2016 Volume 58 Issue 1 Pages 56-65
Objectives: Paraoxonase 1 (PON1) in serum detoxifies organophosphate (OP) insecticides by hydrolysis. The present cross-sectional study aimed to clarify the relationship between PON1 single nucleotide polymorphisms (SNPs) and enzyme activities or OP metabolite concentrations in urine of workers occupationally exposed to low-level OPs. Methods: Among 283 workers in 10 pest control companies located in central Japan who underwent checkups, 230 subjects (male 199, female 31, average age 38.9 ± 11.1 years old) participated in the study. Q192R and L55M polymorphisms were determined by TaqMan assay. PON1 activity was measured using fenitrothion (FNT) oxon, chlorpyrifos-methyl (CPM) oxon, chlorpyrifos (CP) oxon, and phenyl acetate as substrates. Urinary OP metabolite concentrations were measured with gas chromatography-mass spectrometry. Results: The maximum differences in enzyme activities between individuals were 64.6-, 6.3-, 7.7-, and 2.0-fold for FNT oxonase, CPM oxonase, CP oxonase, and arylesterase (ARE), respectively. The activities of CPM oxonase and ARE in workers having the RR genotype were 53.5% and 18.2% lower than in those with the QQ genotype, respectively. CP oxonase activity was 15.0% lower in those having the M allele (LM + MM compared with LL). Urinary metabolite concentrations were not associated with PON1 polymorphisms, but negative associations were observed between the concentrations and activities of FNT oxonase and ARE. Conclusions: While PON1 SNPs can explain differences in catalytic activities toward some OPs, differences in urinary concentrations of OP metabolites are not attributable to PON1 SNPs but instead are attributable to its serum activities. Its serum activities might be more sensitive biomarkers for estimation of individual susceptibility to OP toxicities.
(J Occup Health 2016; 58: 56–65)
Organophosphates (OPs) are insecticides widely used for agriculture, pest control, plant protection, and maintaining the household environment by controlling insect and rodent infestations. Because of their effectiveness against target insects, short half-lives, and low level of accumulation in vivo, OPs constitute a major insecticide class still today, and more than 100 OPs are generally used in agriculture1, 2). In recent years, not only acute toxicities to human health due to occupational exposure3) and suicide attempts and their resulting sequelae4), including delayed polyneuropathy5), but also exposure to much lower doses and the extent of its risk have also drawn attention because low levels of OPs have been detected in house air samples6) and urinary OP metabolites have been ubiquitously detected in the general population7). Thus, the factors that contribute to individual differences in the capacity to metabolize low-level OPs need to be addressed to better estimate the precise health risk due to exposure to them.
Paraoxonase 1 (PON1) in serum detoxifies OPs by hydrolysis8). After phosphorothioate forms of OPs are converted to their toxic oxon forms, which significantly inhibit acetylcholine esterase9, 10), by cytochrome P450, these oxon forms are metabolized to dialkyl phosphates (DAPs), that is, dimethyl phosphate (DMP) and diethyl phosphate (DEP). In addition, phosphorothioate forms are directly hydrolyzed to dimethyl thiophosphate (DMTP) and diethyl thiophosphate (DETP). All DAPs are excreted into urine8, 10, 11). PON1 is encoded as a 355 amino acid protein with a 43-kDa molecular weight8, 12). It is synthesized primarily in the liver, and a portion is secreted into plasma, where it is bound to apo A-1 in high-density lipoprotein particles. It hydrolyzes not only oxon forms of OPs but also phenyl acetate as arylesterase (ARE) and lactones as lactonase13, 14).
There are single nucleotide polymorphisms (SNPs) affecting PON1 enzyme activity. Q192R (glutamine → arginine)15, 16) and L55M (leucine → methionine)17) in the coding region are prominent polymorphisms. The Q192R polymorphism significantly affects the catalytic efficiency of PON1 towards OPs. The RR alloform hydrolyzes paraoxon and chlorpyrifos (CP) oxon more rapidly than QQ, whereas both alloforms have nearly equivalent catalytic efficiencies for metabolizing diazoxon18–20). The L55M polymorphism does not affect catalytic efficiency but has been associated with the plasma PON1 protein level, with the MM alloform being associated with a lower plasma PON1 protein concentration21). Since PON1 activity to hydrolyze OPs depends on the substrates mentioned above, we must further explore the relationship between SNPs and activities towards frequently used OPs. Moreover, the role of individual differences in PON1 activity in OP metabolism needs to be investigated in an exposed population.
In indoor pest control in Japan, pesticide sprayers have mainly used fenitrothion (FNT), dichlorvos, chlorpyrifos methyl (CPM), CP, diazinon, and propetamphos as OPs22). The relationship between SNPs and PON1 activities towards these OPs has not been reported in Asian populations. In addition, there has been no report examining both such activities and urinary OP metabolites in the same study.
The aim of the present study was to clarify the impact of PON1 SNPs on their enzyme activities and the OP metabolites in the urine of Japanese pest control workers whose exposure was well controlled.
This study protocol was approved by the Ethical Review Committee of Human Genome Analysis Research, Nagoya City University Graduate School of Medical Sciences.
The study population comprised indoor pesticide sprayers and clerical workers employed by ten pest control companies located in central Japan. In recent years, the introduction of Integrated Pest Management principles has decreased the use of insecticides, especially OPs, by these companies. In addition, those involved in spraying pesticides wore respiratory protective equipment and gloves as well as workwear. All 283 workers who underwent checkups between 2004 and 2014 were asked to participate in this study. Two hundred thirty subjects (199 males and 31 females, average age 38.9 ± 11.1 years old) gave written informed consent (participation rate 81.3 percent, gender and age distributions were not significantly different between the participation and nonparticipation groups) to participation in the study including determination of their PON1 SNPs. Among them, serum was available for 142 workers (120 males and 22 females, average age 39.7 ± 10.2 years old), and their enzyme activities were measured. This sample size was considered to be appropriate based on calculation using (1) an effect size (ES) (Cohen's f) of 0.25 with a 5% alpha error probability and 80% power and assuming that (2) the frequency of Q192R polymorphism was the same as that previously reported in Japanese23) and that (3) ARE activity varied as in the Han Chinese24). Information on gender, age, smoking and drinking status, class of OPs the workers used, and the elapsed time after the last OP spraying was obtained using a self-administered questionnaire.
Deoxyribonucleic acid (DNA) was extracted with a QIAamp DNA Blood Mini Kit (QIAGEN, Tokyo, Japan) from blood taken in the presence of ethylenediaminetetraacetic acid-2 potassium (EDTA-2K). The quality and quantity of DNA were examined by NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). First void urine was collected for measuring OP metabolites on the day after the last OP spraying operation or was collected on the day of the checkup if the worker was not involved in OP spraying during the preceding week. Blood and urine were stored at −80°C and −30°C until analysis, respectively.
Genotyping analysisWe performed genotype analysis of Q192R polymorphism using allele-specific fluorogenic TaqMan® probes by real-time polymerase chain reaction (PCR) with an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) following the method reported previously25). The PCR reaction was performed with a reaction volume of 24 µl including 12 µl of TaqMan® Universal Master Mix, probes labeled with VIC and FAM, forward and reverse primers, and 100 ng genomic DNA. The final concentrations of each probe were set to 420 and 210 nM, and that of each primer was set to 800 nM, respectively. The PCR cycling conditions were one hold at 50°C for 2 minutes, one hold at 95°C for 10 minutes, and then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute.
L55M SNPs were determined using a TaqMan® SNP Genotyping Assay Kit (SNP ID: rs854560, Drug Metabolism Assays, Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol using an ABI PRISM 7900HT Sequence Detection System.
Enzyme activitiesPON1 enzyme activities towards CP oxon, CPM oxon, and FNT oxon (Wako Pure Chemical Industries, Osaka, Japan) as substrates were measured in serum using a U-3210 Spectrophotometer (Hitachi High-Technologies Corporation, Tokyo, Japan) in accordance with spectrometric methods as described previously26).
The CP oxonase and CPM oxonase activities were determined by measuring the rate of formation of 3, 5, 6-trichloro-2-pyridinol (TCP). Twenty microliters of serum (1 : 20 dilution) was added to a mixture of 10 µl of 30 mmol/l substrate solution (solvent: dimethyl sulfoxide (DMSO)) and 970 µl buffer (2 mol/l NaCl-0.1 mol/l Tris-HCl (pH 8.5)-2.0 mmol/l CaCl2) at 37°C. Absorbance at 320 nm and 310 nm was measured every 12 seconds for 3 minutes, respectively. Likewise, FNT oxonase activity was analyzed every 12 seconds for 10 minutes at 400 nm by measuring the formation of 3-methyl-4-nitrophenol after addition of 15 µl undiluted serum to 10 µl of 3 mmol/l substrate solution (solvent: DMSO) and 975 µl buffer at 37°C.
Initial velocity of enzyme hydrolyses observed as OD/min was converted to nmol/ml/min using the molar extinction coefficient (5.56 l/mol/cm for CP oxon and CPM oxon, and that for FNT oxon was calculated to be 11.09 l/mol/cm from the calibration curve for 3-methyl-4-nitrophenol).
Levels of ARE activity were determined with a SpectraMax 340 (Molecular Devices Corporation, Sunnyvale, CA, USA) using an Arylesterase Assay Kit (Mega Tip Industry Trade Co., Ltd., Gaziantep, Turkey) according to its protocol (substrate was phenyl acetate). Erythrocyte and serum acetylcholine esterases were measured as reported previously27).
Urinary OP metabolitesUrinary concentrations of four DAPs, i.e., DMP, DMTP, DEP, and DETP, were examined according to the method reported by Ueyama et al. (2012)7) using gas chromatography-mass spectrometry (GC-MS; Agilent 5975 inert MSD system, Agilent Technologies, Santa Clara, CA, USA). The results for urinary concentrations were normalized by dividing by the urinary creatinine (cre) concentration, which was simultaneously measured. When the DAP concentrations were below the limit of detection (LOD), the LOD/square root of 2 was assigned for further calculations.
Statistical analysisThe Hardy-Weinberg equilibrium for the study population was tested by Pearson's Chi square test. Quantitative data were expressed as the mean (standard deviation (SD)) or median (range), while qualitative values were expressed as a percentage. Fisher's exact test was used to evaluate the difference in gender frequencies among PON1 genotypes. Differences in parametric variables except for age among PON1 genotypes were determined using the Student's t-test or one-way analysis of variance (one-way ANOVA) with the Bonferroni post hoc test. Age was compared with the Mann-Whitney U test or Kruskal-Wallis test with Holm post hoc test. The Jonckheere-Terpstra trend test was conducted to evaluate the tendencies of enzyme activities and urine metabolites between Q192R SNPs.
Linear regression analysis was performed using log-transformed urinary OP metabolites as objective variables and each enzyme activity and the elapsed time after the last OP spray as major explanatory variables. In the multivariable linear regression analyses, the interactions between SNPs and each of four enzyme activities towards urinary metabolites were examined. P values less than 0.05 were considered to indicate a statistical significance. Statistical analyses were conducted with EZR version 1.27 for Windows28) and G*Power29).
The allele frequencies of 230 participants for 192 Q/R and 55 L/M were 0.34/0.66 and 0.93/0.07, respectively, which were not significantly different from the Hardy-Weinberg equilibrium expectations. Characteristic data, urinary OP metabolite concentrations, PON1 activities, and erythrocyte and serum cholinesterase activities of 142 persons whose sera were available are shown in Tables 1 and 2 according to the Q192R and L55M polymorphisms, respectively. Regarding the L55M polymorphism, there was only one male subject with the MM alloform. Since 1) the serum PON1 concentration in healthy subjects having the MM genotype was closer to that in subjects having the LM genotype rather than to subjects having the LL genotype21) and 2) analyses excluding that participant, i.e., comparisons between the LL and LM genotypes, revealed the same results as those including him in the LM genotype group, the latter results were shown in the present study.
| QQ (n=15) | QR (n=67) | RR (n=60) | p* | p for trend test† | |||||
|---|---|---|---|---|---|---|---|---|---|
| Gender, n (%) | |||||||||
| Male | 13 | (10.8) | 60 | (50.0) | 47 | (39.2) | 0.21 | ||
| Female | 2 | (9.1) | 7 | (31.8) | 13 | (59.1) | |||
| Age, mean (SD) | |||||||||
| 42.3 | (12.3) | 39.1 | (9.8) | 39.8 | (10.1) | 0.55 | |||
| Hours until urine sampling after last pesticide exposure, mean (SD) | |||||||||
| 18.1 | (29.6) | 13.3 | (27.8) | 18.0 | (39.6) | 0.70 | |||
| OP metabolite, median (range) | |||||||||
| DMP | (µg/g cre) | 5.4 | (0.1–473.4) | 8.4 | (ND–1236) | 6.4 | (ND–1711) | 0.86 | 0.98 |
| DMTP | (µg/g cre) | 7.6 | (0.5–128.2) | 4.1 | (0.7–250.6) | 5.4 | (0.4–357.8) | 0.90 | 0.58 |
| DEP | (µg/g cre) | 3.9 | (0.5–22.9) | 3.0 | (0.3–53.5) | 2.7 | (0.4–91.9) | 0.83 | 0.36 |
| DETP | (µg/g cre) | 0.5 | (0.1–13.1) | 0.4 | (ND–49.9) | 0.5 | (ND–10.7) | 0.80 | 0.57 |
| PON1 activity, mean (SD) | |||||||||
| ARE | (µmol/ml/min) | 754.1 | (129.7) | 689.0 | (89.0) | 617.0 | (78.0) | <0.01** | <0.01 |
| CPM oxonase | (nmol/ml/min) | 3.0 | (1.2) | 2.3 | (0.8) | 1.4 | (0.4) | <0.01** | <0.01 |
| [ratio¶] | (×103) | [4.0 | (1.4)] | [3.3 | (1.1)] | [2.3 | (0.5)] | [<0.01** | <0.01] |
| CP oxonase | (nmol/ml/min) | 6.7 | (1.9) | 6.7 | (1.9) | 6.4 | (1.9) | 0.75 | 0.25 |
| [ratio¶] | (×103) | [9.0 | (2.8)] | [9.7 | (2.6)] | [10.4 | (2.4)] | [0.11 | 0.03] |
| FNT oxonase | (nmol/l/min) | 4.3 | (3.4) | 3.6 | (1.5) | 3.5 | (1.6) | 0.30 | 0.74 |
| [ratio¶] | (×103) | [5.6 | (4.0)] | [5.3 | (2.1)] | [5.7 | (2.6)] | [0.68 | 0.15] |
| Erythrocyte cholinesterase activity, mean (SD) | QQ (n=5) | QR (n=32) | RR (n=21) | ||||||
| (nmol/ml/min) | 3.1 | (0.3) | 3.2 | (0.4) | 3.2 | (0.4) | 0.82 | <0.01 | |
| Serum cholinesterase activity, mean (SD) | QQ (n=15) | QR (n=67) | RR (n=59) | ||||||
| (µmol/ml/min) | 344.2 | (81.9) | 359.3 | (68.1) | 377.1 | (84.2) | 0.23 | 0.10 | |
Cre, creatinine; DMP, dimethyl phosphate; DMTP, dimethyl thiophosphate; DEP, diethyl phosphate; DETP, diethyl thiophosphate; ARE, arylesterase; CPM, chlorpyrifos methyl; CP, chlorpyrifos; FNT, fenitrothion; ND, not detected.
| LL (n=118) | LM + MM (n=24) | p* | ||||
|---|---|---|---|---|---|---|
| Gender, n (%) | ||||||
| Male | 98 | (81.7) | 22 | (18.3) | 0.37 | |
| Female | 20 | (90.9) | 2 | (9.1) | ||
| Age, mean (SD) | ||||||
| 40.0 | (10.4) | 38.5 | (9.4) | 0.51 | ||
| Hours until urine sampling after last pesticide exposure, mean (SD) | ||||||
| 15.3 | (32.9) | 17.9 | (35.7) | 0.73 | ||
| OP metabolite, median (range) | ||||||
| DMP | (µg/g cre) | 6.8 | (ND–1,711) | 8.4 | (ND–589.8) | 0.76 |
| DMTP | (µg/g cre) | 4.9 | (0.4–357.8) | 4.6 | (0.7–130.9) | 0.86 |
| DEP | (µg/g cre) | 3.0 | (0.4–91.9) | 2.0 | (0.3–22.9) | 0.37 |
| DETP | (µg/g cre) | 0.5 | (ND–49.9) | 0.4 | (0.1–1.8) | 0.36 |
| PON1 activity, mean (SD) | ||||||
| ARE | (µmol/ml/min) | 668.7 | (103.9) | 649.5 | (78.8) | 0.39 |
| CPM oxonase | (nmol/ml/min) | 2.0 | (0.9) | 2.0 | (0.9) | 0.78 |
| [ratio¶] | (×103) | [2.9 | (1.1)] | [3.1 | (1.2)] | [0.42] |
| CP oxonase | (nmol/ml/min) | 6.8 | (1.8) | 5.7 | (2.0) | 0.02 |
| [ratio¶] | (×103) | [10.1 | (2.5)] | [8.8 | (2.5)] | [0.02] |
| FNT oxonase | (nmol/l/min) | 3.6 | (1.6) | 3.8 | (2.8) | 0.71 |
| [ratio¶] | (×103) | [5.4 | (2.3)] | [5.7 | (3.6)] | [0.59] |
| Erythrocyte cholinesterase activity, mean (SD) | LL (n=47) | LM + MM (n=11) | ||||
| (nmol/ml/min) | 3.2 | (0.4) | 3.3 | (0.5) | 0.70 | |
| Serum cholinesterase activity, mean (SD) | LL (n=117) | LM + MM (n=24) | ||||
| (µmol/ml/min) | 365.8 | (79.4) | 362.1 | (64.5) | 0.83 | |
See Table 1 footnotes for abbreviations.
In this studied population, 107 workers (75.4%) had sprayed some type of pesticides within 1 month before the checkups, and 48 (33.8%) had sprayed OPs during the preceding week before the examination. The most frequently sprayed OP was FNT, which was used by 26 operators (18.3%) during the week. The other major OPs sprayed were CPM, CP, diazinon, propetamphos, dichlorvos, and fenthion. None of the clerical workers (n=13, 9.2%) had used any insecticides during the week, but some of them occasionally washed workwear of the sprayers. The ratios of OP sprayers and clerical workers to subjects classified according to each genotype (QQ, QR, or RR for PON1192 and LL or LM+MM for PON155) were not significantly different between the genotypes (data not shown). The exposure level of all the study subjects was not high enough to inhibit erythrocyte and/or serum cholinesterase activities, and no significant difference in the activities was found between pesticide handlers and clerical workers (data not shown).
The maximum differences in enzyme activities between individuals were 64.6-, 6.3-, 7.7- and 2.0-fold for FNT oxonase, CPM oxonase, CP oxonase, and ARE, respectively. Enzyme activities were not significantly different between smokers and nonsmokers or between drinkers and nondrinkers (data not shown). Workers having the RR genotype showed 53.5% and 18.2% lower CPM oxonase and ARE activities, respectively, than those having the QQ genotype, whereas Q192R polymorphism did not affect CP oxonase (ES (f)=0.08) and FNT oxonase activities (ES (f)=0.14) (Table 1). The Jonckheere-Terpstra trend test indicated that both CPM oxonase and ARE activities exhibited decreasing trends with the increase in number of R alleles (Table 1). Additionally, a significant association was observed between CPM oxonase and ARE activities (Fig. 1; R2=0.231, p<0.05). Similarly, the activities of CP oxonase and FNT oxonase were significantly associated with that of ARE (R2=0.172 and 0.085, respectively, p<0.05, detailed data not shown). Interactions between PON1 polymorphisms and the enzyme activities were not significant.
Relationship between ARE activity (µmol/ml/min) and CPM oxonase activity (nmol/ml/min) according to PON1 Q192R polymorphism. The regression line expresses the association of the CPM oxonase activity with the arylesterase activity without considering SNPs in each subject (R2=0.231; p<0.05).
Table 2 shows that workers having the M allele (LM + MM) had 15.0% lower CP oxonase activity compared with those having the LL genotype, whereas L55M did not affect CPM oxonase, FNT oxonase, and ARE activities.
The ratio of three oxonases to ARE activities are also displayed in Tables 1 and 2. A significant difference between Q192R polymorphisms was observed in CPM oxonase, and the ratio of CP oxonase to ARE activities revealed an increasing trend as the number of R alleles increased. Concerning L55M, the ratio of CP oxonase to ARE activities showed a significant difference between LL and LM+MM polymorphisms, but this was not obsereved for the CPM (ES (d)=0.18) and FNT oxonases (ES (d)=0.13). Relative to the above PON1 polymorphisms, the OP metabolite concentrations in urine did not show a significant difference or trend for each SNP (ES (f) ≤ 0.09 for Q192R and ES (d) ≤ 0.19 for L55M).
Table 3 shows the results of linear regression analyses regarding the relationship between urinary concentrations of DAPs (DMP, DMTP, DEP, and DETP) and each enzyme activity or the time elapsed until urine sampling after the last OP exposure. Univariable analysis revealed that DMP and DMTP significantly decreased as a result of increased FNT oxonase activity and that DMTP and DETP significantly decreased as a result of increased ARE activity (Fig. 2), while the time elapsed after the last exposure was not a significant determinant. Even if one outlier of FNT oxonase activity in Fig. 2A (0.0154 nmol/ml/min) is removed from the analysis of the relationship between FNT oxonase and DAP based on the results of a Smirnov-Grubbs test, both DMP and DMTP were still significantly regressed on the FNT oxonase activity (R2=0.030 and 0.032, respectively; p=0.04 for both). When the analysis was confined to workers who sprayed OPs within a week before the checkups (n=48), negative regressions of urinary DMP and DMTP on the elapsed time after the last OP exposure became significant (R2=0.210 and 0.110, p<0.01 and p=0.02, respectively, detailed data not shown). Multivariable regression analysis including all variables with adjustment for age, gender, and smoking and drinking status showed almost consistent results with those of univariable analyses; that is, DMP was significantly regressed on FNT oxonase, and DMTP was significantly regressed on ARE activity (Table 3).
| DMP | DMTP | DEP | DETP | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| β | 95% CI | β | 95% CI | β | 95% CI | β | 95% CI | |||||
| Univariable analysis (×103) | ||||||||||||
| ARE | −1.1 | −2.5 | 0.3 | −1.9** | −3.0 | −0.9 | −0.2 | −1.0 | 0.5 | −1.0* | −1.8 | −0.1 |
| CPM oxonase | 35.7 | −125.8 | 197.3 | −53.7 | −178.6 | 71.3 | 24.7 | −62.6 | 112.0 | 8.8 | −91.0 | 108.5 |
| CP oxonase | 1.6 | −73.3 | 76.5 | −42.8 | −100.3 | 14.8 | −6.6 | −47.1 | 33.9 | −33.9 | −79.7 | 12.0 |
| FNT oxonase | −98.4* | −174.2 | −22.7 | −73.7* | −132.5 | −14.9 | −22.6 | −64.4 | 19.2 | −34.7 | −82.2 | 12.9 |
| Hours until urine sampling after last pesticide exposure | ||||||||||||
| −0.9 | −5.2 | 3.4 | −0.3 | −3.7 | 3.0 | −2.0 | −4.3 | 0.3 | −0.5 | −3.2 | 2.1 | |
| Multivariable analysis† (×103) | ||||||||||||
| ARE | −1.6 | −3.6 | 0.4 | −2.2** | −3.6 | −0.7 | −0.5 | −1.5 | 0.6 | −1.0 | −2.2 | 0.2 |
| CPM oxonase | 169.7 | −36.0 | 375.3 | 89.5 | −59.6 | 238.6 | 85.5 | −25.1 | 196.0 | 80.8 | −43.7 | 205.4 |
| CP oxonase | 35.1 | −54.2 | 124.4 | 2.7 | −62.0 | 67.4 | −11.1 | −59.1 | 36.8 | −30.0 | −84.0 | 24.1 |
| FNT oxonase | −92.0* | −175.2 | −8.8 | −45.5 | −105.8 | 14.8 | −18.1 | −62.8 | 26.7 | −22.6 | −72.9 | 27.8 |
| Hours until urine sampling after last pesticide exposure | ||||||||||||
| −0.7 | −5.1 | 3.8 | 0.1 | −3.1 | 3.4 | −1.8 | −4.2 | 0.6 | −0.5 | −3.2 | 2.2 | |
Significant associations of A) serum FNT oxonase activity (nmol/ml/min) with DMP/DMTP concentrations (µg/g cre) in urine and B) serum ARE activity (µmol/ml/min) with DMTP/DETP concentrations (µg/g cre) in urine. A regression line is shown without considering PON1 Q192R polymorphism.
When incorporating any of the enzyme activities, a PON1 SNP (either Q192R or L55M), and the interaction between the two variables into a regression model, no significant interaction was detected except for one between CP oxonase activity and Q192R with respect to DETP (Fig. 3). The slope between the CP oxonase activity and urinary DETP concentration in workers having the QQ genotype was greater than that in those having the RR genotype.
Relationship between CP oxonase activity (nmol/ml/min) and urinary DETP concentrations (µg/g cre) according to PON1 Q192R SNPs. The interaction between CP oxonase activity and the Q192R SNPs was significant (p=0.03). The slope of the regression line for workers having the QQ genotype was steeper than that for those having the RR genotype.
Furthermore, examination of the relationships between each enzyme activity and the elapsed time after the last OP exposure or OP spray operation hours revealed that the associations were not significant (ES (f2) ≤ 0.04, data not shown).
The present study first investigated the impact of PON1 SNPs on each enzyme activity and urinary metabolites in a population occupationally exposed to low levels of OPs that did not significantly inhibit cholinesterase activities. CPM oxonase and ARE activities became lower as the number of R alleles increased in the PON1 192 polymorphism, and CP oxonase activity was lower in the worker with the M allele in the PON1 55 polymorphism. As for the impact of the SNPs on urinary DAPs, the metabolite concentrations did not show significant associations with PON1 SNPs, whereas the urinary concentrations tended to decrease in association with the increase in PON1 (ARE and FNT oxonase) activities.
It has been unclear how PON1 SNPs contribute to individual differences in each PON1 activity towards different OPs in different ethnic populations. In the present study conducted in Japanese pest control workers, the PON1 192R allele was associated with lower ARE and CPM oxonase activities, while the 55M allele was associated with lower CP oxonase activity. FNT oxonase activity was not associated with either of the SNPs examined. Thus, PON1 polymorphisms contributed differentially to enzyme activities depending on the substrate. In contrast to our results, Furlong et al. and Zuniga et al. reported that the R allele had higher activity towards paraoxon and CP oxon in Latina mothers and neonates in the US2) and in Chilean agricultural workers30), respectively. The conflicting results might be due to ethnic differences in PON1 expression regulation leading to different PON1 protein levels in blood, which are discussed in the next paragraph.
The next issue is the extent to which PON1 SNPs contribute to the enzyme activities, which were markedly different between individuals in the present study. It is reportedly difficult to attribute differences only to the variation of SNPs. The enzyme activities designated as “PON1 activities” in the present study include those not only of PON1 but also those of other enzymes involved in hydrolysis/O-dearylation, although PON1 plays a major role. In addition, the assessment of PON1 activity is considered to require not only SNP genotyping31–34) but also taking into account of PON1 protein concentrations in the blood to precisely evaluate “PON1 status”35). In this regard, previous studies corrected PON1 activities with the ARE activity (ratio of oxonase to ARE)36) or presented them according to L55M17) or C-108T SNPs37), all of which are correlated with blood PON1 protein levels38). Our approach was to correct PON1 activities with the ARE activity. The Jonckheere-Terpstra trend test showed that the ratio of CP oxonase to ARE activities tended to increase as the number of R alleles increased, which agrees with the findings of the abovementioned reports2, 30). It is thus necessary in future studies to further explore the ethnic differences and commonalities in terms of the contribution of PON1 SNPs to PON1 status.
As for the relationship between PON1 SNPs and urinary OP metabolite concentrations that reflect the metabolized amount between last voiding and urine sampling, the concentrations were not associated with the tested SNPs. Although the elapsed time after the last OP exposure was not associated with the DAP concentrations as a whole, when the analysis was confined to workers who had had direct contact with OPs within 7 days before the checkup, DMP and DMTP showed significantly negative correlations with the time after the last OP exposure. In addition, FNT oxonase activity had a weak negative association with DMP (R2=0.042) or DMTP (R2=0.048) in linear regression analysis. The association became stronger when the analysis was confined to 26 workers who sprayed FNT (R2=0.171 and 0.269, p<0.05, respectively). These results suggested that some PON1 activities, at least that of FNT oxonase, could well explain differences in catalytic capacities toward the corresponding OPs, whereas PON1 polymorphism alone could not; they also suggested that OP metabolites were excreted into urine more quickly as a result of higher PON1 activities. In this regard, the interaction between Q192R and CP oxonase towards DETP was significant. The involvement of the Q192R polymorphism in the difference in individual susceptibility might be relevant for workers extensively exposed to OPs such as CP and parathion, which are metabolized to DETP. This point deserves future study.
The present study has a few limitations. First, the amount of OPs sprayed and the resulting individual exposure levels were assumed to be the same in the present analyses. Detailed individual exposure assessments such as measurements of breathing-zone pesticide concentrations are desirable in future studies. Second, the PON1 status was evaluated based on the enzyme activities corrected with the ARE activity. Correction with the PON1 protein concentration may yield a better estimation. Third, OP metabolites were analyzed with morning spot urine samples collected the day after the last time pesticides were sprayed, which means that measurements of DAPs excreted during the first several hours after exposure were missing. A urine sampling procedure to estimate precisely the total excreted amount needs to be considered to investigate further the impact of PON1 activities on the toxicokinetics of each OP.
Despite the above limitations, a strength of this study was that the participants' allele frequencies of Q192R and L55M were similar to those previously reported for the Japanese population23, 39, 40), thereby making the study findings possibly applicable to exposure settings in the general environment as well as those of pesticide sprayers in a well-controlled working environment.
In conclusion, although PON1 SNPs plays some role in determining PON1 status, urinary metabolite concentrations were not significantly different between the genotypes in a population with low-level occupational exposure to OPs. Serum PON1 activities towards each OP might be more sensitive biomarkers than PON1 SNPs alone for assessment of differences in individual susceptibility to OP toxicities.
Acknowledgments: The authors are greatly indebted to all of the study participants. Kyoko Minato and Mio Miyake are acknowledged for their careful arrangement and support of the checkups. This study was partially supported by JSPS KAKENHI (Grants-in-Aid for Scientific Research) Grant Numbers 20310035, 23689034, 25293151, and 26460799.
Conflict of interest: We declare that there is no conflict of interest.
arylesterase
CPchlorpyrifos
CPMchlorpyrifos-methyl
DAPdialkyl phosphate
DEPdiethyl phosphate
DETPdiethyl thiophosphate
DMPdimethyl phosphate
DMSOdimethyl sulfoxide
DMTPdimethyl thiophosphate
ESeffect size
FNTfenitrothion
LODlimit of detection
OPorganophosphate
PON1paraoxonase 1
SNPsingle nucleotide polymorphism
