Organophosphorous pesticides research in Mexico: epidemiological and experimental approaches

Non-persistent pesticides, such as organophosphorous (OP) insecticides have been extensively used in Mexico, and becoming a public health problem. This review presents data of OP use and related toxicity from epidemiological and experimental studies conducted in Mexico. Studies in agricultural workers from several regions of the country reported moderate to severe cholinergic symptoms, including decreased acetylcholinesterase (AChE) activity (the main acute OP toxic effect that causes an over accumulation of the neurotransmitter acetylcholine), revealing the potential risk of intoxication of Mexican farmers. OP exposure in occupational settings has been associated with decreased semen quality, sperm DNA damage and as endocrine disrupter, particularly in agricultural workers. Alterations in female reproductive function have also been observed, as well as adverse effects on embryo development by prenatal exposure in agricultural communities. This illustrates that OP exposure represents a risk for reproduction and offspring well-being in Mexico. The genotoxic effects of this group of pesticides in somatic and sperm cells are also documented. Lastly, we present data about gene-environmental interactions regarding OP metabolizing enzymes, such as paraoxonase-1 (PON1) and its role in modulating their toxicity, particularly on semen quality and sperm DNA integrity. In summary, readers will see the important health problems associated with OP exposure in Mexican populations, thereby the need of capacitation programs to communicate farmers the proper handling of agrochemicals to prevent their toxic effects and of more well designed human studies to support data of the current situation of workers and communities dedicated to agriculture activities.     

Organophosphorous pesticides research in Mexico: epidemiological and experimental approaches

Non-persistent pesticides, such as organophosphorous (OP) insecticides have been extensively used in Mexico, and becoming a public health problem. This review presents data of OP use and related toxicity from epidemiological and experimental studies conducted in Mexico. Studies in agricultural workers from several regions of the country reported moderate to severe cholinergic symptoms, including decreased acetylcholinesterase (AChE) activity (the main acute OP toxic effect that causes an over accumulation of the neurotransmitter acetylcholine), revealing the potential risk of intoxication of Mexican farmers. OP exposure in occupational settings has been associated with decreased semen quality, sperm DNA damage and as endocrine disrupter, particularly in agricultural workers. Alterations in female reproductive function have also been observed, as well as adverse effects on embryo development by prenatal exposure in agricultural communities. This illustrates that OP exposure represents a risk for reproduction and offspring well-being in Mexico. The genotoxic effects of this group of pesticides in somatic and sperm cells are also documented. Lastly, we present data about gene-environmental interactions regarding OP metabolizing enzymes, such as paraoxonase-1 (PON1) and its role in modulating their toxicity, particularly on semen quality and sperm DNA integrity. In summary, readers will see the important health problems associated with OP exposure in Mexican populations, thereby the need of capacitation programs to communicate farmers the proper handling of agrochemicals to prevent their toxic effects and of more well designed human studies to support data of the current situation of workers and communities dedicated to agriculture activities.     

Paraoxonase 1 (PON1) status and substrate hydrolysis

Paraoxonase 1 (PON1) hydrolyzes a number of organophosphorus (OP) compounds including insecticides and nerve agents. The in vivo efficacy of PON1 to protect against a specific OP exposure depends on the catalytic efficiency of hydrolysis. The Q192R polymorphism affects the catalytic efficiency of hydrolysis of some substrates and not others. While PON1_R192 hydrolyzes paraoxon approximately 9-times as efficiently as PON1_Q192, the efficiency is insufficient to provide in vivo protection against paraoxon/parathion exposure. The two PON1₁₉₂ alloforms have nearly equivalent but higher catalytic efficiencies for hydrolyzing diazoxon (DZO) and provide equivalent in vivo protection against DZO exposures. On the other hand, PON1_R192 is significantly more efficient in hydrolyzing chlorpyrifos oxon (CPO) than PON1_Q192 and provides better protection against CPO exposure. Thus, for some exposures it is only the level of plasma PON1 that is important, whereas for others it is both plasma level and the PON1₁₉₂ alloform(s) present in plasma that are important. In no case is the plasma level of PON1 unimportant, provided that the catalytic efficiency is sufficient to protect against the exposure. Two-substrate enzyme assay/analysis protocols that reveal both PON1 plasma levels and PON1₁₉₂ phenotype (QQ; QR; RR) are designed to optimize the separation of PON1₁₉₂ phenotypes; however, they have not been optimized for evaluating in vivo rates of OP detoxication. This study describes the adaptation of a non-OP, two-substrate determination of PON1 status to the conversion of the PON1 status data to physiologically relevant rates of DZO and CPO detoxication. Conversion factors were generated for rates of hydrolysis of different substrates.     

Gene–environmental interactions and organophosphate toxicity

Organophosphates (OPs) are an important class of insecticides that in the UK have been widely used for treating sheep for ectoparasites as well as in other sectors of the farming industry. Health problems associated with acute OP toxicity are well defined but, ill-health induced by chronic exposures to OPs remains controversial. A substantial number of sheep farmers complain of chronic ill-health which they attribute to repeated exposure to OPs. If OPs were associated with chronic ill-health then individuals with specific defects in OP metabolism might be expected to be at greater risk of ill-health following exposure. To examine such a hypothesis, the characterisation of both OP exposure and those pathways which lead to the formation and removal of the active OP metabolites becomes important. A wide range of OPs have previously been used to treat sheep but currently the only OP licenced for treating sheep is diazinon. Immediately after treatment, farmers’ urines contain detectable levels of OP metabolites but few farmers have a significant decrease in plasma cholinesterase activity. Diazinon, like chlorpyrifos, is an organothiophosphate which is metabolised, particularly by cytochrome p450s, to the corresponding active oxon form. CYP metabolism also leads to the inactivation of the parent compound and the relative balance of inactivation and activation can depend upon the specific OP and the CYP isoform. OP oxons are inactivated by serum paraoxonase (PON1) and mice lacking PON1 activity are susceptible to oxon and parent OP induced toxicity. PON1 polymorphisms at positions 192 (R form with arginine at 192 and Q with glutamine) and 55 (L form with a leucine and a M form with methionine) influence paroxonase activity. The effect of the Q192R polymorphism is substrate specific with reports indicating that diazoxon is metabolised less by the R isoform. In a study of sheep farmers within the UK, the R allele was associated with an increased risk of self-reported chronic ill-health, a result consistent with the hypothesis that this ill-health may have been caused by OPs. Studies in other populations exposed to pesticides also show associations between ill-health and PON1 Q192R polymorphisms but not consistently so. This is not surprisingly given that exposure is often poorly characterised. In vivo models also suggest that PON1 genotypes may have little influence on susceptibility at low doses of the parent OP. Hence further work is required not only to better characterise OP exposure in humans populations but also to identify those populations susceptible to OP toxicity.     

Catalytic efficiency determines the in-vivo efficacy of PON1 for detoxifying organophosphorus compounds

Human paraoxonase (PON1) is a polymorphic, high-density lipoprotein (HDL)-associated esterase that hydrolyzes the toxic metabolites of several organophosphorus (OP) insecticides and nerve agents. The activity polymorphism is determined by a Gln/Arg (Q/R) substitution at position 192. Injection of purified PON1 protects animals from OP poisoning. In the present study, we investigated the in-vivo function of PON1 for detoxifying organophosphorus insecticides in PON1-knockout mice that were challenged via dermal exposure with diazoxon, diazinon and paraoxon. PON1-knockout mice were extremely sensitive to diazoxon. Doses (2 and 4 mg/kg) that caused no cholinesterase (ChE) inhibition in wild-type mice were lethal to the knockout mice, which also showed slightly increased sensitivity to the parent compound diazinon. Surprisingly, these knockout mice did not show increased sensitivity to paraoxon. In-vitro assays indicated that the PON1R192 isoform hydrolyzed diazoxon less rapidly than did the PON1Q192 isoform. In-vivo analysis, where PON1-knockout mice received the same amount of either PON1(192) isoform via intraperitoneal (i.p.) injection 4 h prior to exposure, showed that both isoforms provided a similar degree of protection against diazoxon, while PON1R192 conferred better protection against chlorpyrifos-oxon than PON1Q192. Injection of purified rabbit PON1 or either human PON1(192) isoform did not protect PONI-knockout mice from paraoxon toxicity, nor did over-expression of the human PON1R192 transgene in wild-type mice. Kinetic analysis of the two human PON1(192) isoforms revealed that the catalytic efficiency (Vmax/Km) determines the in-vivo efficacy of PON1 for organophosphorus detoxication. The results indicate that PON1 plays a major role in the detoxication of diazoxon and chlorpyrifos oxon but not paraoxon.     

Effect of statin therapy on plasma high-density lipoprotein-cholesterol levels is modified by paraoxonase 1 in Chinese patients with coronary heart disease

1. We sought to determine the effects of Q192R polymorphism of paraoxonase 1 (PON1) gene on plasma high-density lipoprotein-cholesterol (HDL-C) levels and the response to statin therapy in Chinese patients with coronary heart disease (CHD). 2. Two hundred and thirty-six patients with CHD were treated with simvastatin 20 mg/day. Fasting serum lipids were determined before and after 12 weeks of treatment. 3. No significant differences were detected among the PON1 Q192R polymorphism with respect to plasma lipids. In addition, the effects of simvastatin to increase HDL-C levels were significantly greater in patients with the RR genotype compared with patients with the QR or RR genotypes (P < 0.05). 4. We conclude that the Q192R polymorphism of PON1 significantly modulates the HDL-C response to simvastatin in Chinese patients with CHD.     

山东潍坊地区汉族人群对氧磷酶Q192R基因多态性与冠心病的相关性的研究

目的测定对氧磷酶PON1Q192R基因多态性及在人群中的分布,分析不同基因型与冠心病（CHD）的关系,CHD常见危险因素对基因型亚组的影响。方法冠心病患者128例,男80例,女48例,年龄36～82岁,平均（65．25±9．83）岁;全部病例有明确心梗病史或经冠状动脉（冠脉）造影确诊。正常对照组110例,男66例,女4J4例,年龄31—87岁,平均（63．52±7．91）岁;选自人群健康查体的随机个体。应用聚合酶链反应．限制性片断长度多态性（PCR-RFLP）分析机聚丙稀酰胺凝胶电泳方法检测PON1（对氧磷酶）多态性。结果PONlQl92R有QQ、QR、RR三种基因型,正常人的基因型频率分别为0．2000、0．4909、0．3091,Q、R等位基因频率分别为0．4500、0．5500,冠心病组QQ、QR、RR三种基因频率分别为0．2266、0．5313、0．2421,Q、R等位基因频率分别为0．4921、0．5079。结论潍坊地区汉族人群PON1（对氧磷酶）基因多态性和冠心病无关。     

Effect of organophosphate intoxication on human serum paraoxonase

Recently, interindividual variations in serum paraoxonase (PON1) activity and the differences in its metabolic activity towards different organophosphates (OPs) caused by the coding region polymorphisms L55M and Q192R have been found to be important risk factors in susceptibility to OP poisoning. In this study, we investigated the effect of PON1 on the outcome of acute OP intoxication and the effect of acute OP intoxication on PON1. Twenty-eight OP-poisoned patients and 66 healthy volunteers were studied. Patients were evaluated for the clinical manifestations of OP intoxication as well as PON1 activity, PON1 mass and PON1 polymorphisms. Butyrylcholine-esterase (BChE) activity was 50% lower (2,276 +/- 738 U/L versus 5,037 +/- 1,553 U/L, P<0.01) while PON1 activity was 30% lower [114.2 +/- 67.4 nmol/mL/min versus 152.9 +/- 78.9 nmol/mL/ min, P<0.05) in patients than in controls. We observed that the PON1 and BChE activities of eight of the original subjects returned to normal levels when they were reinvestigated six months after exposure. The frequency of the PON192Q allele was significantly higher in patients than controls (85.7% versus 59.7%, chi2=6.745, P=0.034). QQ/ MM individuals had the lowest activity towards paraoxon, while RR/LL individuals had the highest activity. Our data indicate that interindividual differences in PON1 activity and the PON1-55 and -192 polymorphisms are important risk factors in susceptibility to acute OP poisoning; therefore, identifying an individual's PON1 alloenzymes may play an important role in the treatment of patients suffering from OP intoxication.     

Paraoxonase 1 (PON1) modulates the toxicity of mixed organophosphorus compounds

A transgenic mouse model of the human hPON1_Q192R polymorphism was used to address the role of paraoxonase (PON1) in modulating toxicity associated with exposure to mixtures of organophosphorus (OP) compounds. Chlorpyrifos oxon (CPO), diazoxon (DZO), and paraoxon (PO) are potent inhibitors of carboxylesterases (CaE). We hypothesized that a prior exposure to these OPs would increase sensitivity to malaoxon (MO), a CaE substrate, and the degree of the effect would vary among PON1 genotypes if the OP was a physiologically significant PON1 substrate in vivo. CPO and DZO are detoxified by PON1. For CPO hydrolysis, hPON1_R192 has a higher catalytic efficiency than hPON1_Q192. For DZO hydrolysis, the two alloforms have nearly equal catalytic efficiencies. For PO hydrolysis, the catalytic efficiency of PON1 is too low to be physiologically relevant. When wild-type mice were exposed dermally to CPO, DZO, or PO followed 4-h later by increasing doses of MO, toxicity was increased compared to mice receiving MO alone, presumably due to CaE inhibition. Potentiation of MO toxicity by CPO and DZO was greater in PON1^−/− mice, which have greatly reduced capacity to detoxify CPO or DZO. Potentiation by CPO was more pronounced in hPON1_Q192 mice than in hPON1_R192 mice due to the decreased efficiency of hPON1_Q192 for detoxifying CPO. Potentiation by DZO was similar in hPON1_Q192 and hPON1_R192 mice, which are equally efficient at hydrolyzing DZO. Potentiation by PO was equivalent among all four genotypes. These results indicate that PON1 status can have a major influence on CaE-mediated detoxication of OP compounds.     

Relationship between human paraoxonase-1 activity and PON1 polymorphisms in Mexican workers exposed to organophosphate pesticides

Paraoxonase-1 (PON1) is a serum enzyme which catalyzes the hydrolysis of organophosphate pesticides. In this study we conducted a cross-sectional study and reported on the distribution of three common genetic polymorphisms of the PON1 gene in a population of floriculture workers from Mexico as well as the association between those polymorphisms and other predictors with serum PON1 activity on paraoxon, diazoxon and phenylacetate. The genotype frequencies at position PON1₅₅ were 89% (LL), 10% (LM) and 0.6% (MM), at position PON1₁₉₂ they were 16% (QQ), 47% (QR) and 37% (RR), and 26% (TT), 42% (TC) and 32% (CC) at position PON1_−108. Thus, the frequencies of alleles L, Q and T were 0.94, 0.40 and 0.47, respectively. The PON1₅₅ polymorphism had no significant effect on serum PON1 activity on any substrate. We found a significant association between the PON1₁₉₂ polymorphism and PON1 activity towards paraoxon and diazoxon, which increased in genotypes as follows: 192RR > 192QR > 192QQ for paraoxonase activity and, inversely, 192QQ > 192QR > 192RR for diazoxonase activity. The PON1₋₁₀₈ polymorphism also had a significant effect on PON1 activity level towards paraoxon in the following order among the genotype groups: −108CC > −108TC > −108TT. Serum PON1 activity towards diazoxon was not associated with the PON1₋₁₀₈ polymorphism but it was influenced by the intensity exposure to pesticides at the floriculture industry and the years of the occupational exposure to pesticides. No polymorphism significantly influenced serum PON1 activity on phenylacetate.     

Paraoxonase and susceptibility to organophosphorus poisoning in farmers dipping sheep

OBJECTIVES: Human serum paraoxonase (PON1) hydrolyses organophosphate pesticides (OPs) entering the blood circulation and tissue fluid thus limiting toxicity. The PON1 coding region has two polymorphisms involving the amino acids at position 55 (Lt<--M) and 192 (Qt<--R), giving rise to isoenzymes which differ in their catalytic rate for the hydrolysis of OPs. We therefore hypothesized that individuals inheriting low activity isoforms of PON1 would be more liable to report symptoms of OP toxicity. METHODS: We have therefore investigated the relationship between PON1 genetic polymorphisms and PON1 activity in farmers reporting chronic ill health which they attributed to OP exposure whilst sheep dipping (cases) and farmers who carried out similar activities, but remained well (controls). Diazoxon, paraoxon and phenylacetate were used as substrates for PON1. Diazoxon is the active metabolite of diazinon, the sheep dip most commonly used in the UK. RESULTS: Cases were found to be more likely to have the R192 allele ( 0.01) and to have the L55 allele ( 0.05) than the controls. This combination of R and L genotypes was associated with lower PON1 activity towards diazoxon in both cases and controls. Farmers in the lowest quintile for the rate of serum diazoxon hydrolysis had a greater risk of being a case i.e. of reporting ill health (odds ratio 2.47 (95% CI 1.35-2.82)), than the other four quintiles of diazoxon hydrolysis. The rate of serum hydrolysis of paraoxon was greatest in cases and controls with the R/L haplotype (both 0.001). CONCLUSIONS: The farmers reporting chronic ill health due to organophosphate exposure have a higher proportion of the PON1-192R polymorphism associated with lower rates of diazoxon hydrolysis and lower rates of diazoxon hydrolysis than the controls and that their ill health may be explained by a lower ability to detoxify diazoxon.     

Pharmacogenomic considerations of the paraoxonase polymorphisms

The high density lipoprotein (HDL)-associated enzyme paraoxonase-1 (PON1) exhibits a substrate-dependent activity polymorphism as well as a large variability in plasma levels among individuals. The PON1 activity polymorphism is determined mainly by a glutamine(Q)/arginine(R) substitution at position 192 of PON1 (PON1(Q192R)). There is one additional polymorphism in the coding region at L55M, five in the 5 cent regulatory region and four in the 3' untranslated region. One of the five promoter polymorphisms (C-108T) appears to have a major effect on the levels of PON1 found in plasma. Two factors are important to consider in evaluating the pharmacogenetics of PON1 in an individual, the amino acid present at position 192 and the level of their plasma PON1. 'PON1 status', measured easily via a two-substrate assay, provides data for both of these critical factors. As such, PON1 status will be more useful than genotyping alone for identifying individuals at risk for cardiovascular disease, for predicting sensitivity to OP insecticides, and possibly nerve agents, for predicting the disposition of drugs known to be activated or hydrolyzed by PON1 and for developing robust pharmacokinetic models for each of these roles of these roles of PON1.     

Organophosphorus pesticide exposure decreases sperm quality: association between sperm parameters and urinary pesticide levels

Several studies have suggested that human semen quality has declined over the past decades and some of them have associated it with occupational exposure to pesticides. However, most of these studies have not been associated with a reliable exposure level and have been designed mostly as cross-sectional studies. The present work evaluates, in a longitudinal follow-up study, the effect of organophosphate pesticides (OP) at three occupational exposure levels on semen quality. In addition, the study examined the association between OP urinary levels and sperm parameters in exposed and unexposed workers. A total of 139 semen samples from 52 volunteers were assessed. Urinary OP levels were measured by gas-liquid chromatography.The results revealed that the poorest semen quality was found among the subjects with the highest OP exposure and the highest urinary OP levels. Seasonal variations in sperm concentration and sperm count were registered.The results showed a significant decrease in total sperm count among subjects with the highest exposure to OP. Further studies assessing the effects of OP on male reproductive health should be controlled by the variability in human sperm parameters, sperm seasonality, spermatogenesis time and the changing OP exposure level in men highly exposed to OP.     

Methyl-parathion decreases sperm function and fertilization capacity after targeting spermatocytes and maturing spermatozoa

Paternal germline exposure to organophosphorous pesticides (OP) has been associated with reproductive failures and adverse effects in the offspring. Methyl-parathion (Me-Pa), a worldwide-used OP, has reproductive adverse effects and is genotoxic to sperm, possibly via oxidative damage. This study investigated the stages of spermatogenesis susceptible to be targeted by Me-Pa exposure that impact on spermatozoa function and their ability to fertilize. Male mice were exposed to Me-Pa (20 mg/kg bw, i.p.) and spermatozoa from epididymis-vas deferens were collected at 7 or 28 days post-treatment (dpt) to assess the effects on maturing spermatozoa and spermatocytes, respectively. Spermatozoa were examined for DNA damage by nick translation (NT-positive cells) and SCSA (%DFI), lipoperoxidation (LPO) by malondialdehyde production, sperm function by spontaneous- and induced-acrosome reactions (AR), mitochondrial membrane potential (MMP) by using the JC-1 fluorochrome, and fertilization ability by an in vitro assay and in vivo mating. Alterations on DNA integrity (%DFI and NT-positive cells) in spermatozoa collected at 7 and 28 dpt, and decreases in sperm quality and induced-AR were observed; reduced MMP and LPO were observed at 7 dpt only. Negative correlations between LPO and sperm alterations were found. Altered sperm functional parameters evaluated either in vitro or in vivo were associated with reduced fertilization rates at both times. These results show that Me-Pa exposure of maturing spermatozoa and spermatocytes affects many sperm functional parameters that result in a decreased fertilizing capacity. Oxidative stress seems to be a likely mechanism of the detrimental effects of Me-Pa exposure in male germ cells.     

Genetic polymorphisms and activity of PON1 in a Mexican population

Human paraoxonase (PON1) plays a role in detoxification of organophosphorus (OP) compounds by hydrolyzing the bioactive oxons, and in reducing oxidative low-density lipoproteins, which may protect against atherosclerosis. Some PON1 polymorphisms have been found to be responsible for variations in catalytic activity and expression and have been associated with susceptibility to OP poisoning and vascular diseases. Both situations are of public health relevance in Mexico. Therefore, the aim of this study was to evaluate PON1 phenotype and the frequencies of polymorphisms PON1 -162, -108, 55, and 192 in a Mexican population. The studied population consisted of unrelated individuals (n = 214) of either gender, 18-52 years old. Serum PON1 activity was assayed using phenylacetate and paraoxon as substrates. PON1 variants, -162, 55, and 192, were determined by real-time PCR using the TaqMan System, and PON1 -108 genotype by PCR-RFLP. We found a wide interindividual variability of PON1 activity with a unimodal distribution; the range of enzymatic activity toward phenylacetate was 84.72 to 422.0 U/mL, and 88.37 to 1645.6 U/L toward paraoxon. All four PON1 polymorphisms showed strong linkage disequilibrium (D% >90). PON1 polymorphisms -108, 55, and 192 were independently associated with arylesterase activity; whereas the activity toward paraoxon was related only with PON1 192 polymorphism, suggesting that this polymorphism is determinant to infer PON1 activity. A better understanding of the phenotype and genotypes of PON1 in Mexican populations will facilitate further epidemiological studies involving PON1 variability in OP poisoning and in the development of atherosclerosis.     

Interaction between organophosphate pesticide exposure and PON1 activity on thyroid function

Organophosphate pesticides are widely used in agricultural purposes. Recently, a few studies have demonstrated the ability of these chemicals to alter the function of the thyroid gland in human. Moreover, the paraoxonase-1 enzyme (PON1) plays an important role in the toxicity of some organophosphate pesticides, with low PON1 activity being associated with higher pesticide sensitivity. This study evaluates the interaction between exposure to organophosphate compounds and PON1 enzyme activity on serum levels of TSH and thyroid hormones in a population of workers occupationally exposed to pesticides. A longitudinal study was conducted on a population of floriculture workers from Mexico, during two periods of high and low-intensity levels of pesticide application. A structured questionnaire was completed by workers containing questions on sociodemographic characteristics and other variables of interest. Urine and blood samples were taken, and biomarkers of exposure (dialkylphosphates), susceptibility (PON1 polymorphisms and activity) and effect (thyroid hormone levels) were determined. Interaction between dialkylphosphates and PON1 polymorphisms or PON1 activity on hormone levels was evaluated by generalized estimating equation (GEE) models. A significant interaction was found between serum diazoxonase activity and total dialkylphosphates (ΣDAP) on TSH levels. Thus, when PON1 activity was increased we observed a decrease in the percentage of variation of TSH level for each increment in one logarithmic unit of the ΣDAP levels. This interaction was also observed with the PON1₁₉₂RR genotype. These results suggest a stronger association between organophosphate pesticides and thyroid function in individuals with lower PON1 activity.     

Associations of xenobiotic-metabolizing enzyme genotypes PON1Q192R, PON1L55M and CYP1A1*2A MspI with pathological symptoms of a rural population in south Greece

Paraoxonases and cytochromes P450 constitute two major classes of xenobiotic-metabolizing enzymes involved in the detoxification of pesticide chemicals. In this study, we examined the distribution of two common genetic polymorphisms of the paraoxonase 1 gene and one common polymorphism of the CYP1A1 gene, in relation to pathological diseases occurring in a rural population. Blood and hair samples were collected from 220 participants of an agricultural cohort in the south of Greece for genotype and pesticide analysis. Demographic information and disease status of the participants was obtained by questionnaire, medical examination and medical record. Organochlorine pesticides and metabolites (DDTs, HCHs) were extracted from hair and analyzed using gas chromatography combined with mass spectrometry techniques. Our results indicate exposure of the rural population of Amaliada to organophosphate and past exposure to organochlorine pesticides. Genotypic analysis of PON1Q192R, PON1L55M and CYP1A1*2A MspI polymorphisms was performed using PCR-RFLP. The PON1 192R and 55M alleles absence was significantly associated with hypertension (OR: 2.59; 95% CI: 1.10-6.09) and hepatitis (OR: 21.43; 95% CI: 2.53-181.50), respectively, as indicated from backward logistic regression. Although the presence of PON1 192R allele significantly affected the occurrence of prostate hyperplasia (OR: 0.35; 95% CI: 0.03-0.40), no associations were obtained between the paraoxonase serum activity or the CYP1A1 genotype and the disease status.     

Rabbits possess a serum paraoxonase polymorphism similar to the human Q192R

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect low-density lipoprotein (LDL) against oxidation. These properties are influenced by a well-characterized polymorphism (Q192R) in human PON1. We now report the identification and characterization of a phenotypically similar, but genetically distinct polymorphism in rabbit PON1. This polymorphism in rabbits was detected by phenotyping sera obtained from 16 inbred rabbit strains and 20 outbred New Zealand White rabbits by paraoxonase/arylesterase activity. The genetic basis of the rabbit polymorphism was determined by DNA sequencing and found to reside in a region distinct from the human Q192R and M55L polymorphisms. Three variant nucleotides within exon 4 (corresponding to P82S, K93E and S1O1G) were found to segregate with the observed rabbit PON1 phenotypes (rPON1A and rPON1B). The rPON1A and rPON1B proteins were purified and compared to the two human isoforms (192Q and 192R). The human and rabbit PON1s displayed similar characteristics with respect to physical properties and substrate specificity. However, rPON1A and rPON1B hydrolysed a variety of substrates at different rates. The rPON1A was also at least three-fold more efficient at protecting LDL from oxidation than rPON1B. Our characterization of a rabbit PON1 polymorphism provides useful insights into important functional residues in PON1. In addition, due to the observed similarities between the rabbit and human polymorphisms, the rabbit may serve as a good model to examine the effect of human PON1 polymorphisms in disease development.     

Associations of xenobiotic-metabolizing enzyme genotypes PON1Q192R,PON1L55M and CYP1A1*2A MspI with pathological symptoms of a rural population in south Greece

1. Paraoxonases and cytochromes P450 constitute two major classes of xenobiotic-metabolizing enzymes involved in the detoxification of pesticide chemicals. In this study, we examined the distribution of two common genetic polymorphisms of the paraoxonase 1 gene and one common polymorphism of the CYP1A1 gene, in relation to pathological diseases occurring in a rural population.

2. Blood and hair samples were collected from 220 participants of an agricultural cohort in the south of Greece for genotype and pesticide analysis. Demographic information and disease status of the participants was obtained by questionnaire, medical examination and medical record. Organochlorine pesticides and metabolites (DDTs, HCHs) were extracted from hair and analyzed using gas chromatography combined with mass spectrometry techniques.

3. Our results indicate exposure of the rural population of Amaliada to organophosphate and past exposure to organochlorine pesticides.

4. Genotypic analysis of PON1Q192R, PON1L55M and CYP1A1*2A MspI polymorphisms was performed using PCR-RFLP. The PON1 192R and 55M alleles absence was significantly associated with hypertension (OR: 2.59; 95% CI: 1.10–6.09) and hepatitis (OR: 21.43; 95% CI: 2.53–181.50), respectively, as indicated from backward logistic regression. Although the presence of PON1 192R allele significantly affected the occurrence of prostate hyperplasia (OR: 0.35; 95% CI: 0.03–0.40), no associations were obtained between the paraoxonase serum activity or the CYP1A1 genotype and the disease status.

     

Paraoxonase activity and genetic polymorphisms in greenhouse workers with long term pesticide exposure

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL) associated protein, which plays a critical role in the pathogenesis of atherosclerosis, although it was primarily associated with the hydrolysis of organophosphorus compounds. PON1 was initially thought to be independent from physiological or pathological states, although recently some environmental factors have been reported to modulate its activity. In this study, we have investigated the promoter (PON1 -108C/T and -909 C/G) and coding region (PON1 192Q/R and 55L/M) polymorphisms, as well as PON1 activity towards different substrates (paraoxon, phenylacetate and diazoxon) in 102 individuals with long term low dose exposure to pesticides in a plastic greenhouse setting (sprayers), who are probably the group of agricultural workers with the highest exposure to pesticides. PON1 activity towards paraoxon was nonsignificantly decreased (up to 53.5%) in the sprayers subgroup exposed to organophosphates (n = 41) compared with nonsprayers acting as controls (n = 39). None of the genotypes studied was associated significantly with the subgroup of individuals exposed to organophosphates, although differences between sprayers and nonsprayers were observed in the PON1 -909 G/C polymorphism. Among the environmental factors that significantly predicted lower rates of PON1 activity towards paraoxon are, interestingly, the exposure to organophosphates and current smoking. By contrast, the utilization of protective clothing while spraying pesticides inside the greenhouses was positively associated with PON1 activity, very likely by preventing the pesticides from being absorbed. This study suggests that chronic exposure to pesticides might decrease PON1 activity and pinpoints the potential usefulness of monitoring PON1 activity in occupational settings where exposure to organophosphates occurs.