APPENDIX I-Q: Dunstan, Chemical Exposure-Blood Brain Barrier-Organophosphates.
[ Be sure to see Pesticide files ]
Dr Hugh Dunstan
School of Biological and Chemical Sciences
Discipline of Biology
Fax: 61 2 4921 7281
University Web Page: http://www.newcastle.edu.au/department/bi/birjt/cpruis/
COLLABORATIVE PAIN RESEARCH UNIT
CLINICAL INFORMATION DOCUMENT: 1
By: RH Dunstan D.Phil
2) Consequences of hydrophobic interactions
3) Chronic adverse health effects following organophosphate poisoning
4) Defining organophosphate exposure syndromes
It is necessary to define some basic terms and concepts which are relevant to understanding the impact of organophosphate pesticides on human health.
All OP compounds are structurally and functionally related. The organophosphate pesticides comprising the neutral esters or amides are biologically active by their capacity to interfere with metabolism and they are usually lipid soluble and highly reactive. The inhibition of the enzyme acetylcholinesterase (AChE) results in acute cholinergic over-stimulation at nicotinic and muscarinic synapses of the peripheral, autonomic and central nervous systems. Examples of organophosphate pesticides, Diazinon, Sulfotepp and Monothionotepp are shown below1:
Diazinon is a cholinesterase inhibitor used as an insecticide, particularly in the treatment of ectoparasites in animals. Sulfotepp is an insecticide/miticide. It has potential symptoms from overexposure of eye pain, blurred vision, lacrimation and rhinorrhea; headache; cyanosis; anorexia, nausea, vomiting and diarrhea; local sweating, weakness, twitching, paralysis, Cheyne-Stokes respiration, convulsions, low blood pressure and cardiac irregularities; skin and eye irritation.
Cells The various organs and tissues of the body are composed of cells. Each cell has a specialised structure and function according to its type and location. The "machinery" required for the synthesis of proteins, carbohydrates, hormones and other biological molecules are housed within the cells. The effects of the organophosphate pesticides occur at the surfaces of these cells and within the cells.
Membranes Each cell is surrounded by a membrane.
This membrane represents the barrier between the external environment and the
"cell machinery". Its jobs include selecting which molecules can
travel in and out of the cell, keeping unwanted material out of the cell (such
as toxic waste products), acting as a receptor to chemical messages from other
cells, and preventing infection by pathogens.
The membrane itself is also an important matrix for housing the cell components required to deliver the energy necessary to drive the synthetic machinery inside the cell.
The membrane houses many important proteins involved in energy metabolism, cellular communications, transporting ions (sodium, chlorine…), transporting nutrients and mediating the immune response.
The membrane is a hydrophobic structure, which means that it does not contain water, and provides therefore, an effective barrier around the cell. The proteins which are found in the membrane, have hydrophobic regions which allow the specialised proteins to traverse the membrane.
The membrane is not a rigid structure - in fact it is referred to as having specific "fluid properties" which means that it is flexible and can expand and contract in response to biological stimuli. It is a structure under constant stimulation, regulation and control. It is constantly changing in response to biological demand and environmental changes.
¯ The chemical and physical integrity of this membrane is paramount to the proper function of the cells.
Non-polar Solvent A non-polar solvent is a liquid with a chemical composition with low polarity. Such a solvent would dissolve fats, whereas these fats would not dissolve in water. These non-polar solvents can be used to clean greases or dissolve lipophilic chemicals such as pesticides and herbicides. These solvents are used in the pesticide formulations to provide a matrix for delivering the pesticide to the target sites, provide efficient dispersal during the application process and allow penetration of target of the fatty barriers which surround the target pests. These same properties of the pesticide formulations allow penetration of the pesticides through the skin barriers, respiratory surfaces and alimentary tracts of the humans and animals.
Recalcitrant toxic chemicals Recalcitrant chemicals are those chemicals which are extremely stable and cannot be detoxified by the human liver. These chemicals are usually lipophilic or hydrophobic (i.e. not soluble in water, but soluble in non-polar solvents) and include pesticides such as 1,1-dichloro-2,2-bis (p-chlorophenyl) ethene (DDE), hexachlorobenzene (HCB) and the polychlorinated-biphenyls (PCBs), as well as some of the non-polar halogenated solvents. The organophosphate pesticides are usually not considered as recalcitrant chemicals due to their high reactivity. However, they can be stabilized under certain conditions when introduced to certain matrices such as fine clay particles in the soil.
2) CONSEQUENCES OF HYDROPHOBIC INTERACTIONS
The organophosphate compounds under question are hydrophobic and require the
use of solvents for dissolving and dispersal. These lipophilic
chemicals can be absorbed into the body via a number of routes including
ingestion, dermal absorption and inhalation2,3. Due to
their lipophilic nature, these chemicals can gain
access to the cell membranes where they can alter membrane integrity and
inhibit functional membrane-bound proteins4,5.
The major mechanisms by which these hydrophobic toxic chemicals exert their toxic effects, are via their interactions with the membrane components within this lipophilic domain. These lipophilic molecules can exert their toxic effects by,
1) penetrating the membrane bi-layer,
2) direct interaction with the lipophilic proteins,
3) direct interaction with components of energy metabolism (electron transport chain),
4) direct interaction with signal transduction mechanisms,
5) direct interaction with transport systems, and 6) altering membrane fluidity properties.
These interactions and many others can lead to an overall alteration in cell membrane integrity and function.
The higher turnover contaminants such as the solvents and organophosphate
pesticides can directly exert substantial damage via the mechanisms described
above or indirectly by 1) preventing effective cellular protection by
antioxidant mechanisms normally active in the cell,
2) causing direct denaturing of key structural and functional proteins,
3) enabling the passage into the cells of toxic waste products which would normally be kept outside of the cell (by altering membrane fluidity properties), and 4) enhancing the toxicities of other contaminants by synergistic interactions or by increasing the membrane permeability properties.
The damage caused by the organophosphate pesticides and the non-polar solvents contained within their formulations can therefore persist long after the chemicals have been removed.
Event Related Potentials (ERP)
These are brain potentials which can be recorded from the scalp in response to an external stimulus or event. They occur only when the subject is attentive and only when the subject is required to distinguish one stimulus from a group of other stimuli. It has been used to investigate patients who suffer disorders of cognition and has been used to demonstrate abnormalities in subjects who had an acute poisoning with OP's. The damage to nerve cell membrane integrity and function is implicated by this method of testing.
3) CHRONIC ADVERSE HEALTH EFFECTS FOLLOWING ORGANOPHOSPHATE POISONING Acute organophosphate pesticide poisonings cause substantial morbidity and mortality worldwide6. Organophosphate pesticides are cholinesterase inhibitors. In the poisoned individual, cholinergic synapses cannot degrade acetylcholine, the transmitter released during normal nerve function. This leads to excitation, followed by a degree of paralysis of the extensive peripheral and central cholinergic nervous system. A number of symptoms are usually observed in patients suffering exposure to organophosphates including: flushing, dry mouth, fascilations, tremours, restlessness, agitation, ataxia, weakness, convulsions, and coma. These symptoms develop immediately after exposure, but once the cholinergic imbalance has been corrected , the neurological signs and symptoms disappear7. In vitro experiments showed that after repeated doses of ethylpyrophosphate (TEPP), cholinesterase is irreversibly inactivated, which leads to an accumulative poisoning. It was proposed that repeated inhibition of cholinesterase and interaction with other possible cellular constituents may eventually lead to alteration in biochemical functions and pathological changes.
Behavioural sequelae Evidence has now accumulated over the last 30 years indicating that organophosphorus esters were capable of producing delayed neurological lesions in exposed individuals. The presence of long term neuropsychiatric disturbances in humans following acute exposure to organophosphate compounds has been previously reported in the earlier literature8,9. The subtle effects produced, the trauma of the poisoning, and the latency of appearance of these signs makes it difficult to assess the chronic effects of these chemicals10. While the cholinergic effects of poisonings have been fully documented, the subtle delayed effects on both the central and peripheral nervous systems are neither well known nor understood.
"Partly, this is due to the fact that the signs and symptoms appear after the acute phase of toxicity and, partly, to the fact that most physicians never see the chronic problems arise, the patient having been discharged without any further examination at a later date unless by a family physician who may not comprehend what he is seeing."10
In the category of subtle, delayed adverse health effects, the neurological and neuromuscular deficits, the behavioral effects, and psychiatric sequelae should be included. Many case studies have been reported in the literature, but most of these are poorly documented or anecdotal. However, when these cases are compiled and compared, a pattern begins to merge and these afflicted individuals are no longer so unusual. An example of such a case study is given below which describes a patient exposed to diazinon10.
"Because of a flea infestation in an apartment having a porous brick floor, the tenant sprayed a 1.0% diazinon preparation, avoiding skin contact, holding his breath until he went into the living room intermittently to breathe, and washing thoroughly after using the product. So far, so good! He then slept in this room for about 10 consecutive nights - until the symptoms appeared. When the symptoms started, he thought that he had influenza. However, the symptoms progressed to lacrimation, salivation, physical weakness, impaired speech, mental slowness, and confusion. Within a week, he had figured out what his problem was and had an erythrocytic cholinesterase assay, the result being low but not excessively so. No plasma cholinesterase assay was done. Within two months of exposure, some physical strength had returned, but the neuro-psychiatric symptoms were little improved as were other signs. These included aphasia, slurred speech, difficulty in forming a sentence (and stating it), severe pressure in head with headaches (the pressure sensation has persisted, varying in location), poor memory, episodes of depression, apathy, anger, irritability, nervousness, impaired reading ability, comprehension, and retention of what had been read (he is a proofreader for a publishing house), mental slowness, poor cognitive and problem-solving ability, and impaired vision (corrective glasses but there was "something" that the glassed did not correct). When he contacted me again, some 7 months after his exposure, he felt that he was "recovering", his yardstick of measurement being that he could read almost a whole page of text and retain what he had read (return of cognitive skills). However, it still took almost two years before the patient was of the opinion that he had recovered to some degree of normalcy, even though the quality of his general health was not at his pre-exposed level."
These cases used to be rare, but more and more of these case reports have now been documented7. The literature on potential, suspect, and established sequelae of organophosphorus ester poisoning does not confirm the often read statement the "clinical recovery from nonfatal poisoning is always complete in a few days"11. Easily recognised, serious or permanent symptomatology has never been observed frequently enough to establish a recognisable pattern. Somewhere between the two extremes, there are a variety of suspected or real biological effects occurring in a sizable group of cases11.
There are now a number of detailed epidemiological studies which demonstrate the existence of persistent and serious complaints lasting from 6 months to several years following exposure to the organophosphate pesticides and these symptoms could possibly last forever6,7.
There are several behavioural sequelae
which can be identified with organophosphorus
insecticide poisonings.12 These effects include the following:
1. Impaired vigilance and reduced concentration.
2. Reduced information processing and psychomotor speed.
3. Memory deficit.
4. Linguistic disturbances.
6. Anxiety, irritability.
The cases investigated were exposed to a range of organophosphate insecticides including TEPP. There were no differences between cases exposed to different OP's, but all OP's were cholinesterase inhibitors and all had similar effects. The OP cohort had significantly higher levels of organochlorine residues compared with controls (62.1ppb vs 33.3ppb), but analysis of covariance did not show any significant association of organochlorines with altered neuropsychological function.
A large well designed study by Savage et al7 was conducted to determine whether persons with previous documented acute organophosphate pesticide poisonings exhibited covert manifestations of latent chronic neurological deficits. The study investigated 100 cases of previously poisoned subjects and 100 matched controls.
There were clear chronic neurological sequelae to acute organophosphate poisoning. The sequelae can be subtle and neurological examination, clinical EEG and ancillary laboratory tests may not discriminate poisoned subjects from control.
The subjects had significantly worse scores in:
Evaluation of subjective assessments of functioning found significant differences in aspects of language and communication, memory, cognitive intellectual functioning and perceptual functions.
Evaluation of personality attributes revealed significant problems in depression, irritability (similar findings to Gershan et al 19618), confusion and social withdrawal. These data suggest that the findings of the objective testing regimes were reflected in the every day functioning of the OP exposed cases. The authors concluded:
"When confronted with a patient who has been poisoned by organophosphates, the clinician cannot rely solely on the standard examination or on clinical intuition to decide which patients need further evaluation. The clinical neurological examination focuses primarily on sensory and motor functioning and is relatively insensitive to higher level cognitive skills and activity, which are best assessed by the neuropsychologist. Although the neuropsychological evaluation demonstrated some impairment of fine coordination and motor speed of the upper extremities in the poisoned subjects, the major deficits were cognitive and appeared on tests of abilities that receive limited evaluation in the clinical neurological examination."
A retrospective study was performed by Rosenstock et al6 on a population of Nicaraguan agricultural workers poisoned by organophosphorus insecticides, to determine whether single episodes of acute intoxication could lead to neuropsychological dysfunction, as seen reported in isolated case studies. The 36 poisoned males were examined approximately 24 months after poisoning and, with matched controls, were subjected to a neuropsychological assessment, which was based on the World Health Organisation core test battery with an additional six tests.6,13
The poisoned group was significantly below the performance of the controls in neuropsychological functions including: auditory attention, visual memory, visuomotor speed, sequencing, and problem solving, motor steadiness, reaction, and dexterity.
The patients also reported symptoms consistent with central nervous system involvement. The findings were consistent with those of Dille and Smith,14 Savage et al.,7 and Midtling et al15.
The conclusions of this study suggested that single episodes of clinically significant organophosphorus ester intoxication can be associated with a persistent decline in neuropsychological functioning. This study and that of Savage et al7 confirm the observations of the many anecdotal studies reported in earlier literature.
An early study by Gershon and Shaw (1961)16 investigated 14 men and 2 women who had been exposed for between 1.5 and 10 years to organophosphorus insecticides. Schizophrenic and depressive reactions were observed with severe impairment of memory and difficulty in concentration. These were similar findings to those reported in an earlier study17. It was proposed the activation of the depression and schizophrenia may be related to the anticholinesterase activity of the organophosphorus insecticide. In this investigation, 8 out of 16 subjects exposed to organophosphorus insecticides reported impaired memory, 5 of which developed depression and 3 expressed irritability. Of the remaining 8 subjects, 4 suffered schizophrenia, and 2 suffered depression. A number of papers further support the findings that acute exposure causes chronic adverse effects on health18-23.
4) DEFINING ORGANOPHOSPHATE EXPOSURE SYNDROMES There are several subacute and chronic syndromes which can result from OP
exposure and these have been summarised by Jamal24:
1. The Intermediate Syndrome (IS)
2. The Organophosphate Induced Delayed Neuropathy (OPIDN)
3. The Chronic Organophosphate Induced Neuropsychiatric Disorder (COPIND)
The Intermediate Syndrome (IS)
Any OP can cause this syndrome and it can occur approximately 3 days after poisoning. It is not responsive to atropine or oxime therapy. The patient usually recovers within 2-3 weeks, but this is usually an intermediary stage occurring prior to the delayed effects described below.
The Organophosphate Induced Delayed Neuropathy (OPIDN)
This occurs usually 2-3 weeks after exposure and is serious and irreversible24. It consists of a distal symmetrical sensori-motor mixed peripheral neuropathy mainly affecting the lower limbs. It is not related to the effect of OP's on acetylcholine esterase. It is thought to act by causing an aging effect (by dealkylation) of a protein enzyme in nerve cells called neuropathy target esterase (NTE). This is only induced in certain species including humans and chickens. Other induced abnormalities include axon transport, physicochemical changes of proteins and axon membrane integrity24. The exact mechanism(s) are not fully understood.
The Chronic Organophosphate Induced Neuropsychiatric
Evidence indicates that OP's can induce chronic effects on both the peripheral and central nervous systems following acute intoxication24. The mechanisms for this condition are not related to the inhibition of acetylcholinesterase or NTE. There have been 2 types documented: Type I representing COPIND following an acute poisoning episode(s) and Type II representing COPIND following an chronic long term exposure to low level subclinical doses. There is no distinction between OP's in their apparent ability to cause COPIND.
Type I Twelve well designed studies performed from 1956 through to 1996 have shown repeatedly that chronic damage to central and peripheral nervous systems occurred following acute poisonings of OP compounds (eg Savage et al7, Steenland et al22). These findings have not been contradicted by any controlled studies. There are also numerous case study reports which support the larger studies.
The components of COPIND There have been several components of COPIND described24, including neurobehavioural and cognitive changes, psychiatric and mental manifestations, chronic fatigue, peripheral neuropathy, neuromuscular junctional dysfunction, electroencephalogical changes, autonomic nervous system disturbances, frontal lobe syndrome and abnormalities of cognitive evoked potentials. Some patients have all markers, while others only show a variety of combinations. Chronic fatigue and excessive tiredness have been described very frequently in patients with COPIND with over 10 publications cited in support of this statement24.
Recent findings indicate stress undermines the integrity and the protective role of the blood brain barrier so that the penetration of toxic substances such as AchE toxins, drugs and viruses increases up to 100 fold25. Another recent study indicates that physical exertion following exposure substantially increases muscle damage in animal models26.
1) The Merck Index, 12th Edition, Editors: S Budavari et al (1996), Merck Co Inc, NJ.
2) Joy, R.M. (1993) Chlorinated hydrocarbon pesticides, in: Pesticides and neurological diseases, 2nd edition, (Ed. D.J. Ecobichon and R.M. Joy), CRC Press, London.
3) Srivastava, A.K., Gupta, B.N., Mathur, A.K., Mathur, N., Mahendra, P.N. and Bharti, R.S. (1991) The clinical and biochemical study of pesticide sprayers. Human and Experimental Toxicology, 10, 279-283.
4) Cascorbi, I. and Foret, M. (1990) Interaction of Xenobiotics on the glucose-transport system and the Na+/K+-ATPase of human skin fibroblasts. Ecotoxicology and Environmental Safety, 21, 38-46.
5) Foret, M. and Ahlers, J. (1988) Effects of phenol's on growth rate and adenosine uptake of CHO cells. Ecotoxicology and Environmental Safety 16, 303-309.
6) Rosenstock L et al (1991) Chronic central nervous system effects of acute organophosphate pesticide intoxication. Lancet 338:223.
7) Savage EP et al (1988) Chronic neurological sequelae of acute organophosphate pesticide poisoning Arch Environ Health 43:38.
8) Gershon S and Shaw FH (1961) Psychiatric sequelae of chronic exposure to organophosphorus insecticides. Lancet 1 1371-74.
9) Dille JR and Smith PW (1964) Central nervous system effects of chronic exposure to organophosphate insecticides. Aerosp Medicine 35:475.
10) Ecobichon DJ (1994) Organophosphorus ester insecticides, in "Pesticides and neurological diseases", (Ed. D.J. Ecobichon & R.M. Joy) second edition, pp171-249. CRC press, London ISBN 0-8493-4361-5.
11) D.J. West I (1968) Sequelae of poisoning from phosphate ester pesticides, Ind Med Surg. 37:832.
12) Levin HS and Rodnitzky RL (1976) behavioural aspects of organophosphate pesticides in man. Clin Toxicol. 9:391.
13) Operational guide for the W.H.O. Neurobehavioural Core Test Battery, World Health Organisation, Geneva (1986).
14) Dillie JR and Smith PW (1964) Central nervous system effects of chronic exposure to organophosphate insecticides. Aerospace Med 35:475.
15) Midtling JE et al (1985) Clinical management of field worker organophosphate poisoning West J Med 142:514.
16) Gershon S & Shaw FH (1961) Psychiatric sequelae of chronic exposure to organophosphorous insecticides. Lancet June 24, 1371-1374
17) Rowntree DW, Nevin S, Wilson A. (1950) J Neurol. Psychiat. 13: 47.
18) Davies JE Neurotoxic concerns of human pesticide exposures. Am J Ind Med (1990) 18:327-31
19) Stephans R et al Neuropsychological effects of long term exposure to organophosphates in sheep dip. Lancet (1995) 354:1135-9
20) Rosenstock, L et al Chronic neuropsychological sequelae of occupational exposure to organophosphate insecticides. Am j Ind Med (1990) 18:321-5
21) Levin et al Anxiety associated with exposure to organophosphorous compounds. Arch Gen Psychiatry (1976) 33:225-8
22) Steenland K et al Chronic neurological sequelae to organophosphate pesticide poisoning Am J Public Health (1994) 84:731-6)
23) London L (1998) Effects of long term organophosphate exposures on neurological symptoms, vibration sense and tremor among South African farm workers. Scan J Work Environ Health 24:18-29.
24) Jamal GA Neurological syndromes of organophosphorus compounds. Adverse Drug React. Toxicol Rev. (1997) 16:133-170.
25) Friedman A et al (1996) Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response. Nature medicine 1996 2:1382-5
26) Hurbert M and Lison D (1995) Study of muscular effects of short term pyridostigmine treatment in resting and exercising rats. Human Environ Toxicol 14:49-54
Authors Declaration Whilst every effort has been taken to ensure that all the material presented herein is factually accurate, this report expresses the authors opinion as to the potential effects of organophosphate exposure. No liability is assumed if this advice or service does not provide useful or effective.
Signed: Date: 14 August, 2001
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