Paraoxonase (PON1) polymorphism and activity as the determinants of sensitivity to organophosphates in human subjects

Chemico-Biological Interactions 168 (2007) 184–192

Paraoxonase (PON1) polymorphism and activity as the determinants of sensitivity to organophosphates in human subjects Jintana Sirivarasai a,∗ , Sming Kaojarern a , Krongtong Yoovathaworn b , Thanyachai Sura c a

Division of Clinical Pharmacology and Toxicology, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand b Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand c Division of Medical Genetics, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand Received 11 January 2007; received in revised form 12 April 2007; accepted 12 April 2007 Available online 21 April 2007

Abstract Paraoxonase (PON1) plays an important role in mechanism of organophosphorus compound (OP) toxicity, as seen both in vitro and in vivo studies. Polymorphisms of PON1 gene at coding and promoter regions have also been to affect on the hydrolytic activity and PON1 level. The objectives of this study were to determine PON1 polymorphism and activity in an OP-exposed population and the effects on inhibition of cholinesterase activity. The studied population consisted of control (n = 30) and exposed groups (n = 90). All enzyme activities (AChE, BuChE, paraoxonase, arylesterase and diazonase) were measured once for control group and two periods of exposure for exposed group. Three polymorphisms of PON1 (Q192R, L55M and T−108C) were identified only in the exposed subjects. The results demonstrated that AChE activity in both high (345.5 ␮kat/gHb) and low exposure periods (496.9 ␮kat/gHb) of the exposed group were significantly different from control group (649.7 ␮kat/gHb, p < 0.01). For BuChE activity, the exposed group also showed the statistically lower level in both periods (high exposure period: 62.17 ␮kat/L and low exposure period: 81.84 ␮kat/L) than those in the control group (93.35 ␮kat/L). Serum paraoxonase activity was significantly different among individual genotypes, RR > QR > RR, LL > LM and −108CC > −108CT > −108TT, but this was not found for those of arylesterase and diazonase activities. Q192R and L55M as well as Q192R and T−108C also presented substantial linkage disequilibrium. Further analysis was performed with haplotypes and various enzyme activities. AChE activity was not affected by haplotypes. Individuals with “211” haplotype showed significantly higher paraoxonase activity and BuChE activity than other haplotypes but not in diazonase activity. In conclusion, PON1 gene exhibited a wide variation in enzyme activities both within and between genotypes which implied insights of a potentially difference in sensitivity to OP toxicity. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Paraoxonase; Polymorphism; Acetylcholinesterase; Butyrylcholinesterase

1. Introduction

∗

Corresponding author. Tel.: +66 2 2011628; fax: +66 2 2011084. E-mail address: [email protected] (J. Sirivarasai).

In Thailand, organophosphorus (OP) compounds are extensively used in agricultural purposes. Pressures to sustain high crop yields have led to heavy

0009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2007.04.006

J. Sirivarasai et al. / Chemico-Biological Interactions 168 (2007) 184–192

usage of pesticides. Residue, especially organochlorines and OPs, have been found in soil, water and agricultural products throughout the country [1]. For example, the maximum level of methyl parathion and mevinphos in water from agricultural areas are 8.72 and 47.50 ␮g/L, respectively, which are higher than other pesticides [2]. Occupational exposure and suicide are the main causes of OPs poisoning to Thailand’s residents [1]. Organophosphates can cause various health problems due to the inhibition of acetylycholineseterase (AChE) at nerve endings. AChE at nerve ending represents the molecular target of OP toxicity. When the enzyme is inhibited, it cannot hydrolyze acetylcholine, leading to the neurotransmitter accumulation [3]. The toxic effects involve the parasympathetic, sympathetic, motor, and central nervous systems. Biomarkers of these exposures have been developed and include measurements of AChE in erythrocytes and butyrylcholinesterase (BuChE) in plasma [4]. The hydrolysis of toxic oxon metabolites of various OPs such as diazinon, parathion and chlorpyrifos by serum paraoxonase is a major factor determining their toxicity to human. Human serum paraoxonase (PON1) is a calcium-dependent esterase predominantly synthesized in liver. It closely associates with the high-density lipoprotein (HDL) particles that contain apolipoprotein AI [5]. Genetic polymorphisms of PON1 exhibit a large variation in activity and concentration among individuals. Two polymorphisms at coding region due to amino acid substitution are a Gln/Arg (Q/R) at position 192 and a Leu/Met (L/M) at position 55 affect the catalytic activity of paraoxonase enzyme [6–9]. A T−108C is a polymorphism of thymidine/cytocine in the promoter region of the PON1 gene that displays substantial variation in PON1 concentration [10,11]. Studies with PON1-knockout mice demonstrated that PON1 is critical for the in vivo detoxification of chlorpyrifos-oxon [12]. Furthermore, experiments where purified or partially purified PON1 was injected into rats or mice showed that increasing PON1 in the plasma leads to an increase in resistance to specific OPs [13–15]. From these evidence, it has been suggested that the polymorphisms affecting PON1 activity at positions 192 and 55, together with the large variability in PON1 concentrations might result in potential large individual differences in the ability to detoxify OPs metabolized via the cytochrome P450/PON1 pathway. Little information is available concerning these factors in Thai population, especially in high-risk group. Therefore, the objectives of this study were to determine PON1

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polymorphism and activity in OP-exposed population and ascertain the effects on inhibition of cholinesterase enzyme. 2. Materials and methods 2.1. Subjects The study was approved by The Ethical Clearance Committee on Human Rights Related to Researches Involving Human Subjects, Faculty of Medicine, Ramathibodi Hospital, Mahidol University. The exposed group consisted of 90 Thai ethnic individuals (age 16–65 years) that mainly used organophosphate pesticides during application periods (at least 1 year prior to the commencement of this study) at Rachaburi province in Thailand. The organophosphates used most often were dichlovos, chlorpyrifos and dimethoate. To avoid and minimize any interference with the biochemical parameters, individuals presenting diabetes, hypercholesterolemia, hypertension, liver and kidney dysfunctions, infection, hematological diseases and any other chronic diseases were excluded. The control group consisted of 30 healthy volunteers with no occupational exposure to pesticides. Both exposed and control groups were offered health examination consisting of medical history, physical examination and laboratory tests. For the exposed group, the study period was divided into low (the pesticides use was lowest, considered as baseline) and high (the spraying process peak) exposure periods. The time between exposures was 3 months. 2.2. Laboratory analysis After an 8–12 h overnight fasting, venous blood was drawn by venipuncture from all subjects into tubes containing sodium heparin or EDTA as an anticoagulant and plain tubes. Each blood sample was centrifuged for 15 min at 1200 × g to remove the plasma and/or serum. 2.3. Erythrocyte acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) Erythrocytes were pelleted by centrifugation, washed twice in NaCl 0.9% and diluted in an equal volume of saline. AChE activity was analyzed by the method of Ellman et al. [16], using acetylthiocholine as substrate. Thiocholine produced was coupled with dithiobis nitrobenzoic acid and was quantified at 412 nm using a spectrophotometer. Activity was expressed as ␮kat/gHb. Plasma BuChE activity was determined by measuring

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the hydrolysis of butyrylcholine iodide at 240 nm and 25 ◦ C [17]. Data were expressed as the SI-unit katal (␮kat/L). 2.4. Serum paraoxonase (PON1) activity [18] PON1 arylesterase activity was measured by adding 2.5 ␮L of serum to 100 ␮L of 13.62 mM phenylacetate solution and 900 ␮L of 100 mM Tris–HCl buffer containing 2 mM CaCl2 at pH 8.0 (27 ◦ C). The rate of phenol generation was monitored at 270 nm, and a molar extinction coefficient of 1310 was used to calculate the enzyme activity. PON1 paraoxonase activity was measured by using 25 ␮L of serum to 500 ␮L of paraoxon solution (5.5 mM paraoxon in 100 mM Tris–HCl buffer containing 2 mM CaCl2 , pH 8.0) at 25 ◦ C. Reaction was monitored at 405 nm, and an extinction coefficient of 17,100 M−1 cm−1 was used for activity calculation. PON1 activity for hydrolyzing diazoxon was determined by using 5 ␮L of serum to 1000 ␮L of diazoxon (3.52 mM paraoxon in 100 mM Tris–HCl and 2 mM NaCl buffer containing 2 mM CaCl2 , pH 8.0) at 25 ◦ C. Reaction was monitored at 270 nm, and an extinction coefficient of 3080 M−1 cm−1 was used for calculation. Initially, PON1 activity was expressed as micromoles of hydrolysis products formed per minute per milliliter, after that the enzyme activity was recalculated to the SI-unit katal (␮kat/L). 2.5. PON1 genotype The genomic DNA was extracted from whole blood by MasterPureTM DNA Purification Kit (EPICENTER® , USA). All genotyping was conducted by polymerase chain reaction amplification followed by specific restriction digestion and gel electrophoresis. The PON1 Q192R polymorphism was detected by AlwI digestion, the PON1 L55M by NlaIII digestion and the PON1 T−108C by BsrBI digestion as described previously [19]. 2.6. Statistical analysis All data were presented as the mean ± S.E. Statistical evaluations were performed by Student’s t-test and the analysis of variance (ANOVA). Hardy–Weinberg equilibrium was analyzed by the Fisher’s exact test. Analysis of multiple single nucleotide polymorphism (SNP) was performed by SNPStats, a free web-based tool [20]. The linkage disequilibrium (LD) statistics D and r2 were computed for each pair of SNP. Haplotypes were inferred by EM (Expectation Maximization) algorithm method.

3. Results Table 1 shows the mean differences in the study variables between control and exposed groups. No significant differences in mean BMI, blood pressure and other biochemical parameters were found between the two groups. The mean time of OPs use in the exposed group was 9.3 years. Enzyme activities were compared between control group and exposed population (Table 2). The results demonstrated that both AChE activity in high (345.56 ␮kat/gHb) and low exposure periods (496.93 ␮kat/gHb) of exposed group were significantly different from that of control group (649.79 ␮kat/gHb, p < 0.01). For BuChE activity, the exposed group also showed the statistically lower level in both periods (high exposure period: 62.17 ␮kat/L and low exposure period: 81.84 ␮kat/L) than those in control group (99.35 ␮kat/L). There was no significant difference in paraoxonase, arylesterase and diazonase activities between the control and the exposed groups. For biological monitoring of OP exposure, the determination of cholinesterase activity is well known to expressed as percentage of enzyme inhibition (% inhibition) at the high exposure period with respect to that of low exposure period. AChE and BuChE activities showed 30 and 26% inhibition, respectively (Table 2). However, paraoxonase, arylesterase and diazonase activities revealed very little change between these two different periods of exposure. Only PON1 genotype and allele distribution of exposed group was shown in Table 3. The frequencies of PON1 genotypes were in Hardy–Weinberg equilibrium. Allele frequency in our study population was similar to that of the general Thai population for PON1 L55M [18]. There were higher frequencies of the PON1 192R and −108C alleles in our study than another study in Thai population [18] (0.61 versus 0.29 and 0.53 versus 0.24, respectively). Different results were, however, obtained in the western population [10,30]. Table 4 shows PON1 genotype and enzyme activity of the exposed group. Measurements of enzyme activities in both high and low exposure periods revealed variability in paraoxonase (up to 8-fold), diazonase (up to 5.5-fold) and arylesterase (up to 3-fold). In addition, AChE and BuChE in the high exposure period demonstrated high variability (over 17.5- and 13.6-fold, respectively) in contrast to moderate variability in low exposure period (9.5- and 8.7-fold, respectively). For each genotype, 192RR showed statistically higher paraoxonase activity (4.84 ␮kat/L) than QR and QQ (2.86 and 1.36 ␮kat/L, respectively) but in contrast to

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Table 1 Characteristics and laboratory profile of the study population (mean ± S.E.) Variable

Exposed group (n = 90)

Control group (n = 30)

Age (years) Sex (male) Alcohol consumption Smoking Year exposed BMI (kg/m2 ) SBP (mmHg) DBP (mmHg) Hb (g/dL) Serum AST (U/L) Serum ALT (U/L) Serum calcium (mmol/L) Blood glucose (mmol/L) Blood urea nitrogen (mmol/L) Blood creatinine (␮mol/L) Total cholesterol (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L) Serum APO A–I (g/L)

39.9 ± 1.13 (18–61) 54.4% 27.8% 26.7% 9.3 ± 0.74 (2.0–30.0) 23.3 ± 0.4 (15.9–26.3) 124.0 ± 1.2 (99.0–139.0) 77.84 ± 8.45 (54.00–90.00) 13.83 ± 0.16 (10.00–17.30) 22.30 ± 0.86 (11.00–38.00) 41.22 ± 1.44 (21.00–65.00) 2.33 ± 0.01 (2.06–2.86) 5.07 ± 0.06 (4.00–6.10) 3.93 ± 0.11 (2.10–6.50) 71.58 ± 1.80 (54.0–85.00) 4.84 ± 0.06 (3.25–5.37) 1.23 ± 0.02 (1.03–2.11) 3.08 ± 0.05 (1.47–3.48) 1.68 ± 0.02 (1.11–2.44)

27.9 ± 0.6 (21–35) 56.6% 0 0 0 22.2 ± 0.3 (18.3–25.4) 115.3 ± 1.1 (104.0–128.0) 67.16 ± 1.19 (54.0–81.0) 14.03 ± 0.58 (11.20–16.30) 22.63 ± 2.30 (11.0–37.0) 30.6 ± 2.14 (12.0–59.0) 2.36 ± 0.02 (2.18–2.78) 5.21 ± 0.11 (4.00–5.89) 3.68 ± 0.18 (2.10–6.40) 72.53 ± 2.67 (46.0–81) 4.79 ± 0.10 (3.46–5.30) 1.22 ± 0.04 (1.03–1.81) 3.05 ± 0.08 (1.73–3.20) 1.72 ± 0.45 (1.25–2.69)

Table 2 Enzyme activities in the population exposed to OPs at two different times of the spraying and control group (mean ± S.E.) Enzyme activity

Exposed population (n = 90) High exposure period

AChE (␮kat/gHb) BuChE (␮kat/mL) Paraoxonase (␮kat/mL) Arylesterase (␮kat/mL) Diazonase (␮kat/mL) a * #

345.5 62.17 3.44 930.6 158.5

± ± ± ± ±

2.3*,# 3.16*,# 0.15 20.3 5.3

Δa (%)

Control group (n = 30)

−30 −26 −0.02 +0.02 −0.02

649.7 93.35 3.45 906.6 187.8

Low exposure period 496.9 81.84 3.50 907.1 162.8

± ± ± ± ±

19.8* 3.16* 0.15 18.1 9.1

± ± ± ± ±

30.0 5.16 0.26 42.0 10.6

Percentage variation or enzyme activities at the high exposure period with respect to that of low exposure period. Significantly different from control group (p < 0.01). Significantly different from low exposure period (p < 0.01).

diazonase activity with lowest value in RR genotype (127.1 ␮kat/L). The L55M polymorphism also demonstrated a significant difference in paraoxonase activity between LL and LM (both exposure periods). In addi-

tion, this study revealed that −108C allele appeared with high and −108T with low paraoxonase activity, comparable to inverse association in diazonase activity. Arylesterase activity was not affected by PON1

Table 3 Allele frequency of PON1 in this study and other studies PON1: position, allele

This study

Phuntuwate et al. [18]

Brophy et al. [9]

Leviev and James [29]

Suehiro et al. [11]

Q192R Q R

0.31 0.61

0.71 0.29

0.73 0.27

0.69 0.31

0.40 0.60

L55M L M

0.94 0.06

0.95 0.05

0.64 0.36

0.65 0.35

0.94 0.06

T−108C T C

0.47 0.53

0.76 0.24

0.50 0.50

0.54 0.46

0.52 0.48

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Genotype

High exposure period

Low exposure period

AChE (␮kat/gHb)

BuChE (␮kat/L)

PON (␮kat/L)

Arylesterase (␮kat/L)

Diazonase (␮kat/L)

AChE (␮kat/gHb)

BuChE (␮kat/L)

Q192R QQ (n = 15) QR (n = 41) RR (n = 34)

398.0 (38.3) 354.7 (25.6) 328.2 (24.8)

48.01 (7.00) 60.18 (4.33) 70.85 (5.83)

1.36 (0.07) 2.86a (0.08) 4.84a,b (0.15)

908.8 (36.0) 898.8 (28.0) 916.6 (31.3)

200.3 (16.6) 169.2 (9.8) 127.1a,b (5.8)

532.2 (41.1) 507.4 (32.6) 468.7 (19.8)

68.68 (7.83) 83.02 (4.50) 86.35 (5.50)

1.43(0.07) 2.98a (0.08) 4.92a,b (0.14)

944.0 (45.8) 933.5 (30.1) 921.5 (35.5)

207.5 (17.8) 175.5 (7.0) 128.0a,b (6.3)

L55M LL (n = 80) LM (n = 10)

341.5 (17.1) 377.5 (57.3)

64.85 (3.50) 40.34c (8.50)

3.85 (0.12) 2.20c (0.21)

917.0 (19.3) 829.6 (51.3)

157.8 (5.6) 163.7 (16.3)

499.9 (20.8) 472.5 (67.5)

84.68 (3.33) 59.18c (8.84)

3.93 (0.12) 2.32c (0.21)

940.8 (21.3) 849.3 (65.1)

162.2 (6.0) 168.3(19.6)

T−108C TT (n = 23) TC (n = 39) CC (n = 28)

358.7 (31.8) 325.7 (25.6) 362.4 (29.5)

63.35 (5.83) 66.18 (5.83) 55.51 (4.67)

1.60 (0.09) 3.21d (0.18) 4.23d,e (0.18)

957.5 (25.5) 863.6 (24.0) 927.1 (42.1)

180.7 (13.3) 155.3 (6.3) 144.8d (9.5)

499.7 (37.1) 482.1 (33.1) 515.2 (33.1)

82.52 (5.83) 86.02 (5.67) 75.52 (5.00)

1.66 (0.09) 3.31d (0.19) d,e 434 (0.17)

996.5 (33.1) 890.0 (28.1) 933.1 (43.6)

187.8 (14.1) 160.5 (7.3) 145.5d (9.5)

a b c d e

Significantly different from QQ, p < 0.001. Significantly different from QR, p < 0.001. Significantly different from LM, p < 0.001. Significantly different from TT, p < 0.001; Significantly different from TC, p < 0.001.

PON (␮kat/L)

Arylesterase (␮kat/L)

Diazonase (␮kat/L)

J. Sirivarasai et al. / Chemico-Biological Interactions 168 (2007) 184–192

Table 4 PON1 genotype and enzyme activity of exposed group

J. Sirivarasai et al. / Chemico-Biological Interactions 168 (2007) 184–192 Table 5 Linkage disequilibrium for PON1 polymorphism in exposed group, shown as D and r2 Genotype

Q192R

L55M

T−108C

Q192R L55M T−108C

– 0.7758 0.7443

0.7758 – 0.1196

0.7443 0.1196 –

Q192R L55M T−108C

– 0.245 0.562

0.245 – 0.028

0.562 0.028 –

D

r2

polymorphisms. Our results indicated that subjects with 55LM displayed significantly lower BuChE activity than those with 55LL, both in high (40.34 ␮kat/L versus 64.85 ␮kat/L) and low exposure periods (59.18 ␮kat/L versus 84.68 ␮kat/L). However, there was no correlation between AChE activity and each PON1 genotype. Analysis for multiple polymorphisms was performed in our study. Linkage disequilibria D and r2 correlation values were calculated for the PON1 192, 55 and −108 polymorphisms. The two coding region polymorphisms in PON1 expectedly presented substantial linkage disequilibrium (r2 = 0.245). The promoter region polymorphism −108 also displayed a significant D value for PON1 192 but not for PON1 55 (Table 5). Haplotypes with frequencies ≥5% in a specific ethnic population were considered “common” haplotypes whereas those with <5% frequency were combined into one group, defined as rare haplotype, for subsequent analysis. Table 6 shows the distribution of haplotypes, both common and rare haplotypes. Among our population, the most prevalent haplotype (40.8%) was the haplotype “212” and the total frequency of all rare haplotypes (121, 112, 122 and 222) was 9.8%. Further analysis was performed with haplotypes and various enzyme activities, as shown in Fig. 1. AChE activity Table 6 Haplotypes of three PON1 polymorphisms in exposed group Actual haplotype

Grouped haplotype

Haplotypea

Frequency (%)

Haplotypea

Frequency (%)

212 111 211 121 112 122 222

40.82 30.70 18.67 3.96 3.70 1.09 1.06

212 111 211 Rare

40.82 30.70 18.67 9.81

a The SNP positions within a haplotype are the following: Q192R, L55M, T−108C; 1 = wild type; 2 = variant.

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was not affected by haplotypes. In high and low exposure periods, individuals with “211” haplotype showed significantly higher paraoxonase activity (4.72 ± 0.23 and 4.60 ± 0.22 ␮kat/L, respectively) and BuChE activity (81.01 ± 5.33 and 97.85 ± 4.50 ␮kat/L, respectively) than other haplotypes. For diazonase activity, individuals with “111” haplotype displayed significantly higher value (199.7 ± 7.33 ␮kat/L) than haplotypes “212” (143.1 ± 4.6 ␮kat/L) and “211” (143.5 ± 8.5 ␮kat/L) in high exposure period, and there was also a similar trend in low exposure period. When BuChE and diazonase activities in each haplotype were considered, the results showed that the exposed subjects who exhibited highest diazonase activity did not present highest BuChE activity as seen in those of highest paraoxonase activity. 4. Discussion To our knowledge, this is the first investigation in Thailand where PON1 multiple polymorphisms and activities toward various substrates are determined for sensitivity to OP toxicity. OPs act as cholinesterase inhibitors and PON1 detoxifies OPs by cleavage of active oxons that are potential cholinesterase inhibitors in the peripheral and central nervous system [3]. Consequently, the good biological monitoring of these exposure and effects are measurements of AChE and BuChE, and they have generally been accepted that the use of these biomarkers rely upon comparison of post-exposure data with individual pre-exposure measurements [21]. Simple biochemical principles dictate that rates of detoxification of substrates are dependent on enzyme activity and its level. Here, we performed biochemical assays for AChE, BuChE, paraoxonase, diazonase and arylesterase activities in exposed individuals both in high and low exposure periods compared to those in control group. The significant decreases in both AChE and BuChE activities over the high exposure period and significant lower from the control group pinpoint certain inhibitory effects of OP pesticides on these esterases. These data were consistent with those published in farmworkers in North Carolina (AChE 503.1 ␮kat/gHb for farmer workers and 536.7 ␮kat/gHb for non-farmworkers, p < 0.05) [22] and in Kenya agricultural workers (AChE 69.5 ␮kat/L for the exposed group and 97.2 ␮kat/L for the control, p < 0.05) [23]. WHO guidelines for interpreting erythrocyte AChE measurements state that a 20–30% inhibition indicates evidence of exposure, 30–50% inhibition indicates hazard and 50% or greater suggests poisoning [21]. The average inhibition level of AChE and BuChE following OP exposure were 30 and 26%, respectively, which was similar to the study in Kenya

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Fig. 1. PON1 haplotypes and enzyme activities both in high (A) and low (B) exposure periods (*: significant different from h.211; a: significant different from h.111; b: significant different from h.212; c: significant different from h.211; p < 0.05).

agriculture workers with 35% inhibition of AChE [23]. Percentages of both enzyme inhibitions in this exposed population provided evidence of exposure to OPs. Smoking cigarette and alcohol consumption are mentioned as predictors of PON1 activity in previous studies [24,25] but in this study (data did not show), these factors did not relate to variation of enzyme activity. Based on PCR-RFLP method, the three PON1 polymorphisms in a population of 90 exposed individuals have been identified within Hardy–Weinbergequilibrium expectation. The PON1 192R allele frequency (R = 0.61) determined was also consistent with that published by Suehiro et al. [11] in a Japanese population with a frequency of 0.60 for the R allele. Different results were, however, obtained in a Thai population by Phuntuwate et al. [18] with R = 0.29 and in a western population with R = 0.31 [30]. For L55M and T−108C, allele frequencies were similar to Asian population [11,18] and different from some European population [10,30]. The allele frequencies for PON1 polymorphisms difference among ethnic groups might be a coincidence, or selection pressure that acts on these polymorphisms to maintain specific allele frequencies across different ethnic groups [10]. In human population, PON1 exhibits genetic polymorphisms, both in coding and promoter regions, leading to changes in PON1 activity and level [6–11]. The present study evaluated the contribution of the PON1 coding region Q192R and L55M and promoter T−108C on the effect of OP exposure (AChE and BuChE activities).

Firstly, we analyzed each genotype with enzyme activities. The Q192R substitution has been shown to be responsible for the substrate-dependent activity polymorphism. The PON1 192R isoform hydrolyzes paraoxon more rapidly than the PON1 192Q isoform, whereas the PON1 192Q isoform hydrolyzes diazoxon, soman and sarin more rapidly than the PON1 192R [15]. Our results were in agreement with other studies [26–29] in that individuals with 192RR showed higher paraoxonase activity than QR and QQ genotypes but in contrast to highest diazonase activity with 192QQ genotype. There was a gene-dependent increase in PON activity correlated with the presence of the R allele, suggesting a biosensor effect of the R allele in anti-AChE exposure. The polymorphism at position 55 (L55M) has not been demonstrated to affect the rates of hydrolysis of different substrates, as seen in Q192R polymorphism. However, previous studies [8,9] reported that PON1 55M is associated with lower PON1 activity levels and lower PON1 protein as well as mRNA. Other possibility may be that this amino acid substitution leads to protein instability or the association of PON1 with HDL. Our data presented here on the relationship between PON1 55 genotype and PON1 activity clearly showed that PON1 55M was associated with lower paraoxonase activity. These data thus agree with the published observations [28,29] in that on average L allele produces more PON1 mRNA and that the plasma from LL individuals has significantly more PON1 activity and higher PON1 enzyme concentration than MM individuals.

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In this study, it was found that the −108C variant had ≈2.6 times higher paraoxonase activity than did the −108T variant, similar to study of Leviev and James [29]. The polymorphic position T−108C lies within the GGCGGG (the polymorphic site is italicized) consensus sequence of the binding site for the transcription factor Sp1. Mutations within this site have previously been shown to affect the promoter activity of other sites. Levels of paraoxonase, aryleseterase and diazonase activities in each polymorphism could provide insights into the range of OP sensitivity by considering the cholinesterase activity. However, we did not find any significant difference except for 55LM individual with low paraoxonase activity who demonstrated lower BuChE activity than those of LL genotype. Genotype information alone might distort the underlying contributions from combinations of SNPs to PON1 activity. Phase information (alignment information of multiple SNPS along one chromosome) may help explain some portion of the overlapping activity, strengthen the genotype–phenotype relationship and association between gene and disease or adverse health effects [30]. We employed statistical methods to calculate LD and inferred haplotype. As observed previously, the PON1 192 was in high degree of linkage disequilibrium with L55M and T−108C (D = 0.78 and 0.74, respectively). There is no LD between L55M and T−108C that have already found in some studies [26–28]. Seven of eight possible haplotypes were demonstrated in our population with the most prevalent haplotype “212”. For further analysis, we have rearranged all haplotypes to four haplotypes (“212, 111, 211 and rare”) and the results revealed that individuals with “211” haplotype showed significantly highest paraoxonase activity. When AChE and BuChE activities, the surrogate of OP sensitivity, were evaluated, only BuChE activity showed interesting results among different haplotypes. Haplotype “211” with highest paraoxonase activity also presented highest BuChE activity. These findings indicated effects of PON1 haplotypes on BuChE activities; however, we also concerned with the possible factors, resulting from either an effect of haplotype on the baseline BuChE activity or on the decrease in enzyme activity seen with pesticide exposure. Based on haplotype analysis, it can indicate that the variant genotypes residing on the same chromosome might have synergistic effects on PON1 activity. In addition, these results gave rise to the health benefits for individual haplotype with high paraoxonase activity to be resistant to OP intoxication. No association between diazonase and BuChE activity in the exposed subjects might be partly explained by a number of factors, as described by O’Leary et al. [31],

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including the assay conditions, substrate concentration, the source of the substrate and the degree of spontaneous hydrolysis of the diazoxon substrate to pirimidinol varied upon storage. The outcomes from the present study also demonstrated some views different from previous data. This may be due to a large number of possible factors affecting adverse health effects due to OPs or oxon forms, and these include physio-chemical properties of OP, duration and routes of exposure, individual variables (age, gender, environmental factors, concurrent medications, cholinergic status) and genetic factors influencing other polymorphisms in the major metabolizing enzymes of OP (cytochrome P450, carboxylesterase). Consequently, additional researches concerning these factors as well as the haplotyping method are also needed for further understanding the evidence related to various sensitivity to OP intoxication. In conclusion, our findings have defined the interaction between PON1 polymorphisms and OP sensitivity with genotype-dependent relationship among PON1 polymorphisms and cholinesterase activities. These associations may potentially affect the susceptibility to OPs or other anticholinesterase toxicity, especially in occupational exposed population. Acknowledgements We are grateful to Prof. Surat Komind, Dr. Daruneewan Warodomwichit, and Dr. Orawan Phuchiwattananon, Division of Nutrition and Biochemistry, Faculty of Medicine, Ramathibodi Hospital, Mahidol University for their valuable assistance and facilities with all enzyme activity measurements. The authors also wish to thank Prof. Amnuay Thithapandha, Faculty of Medicine, Ramathibodi Hospital, Mahidol University for his editing of this manuscript. This study was supported by a grant from the Post-Graduate Education, Training and Research Program in Environmental Science, Technology and Management under Higher Education Development Project of the Commission on Higher Education, Ministry of Education, Thailand. References [1] A. Thapinta, P.F. Hudak, Pesticide use and residual occurrence in Thailand, Environ. Monit. Assess. 60 (2000) 103–114. [2] PCD (Pollution Control Department), Pollution in Thailand, 1996, Bangkok, Ministry of Science, Technology and Environment, 1997. [3] M. Maroni, C. Colosio, A. Ferioli, A. Fait, Organophosphorus pesticides, Toxicology 143 (2000) 9–37.

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