 50°C x 2 min; 95°C x 10 min; (95°C x 15 sec + 58°C x 1 min) x 45 cycles. Then, Sanger sequencing was performed by using CEQ 8800 Sequencer System (Beckman Coulter, USA) to compare CYP1A1 rs4646903 polymorphism results that were determined by Real-time PCR. Genetic analysis were performed at the Department of Molecular Biology, 108 Military Central Hospital. Journal of military pharmaco-medicine n 0 1-2020 228 RESULTS AND DISCUSSION 1. The dioxin concentrations in blood of Vietnamese victims exposed to chemical warfare/dioxin. Table 1: Analysis of dioxin concentrations in the blood of Vietnamese victims exposed to chemical warfare/dioxin. Concentrations (ppt) Min - max Median (25 - 75%) 2,3,7,8-TCDD 3.32 - 858.33 38.56 (30.41 - 51.35) 2,3,7,8-TCDF 0.12 - 177.35 2.38 (2.02 - 2.88) PCDD TEQ 6.94 - 981.3 55.44 (46.48 - 67.59) PCDF TEQ 3.2 - 229.29 11.15 (10.64 - 13.13) WHO-PCDD/F TEQ 11.4 - 1,080 62.70 (54.40 - 75.80) (TCDF: Tetrachlorodibenzofuran) The analysis showed that all subjects in the study group had high concentrations of 2,3,7,8-TCDD and congeners in blood. The TEQ of all subjects exceeded the permitted standards in the world. According to Nguyen Hoang Thanh (2010), in a survey of exposed people in “hot pot” of dioxin-contaminated areas in Da Nang and Bien Hoa, all samples were detected to have 2,3,7,8-TCDD in blood and TEQ of WHO-PCDD/F ranged from 13 - 39 ppt [2]. US Air Force veterans who involved in the spraying of agent orange during Ranch Hand Operation in Vietnam from 1962 - 1971 had CDD in serum ranged from 10 - 521 ppt [3]. In our study, the TEQ values of 100 people in Da Nang and Bien Hoa ranged 11.4 - 792 ppt. 2. The distribution of CYP1A1 rs4646903 genotypes in Vietnamese victims exposed to chemical warfare/dioxin (According to the Hardy - Weinberg equilibrium). Table 2: CYP1A1 rs4646903 polymorphisms in Vietnamese victims exposed to chemical warfare/dioxin. Genotype Allele A/A 29% Allele A 51.5% A/G 45% Allele G 47.5% G/G 24% Allele T 1% T/G 2% χ2 = 0.615, p = 0.893. The rate of allele G carriers in our study was higher than in the 1000 Genomes Project Phase 3 study on 99 healthy Kinh people living in Ho Chi Minh City (the percentage of allele G carriers was 43.4% and allele A was 56.6%, no person carried allele T) [11, 12]. In the study by Kobayashi S et al (2013) on 421 pregnant women exposed to dioxin in Sapporo, Hokkaido, Japan, the recessive allele ratio of polymorphic CYP1A1 MspI rs4646903 was 34.4% and only 2 types of alleles were detected at this polymorphic location [10]. Journal of military pharmaco-medicine n 0 1-2020 229 3. The association between dioxin concentrations in blood and CYP1A1 rs4646903 polymorphisms of Vietnamese victims exposed to chemical warfare/dioxin. Table 3: Concentrations of 2,3,7,8-TCDD (ppt). Genotype n Min - max Median (25 - 75%) A/A 29 4.92 - 858.33 61.28 (36.21 - 213.92) A/G 45 3.69 - 203.51 44.4 (34.35 - 59.14) G/G 24 3.32 - 621.71 12.03 (5.99 - 24.02) T/G 2 34.09 - 34.3 34.2 (34.09 - 34.3) Pmedian test < 0.05 Contain allele A (1) 74 3.69 - 858.33 48.37 (37.08 - 60.23) Without allele A (2) 26 3.32 - 621.71 14.7 (6.35 - 28.16) p(1-2) < 0.05 Contain allele G (3) 71 3.32 - 621.71 34.3 (21.63 - 47) Without allele G (4) 29 4.92 - 858.33 61.28 (36.21 - 213.92) p(3-4) < 0.001 There was no similarity in the concentration of 2,3,7,8-TCDD between the genotypes of CYP1A1 rs4646903 (p < 0.05). Furthermore, 2,3,7,8-TCDD concentration in genotypes containing allele A/without allele A and containing allele G/without allele G of CYP1A1 rs4646903 were statistically different (p < 0.05). Table 4: The TEQ of PCDD (ppt). Genotype n Min - max Median (25 - 75%) A/A 29 10.05 - 981.3 86.9 (56.46 - 270.79) A/G 45 14.35 - 226.33 56.11 (48.09 - 80.66) G/G 24 6.94 - 731.62 22.08 (14.81 - 36.43) T/G 2 48.32 - 50.79 49.56 (48.32 - 50.79) Pmedian test < 0.001 Contain allele A (1) 74 10.05 - 981.3 62.59 (53.74 - 81.61) Without allele A (2) 26 6.94 - 731.62 23.7 (17.22 - 42.38) p(1-2) < 0.05 Contain allele G (3) 71 6.94 - 731.62 48.32 (33.86 - 57.29) Without allele G (4) 29 10.05 - 981.3 86.9 (56.46 - 270.79) p(3-4) < 0.05 There was no similarity in the TEQ of PCDD between the genotypes of CYP1A1 rs4646903 (p < 0.001). Furthermore, the TEQ of PCDD in genotypes containing allele Journal of military pharmaco-medicine n 0 1-2020 230 A/without allele A and containing allele G/without allele G of CYP1A1 rs4646903 were statistically different (p < 0.05). Table 5: The TEQ of PCDF (ppt). Genotype n Min - max Median (25 - 75%) A/A 29 10.05 - 981.3 86.9 (56.46 - 270.79) A/G 45 14.35 - 226.33 56.11 (48.09 - 80.66) G/G 24 6.94 - 731.62 22.08 (14.81 - 36.43) T/G 2 48.32 - 50.79 49.56 (48.32 - 50.79) pMedian test < 0.001 Contain allele A (1) 74 3.61 - 229.29 11.45 (10.68 - 13.25) Without allele A (2) 26 3.2 - 76.6 10.74 (9.1 - 14.58) p(1-2) = 0.427 Contain allele G (3) 71 3.2 - 76.6 10.73 (10.35 - 11.47) Without allele G (4) 29 6.3 - 229.29 13.43 (11.62 - 18.31) p(3-4) < 0.05 There was no similarity in the TEQ of PCDF between the genotypes of CYP1A1 rs4646903 (p < 0.001). Furthermore, the TEQ of PCDF in genotypes containing allele A/without allele A were not statistically different (p > 0.05), but the genotypes containing allele G/without allele G of CYP1A1 rs4646903 were statistically different (p < 0.05). The results showed that the dioxin concentrations and TEQ were significantly higher in people carrying allele A compared to those carrying allele G or allele T of CYP1A1 rs4646903 polymorphism. Our result was similar to several studies by Japanese scientists. Tsuchida et al (2003) studied on 28 healthy Japanese men aged 50 - 59 with dioxin in the blood, people with genotype CYP1A1 MspI A (gene containing dominant allele) had higher PCDD, PCDF, and co - PCB concentrations in blood than people with genotype B (gene containing recessive allele), in which the total concentration of non - ortho - PCBs in genotype A was significantly higher than genotype B [6]. In the study by Kobayashi S et al (2013) on 421 Japanese pregnant women, there was a significant association in the dominant genotype model in CYP1A1 rs4646903 (TT + TC vs. CC) (p = 0.048 for PCDD TEQ and p = 0.035 for PCDF TEQ). Furthermore, regarding the concentration of each substance, 2,3,4,7,8-pentachlorinated dibenzofuran (PeCDF) concentrations in genotype model and dominant model were significant different (genotype model TT vs. CC and dominant model [TT + TC] vs. CC) (p = 0.049 and 0.028, respectively) [5]. Journal of military pharmaco-medicine n 0 1-2020 231 Table 6: Correlation analysis between dioxin concentrations in blood and CYP1A1 rs4646903 polymorphisms. CYP1A1 rs4646903 (A/T/G) Variable n r p TEQ of PCDD 100 -0.345 0.000 TEQ of PCDF 100 -0.199 0.048 2,3,7,8-TCDD 100 -0.332 0.001 2,3,7,8-TCDF 100 -0.241 0.016 The variables were significantly correlated with the genotypic distribution (p < 0.05). Figure 1: ROC curve in evaluating the sensitivity, specificity with allele A distribution. Based on the ROC curve, it could be seen TEQ of PCDD; 2,3,7,8-TCDD; 2,3,7,8-TCDF had the value of defining allele A with the area under the curve > 0.6. In particular, 2,3,7,8-TCDF had an area under the curve = 0.833; cut-off point at position 1.17 ppt had the highest Youden J index (sensitivity 89.2%; specificity 76.9%). 2,3,7,8-TCDD had an area under the curve = 0.743; the cut-off point at position 34.3 ppt had the highest Youden J index (sensitivity 67.6%; specificity 76.9%). TEQ of PCDD had an area under the curve = 0.756; the cut point at 37.39 ppt had the highest Youden J index (sensitivity 73%; specificity 69.2%). Journal of military pharmaco-medicine n 0 1-2020 232 CONCLUSIONS Through the study on 100 Vietnamese victims exposed to chemical warfare/dioxin, we found that: - All the victims had high level of dioxins including 2,3,7,8-TCDD and TEQ in the blood. - The distribution of CYP1A1 rs4646903 genotype: Among the research group, the percentage of allele A carrier was 51.5%, allele G 47.5% and allele T 1%. The A/G genotype accounted for the highest proportion (45%), the T/G genotype accounted for the lowest rate (2%), whereas the proportions of A/A genotype was 29% and G/G genotype was 24%. - There were significant differences of 2,3,7,8-TCDD concentrations, TEQ of PCDD and PCDF in the CYP1A1 rs4646903 genotype model, dominant model, and recessive model. The dioxin concentrations and toxic equivalencies were higher in those with dominant allele genotype (A) than those with recessive alleles only (G, T). - There were significant correlations between the genotypic distribution and dioxin concentration. 2,3,7,8-TCDF concentration had the highest sensitivity of defining allele A with the area under the curve = 0.833; cut-off point at position 1.17 ppt had the highest Youden J index (sensitivity 89.2%; specificity 76.9%). REFERENCES 1. Bộ Tài nguyên và Môi trường. Tác hại của dioxin đối với con người Việt Nam. Nhà xuất bản Y học. 2008. 2. Nguyễn Hoàng Thanh. Thu dung, chẩn đoán và điều trị nạn nhân chất độ hóa học/dioxin. Dự án cấp Bộ Quốc phòng. 2010. 3. Institute of Medicine Committee to review the health effects in Vietnam veterans of exposure to herbicides. Veterans and Agent Orange: Update 2012. National Academies Press (US), Washington (DC). 2012. 4. The United States Environmental Protection Agency's Office of Science and Technology. Method 1613: Tetra- through Octa-chlorinated dioxins and furans by isotope dilution HRGC/HRMS. Washington, D,C. 1994. 5. Kishi R, Kobayashi S, Ikeno T et al. Ten years of progress in the Hokkaido birth cohort study on environment and children's health: Cohort profile. Environ Health Prev Med. 2013, 18 (6), pp.429-450. 6. Tsuchiya Y, Nakai S, Nakamura K et al. Effects of dietary habits and CYP1A1 polymorphisms on blood dioxin concentrations in Japanese men. Chemosphere. 2003, 52 (1), pp.213-219. 7. Chen H.L, Su H.J, Wang Y.J et al. Interactive effects between CYP1A1 genotypes and environmental polychlorinated dibenzo-p- dioxins and dibenzofurans exposures on liver function profile. J Toxicol Environ Health A. 2006, 69 (3-4), pp.269-281. 8. Van den Berg Martin, Birnbaum Linda S, Denison Michael et al. The 2005 World Health Organization re-evaluation of human and hammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicological sciences: An Official Journal of the Society of Toxicology. 2006, 93 (2), pp.223-241. 9. Mescher M, Haarmann-Stemmann T. Modulation of CYP1A1 metabolism: From adverse health effects to chemoprevention Journal of military pharmaco-medicine n 0 1-2020 233 and therapeutic options. Pharmacol Ther. 2018, 187, pp.71-87. 10. Kobayashi S, Sata F, Sasaki S et al. Genetic association of aromatic hydrocarbon receptor (AHR) and cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) polymorphisms with dioxin blood concentrations among pregnant Japanese women. Toxicol Lett. 2013, 219 (3), pp.269-278. 11. Sudmant Peter H, Rausch Tobias, Gardner Eugene J et al. An integrated map of structural variation in 2,504 human genomes. Nature. 2015, 526 (7571), pp.75-81. 12. Auton Adam, Abecasis Gonçalo R, Altshuler David M et al. A global reference for human genetic variation. Nature. 2015, 526 (7571), pp.68-74.