COLLABORATIVE PAIN RESEARCH UNIT
ORGANOPHOSPHATES CLINICAL INFORMATION DOCUMENT

By: RH Dunstan D.Phil
CHEMICAL EXPOSURE

Dr Hugh Dunstan
Senior Lecturer
School of Biological and Chemical Sciences
Discipline of Biology
The University of Newcastle
Fax: 61 2 4921 7281

University Web Page:
http://www.newcastle.edu.au/department/bi/birjt/cpruis/

Email: birhd@alinga.newcastle.edu.au

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 inhalation^2,3.

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 proteins^4,5.

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 literature^8,9.

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 forever^6,7.

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.
5. Depression.
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:

*Intellectual functioning,
*academic skills,
*abstraction and flexibility of thinking, and
*simple motor skills (speed & coordination).

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 al ⁶ 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 health^18-23.

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 Disorder (COPIND)

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
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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)
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13) Operational guide for the W.H.O. Neurobehavioural Core Test Battery,
World Health Organisation, Geneva (1986).

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23) London L (1998) Effects of long term organophosphate exposures on
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Signed: Date: 14 August, 2001