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* Departments of Physiology and Neurosurgery, Soroka University Medical Center,
The Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel;
Krembil Neuroscience Centre, Toronto Western Hospital, University of Toronto, Toronto, Canada; and
Department of Behavioral Sciences, Zlotowski Center for Neurosciences, Ben-Gurion University of the Negev, Beersheva, Israel
1Correspondence: Department of Physiology, Faculty for Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel. E-mail: alonf{at}bgu.ac.il
SPECIFIC AIMS
Organophosphate compounds (OPs) are commonly used as agricultural pesticides and household insecticides throughout the world. They act by inhibiting the acetylcholine (ACh) hydrolyzing enzyme acetylcholinesterase (AChE). Humans can mount a variety of responses to OP exposure. Several enzymes, including AChE, butyrylcholinesterase (BChE), and paraoxonase1 (PON1), are known to protect against OP exposure. Acute poisoning by OPs leads to accumulation of ACh at cholinergic synapses in the peripheral and central nervous systems, potentially causing nausea, excessive salivation, incontinence, bradycardia, headache, fatigue, seizures, coma, and death. Although the short-term effects of acute OP poisoning are understood to a great extent, the long-term consequences of acute poisoning and chronic, subthreshold exposure are still not clear. Several studies have reported restlessness, forgetfulness, and other neuropsychiatric symptoms as common complaints in exposed human populations. However, few significant changes in cognitive function have been detected by neuropsychological testing in populations exposed to low levels of OPs without any history of acute poisoning. The aim of the study was to explore gene-environment interactions involving functional changes in brain activity following chronic, subthreshold organophosphate exposure. Our candidate gene was the ACHE/PON1 locus on chromosome 7, and the physiological response was studied by combining neurophysiologic and neuropsychological approaches with biochemical and genetic tests.
PRINCIPAL FINDINGS
1. Quantitative EEG analyses and source localization methods revealed significant exposure-induced changes in EEG activity in specific cortical regions
We recorded EEG from 19 exposed and 9 control subjects with a similar age and gender distribution. Calculation of the average power spectrum revealed significantly lower power in the theta band and increased power in the beta 3 band (P=0.04 and P=0.03, respectively) of exposed compared with non-exposed groups, (Fig. 1
A–D). Such differences suggest that changes in cortical activity may differ in different brain regions. We used LORETA to determine the cortical regions responsible for generating site-specific differences in the power spectra. Visualization of activity computed by LORETA shows significant differences in the localization of activity in all seven frequency bands of exposed subjects compared with controls. Interestingly, exposed subjects showed significantly decreased activity in the limbic system and cingulate cortex compared with controls. Additionally, they displayed significantly increased prefrontal activity in the delta and beta3 bands (Fig. 1E-F
).
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2. Neuropsychological testing identified a significant deficit of visual recall in exposed individuals
We conducted neuropsychological tests and compared the results for 24 of the exposed subjects and 24 controls matched for age, sex, and education, using a t test for dependent measures. The delayed memory (20 min) portion of the visual reproduction subtest of the Wechsler Memory Scale indicated significant differences.
3. Chronic, subthreshold doses of organophosphate pesticides were shown to cause significant reductions in serum acetylcholinesterase activity and increase in serum paraoxonase and arylesterase activities
Biochemical assays for AChE, BChE, PON, and arylesterase activity were performed for 30 exposed subjects. Serum AChE activity was found to be significantly lower in exposed individuals compared with controls. There was no significant difference in BChE activity. Both PON and arylesterase activity were significantly higher than predicted in exposed subjects (447 and 441% of the control, respectively, P <0.001) (Fig. 2
A). Alterations in biochemical activity reflect, besides exposure levels, also the individual’s reaction to the chemical stress manifested by modified gene expression. This, in turn, is a composite effect of inheritance and environmental status (i.e., exposure). Therefore, as a follow-up to the biochemical investigation, we searched for genetic variation.
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4. Specific paraoxonase (PON1) genotypes (polymorphisms encoding the L55M and Q192R substitutions) are associated with the exposure-related accentuation of the exposure-induced biochemical and EEG changes
Biochemical analysis in carriers of specific PON1 55, 192, and –108 genotypes revealed several significant trends. In general, AChE activity did not vary by PON1 genotype except for a slight decrease in LLRR compared with MMQQ individuals (P =0.04) (Fig. 2
B). In contrast, different PON1 genotypes were correlated with significant alterations in paraoxonase activity. The presence of the PON1 L55M substitution (TTG to ATG) was consistent with a decrease in PON activity, presumably related to reduced PON1 mRNA and protein levels. We controlled for the effects on PON activity associated with the PON1 Q192R substitution by selecting individuals without the R allele. Interestingly, in the non-exposed population, MMQQ individuals (n=10) exhibited about one-third of the PON activity of LLQQ individuals (n =14) (MMQQ/LLQQ=0.36, P<0.0001). In contrast, in exposed subjects MMQQ (n =6) and LLQQ (n =7) individuals exhibited almost equal PON activity (MMQQ/LLQQ =1.3), suggesting that the M allele is associated with a greater increase in PON activity in exposed individuals. This is supported by our data that show 8.1 times more PON activity in exposed MM individuals than in controls and only 3.8 times more activity in exposed LL individuals than in controls. PON enzyme levels can be assessed by measuring the arylesterase activity. Indeed, arylesterase activity in individuals with the M allele was 6.0 times greater in exposed individuals than in non-exposed controls, whereas arylesterase activity in the exposed population without the M allele was only 3.5 times greater than in non-exposed individuals (Fig. 2)
.
The PON1 Q192R (CAA to CGA) substitution, which affects catalytic efficiency, was associated with increased PON activity. In controls there was a gene-dependent increase in PON activity correlated with the presence of the R allele (Fig. 2B
). Interestingly, changes in PON activity in exposed subjects were characterized by a much more robust change associated with the R allele. In addition, we could identify an important difference between exposed and non-exposed groups. Non-exposed LLRR individuals (n =13) showed 
2 times more PON activity than non-exposed LLQQ individuals (n =10) (LLRR/LLQQ =2.09, P <0.0001), whereas in exposed subjects LLRR individuals (n =3) showed almost 3 times more PON activity than LLQQ individuals (n =7) (LLRR/LLQQ =2.86, P <0.0001), suggesting an exposure-dependent response associated with the PON1 Q192R substitution and a potential biosensor effect of the R allele in anti-AChE exposure.
Our biochemical data showed significant differences in the individual responses to exposure, associated with the presence or absence of the M (L55M) and R (Q192R) alleles in the PON1 gene. Thus, we hypothesized that the increase in beta3 activity observed in the EEG of exposed individuals may also differ between exposed individuals with different genetic profiles. We found significantly increased beta3 activity in the frontal cortical regions (decreased in the temporal regions) of exposed individuals with the R allele compared with controls and with exposed individuals without the R allele. The M allele had no such effect. Comparing the LORETA values (representing the current source densities) in these brain regions revealed that exposed individuals with the R allele showed significantly increased frontal activity and decreased temporal activity (P <0.03).
CONCLUSIONS AND SIGNIFICANCE
These findings relate to the state of the field by addressing four major questions relevant to genome-environment interactions: 1) What, if any, neurological/neuropsychological effects result from chronic, subthreshold organophosphate exposure? 2) Do gene-environment interactions play a significant role in determining increased susceptibility to organophosphate exposure? 3) Is cholinergic signaling involved? 4) Can such increased risks be identified in EEG and blood test analyses?
Previous studies have suggested that chronic, subthreshold organophosphate exposure in humans may cause cognitive deficits such as decreased attention and impaired memory. Our study adds to the growing body of knowledge by identifying specific brain regions affected by such exposure. Additionally, we present findings that link genetic polymorphisms, previously identified as potential candidates for conferring susceptibility to organophosphates, to both functional alterations in brain activity and blood biochemistry measurements.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5576fje
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