studies on the correlation between blood cholinesterase inhibition and ‘target tissue’...

E L S E V I E R Toxicology 92 (1994) 11-25

IOXICOLOGY

Studies on the correlation between blood cholinesterase inhibition and 'target tissue'

inhibition in pesticide-treated rats

Stephanie Padilla *a, Valerie Z. Wilson b, Philip J. Bushnell c

aCellular and Molecular Toxicology Branch, CNeurobehavioral Toxicology Branch, Neurotoxicology Division (MD-74B), Health Effects Research Laboratory, United States Environmental Protection

Agency, Research Triangle Park, NC 27711. USA bManTech Environmental Technology, Inc. #2 Triangle Drive, Research Triangle Park, NC 27709,

USA

Received 15 June 1993; accepted 14 April 1994

Abstract

Inhibition of cholinesterase activity in the blood has been proposed as an index of ChE activity in tissues targeted by ChE-inhibiting pesticides, including the muscle end-plate region and the central nervous system (CNS). While opinions vary regarding the utility of blood ChE activity in predicting ChE activity in the target tissues, there appear to be no comprehensive studies designed to assess this possible correlation in a time- and dose-dependent manner. We undertook this type of study by administering a single dose of an organophosphate, chlorpyrifos (0, 30, 60 or t25 mg/kg in corn oil, s.c.) to rats and then sacrificing animals at 1, 4, 7, 21 or 35 days after dosing. Whole blood, plasma, erythrocytes, frontal cortex, hippocampus, striatum, hypothalamus and diaphragm tissue were collected and assayed for ChE activity. Collapsed across dosages, optimal correlations of blood ChE activity with brain or muscle activity occurred 7-21 days after dosing (when ChE inhibition was maximal and most stable). At all times after dosing, there was a high correlation among ChE activity in the hippocampus, striatum and frontal cortex. Generally, ChE activity in whole blood and erythrocytes correlated better with the activity in brain and muscle than did activity in the plasma (whole blood _> erythrocytes >> plasma). Similar relationships were also observed in a more abbrevi- ated study using a direct acting organophosphate, paraoxon. ChE activity was determined in blood components, brain and muscle at the time of maximal inhibition (4 h after injection) and during recovery (24 hrs after injection) using two dosage levels (0.17 or 0.34 mg/kg, s.c.).

* Corresponding author.

Elsevier Science Ireland Ltd. SSDI 0300-483X(94)02866-S

12 S. Padilla et al. / Toxicology 92 (1994) 11-25

Taken together, these data indicate that the level of ChE activity in the blood may accurately reflect activity in other tissues, but that this correlation is tissue- and time-specific.

Keywords: Organophosphate pesticide; Rat; Cholinesterase activity; Chlorpyrifos; Paraoxon

I. Introduction

Organophosphate and carbamate pesticides are designed to inhibit acetylcholinesterase (E.C. 3.1.1.7), the enzyme which normally hydrolyses the neurotransmitter acetylcholine. In the presence of an inhibitor of cholinesterase (ChE) activity, synap- tic acetylcholine may escalate to abnormally high levels. In mammals, this rise in acetylcholine levels in the nervous system and at the muscle motor end-plate is postulated to precipitate a 'cholinergic crisis' which can be debilitating and possibly fatal (Ecobichon, 1991).

Because nervous system or muscle ChE is known to participate in neurotransmission, it is accepted by the scientific and regulatory community that ChE inhibition in these 'target tissues' may adversely affect the animal. Because, however, blood ChE activity does not appear to take part in neurotransmission, inhibition of the circulating ChE activity is classified only as a marker of exposure, not as a definitive neurotoxic effect. From a more practical point of view, it is not known whether, after dosing animals with an antiChE compound, the inhibition of the circulating ChE activity accurately reflects ChE inhibition in 'target tissues' (US Environmental Pro- tection Agency, 1990). This is an important regulatory question because often ChE activity is measured in blood samples obtained from agricultural workers, pesticide handlers and applicators, and pesticide-dosed laboratory animals; in all of these cases, one would like to know whether this residual blood ChE activity reflects ChE inhibition in the more relevant, target tissues.

It is not clear from the literature whether the inhibition of circulating (i.e., blood) ChE mirrors ChE inhibition in the nervous system or other target tissues such as muscle. The relationship between blood ChE activity and other tissues has for the most part been gleaned from studies in which this relationship was not the focus of study. A reasonable correlation using the means of the groups, rather than individu- al animals, however, does seem to be apparent between blood cholinesterase inhibition and inhibition in the so called 'target' tissues in many of those studies (Miyamoto, 1969; Sue t al., 1971; Koshakji et al., 1973; Reiter et al., 1973; Anand et al., 1977; Pope and Chakraborti, 1992; Pope et al., 1991), while other reports, both in the clinical setting and the laboratory setting, question this correlation (Karnik et al., 1970; Endo et al., 1988; Jimmerson et al., 1989). In one of the more recent, detailed studies which doubt this correlation, Jimmerson and co-workers (Jimmer- son et al., 1989) analysed a limited range of ChE inhibition in rats administered Soman, a highly toxic and reactive non-pesticide organophosphate, at one time point and concluded that the correlation was not dependable. Conversely, some reliable correlations between the blood ChE activity and brain levels of ChE were reported recently by Pope and co-workers using animals dosed with environmentally-relevant

S. Padilla et al. / Toxicology 92 (1994) 11-25 13

compounds; however, their studies were limited either to multiple dosages at one time point (Pope and Chakraborti, 1992) or to one dose at multiple time points (Pope et al., 1991).

We designed the present study to investigate one commonly used pesticide, chlorpyrifos, in greater detail. Animals were given a single injection of one of three dosage levels of chlorpyrifos, and ChE activity was measured in several tissues at five time points after dosing. In addition, we analysed the blood-target tissue relationship using an acute dose of the active metabolite of another pesticide, parathion, at two dosage levels, during the time of peak inhibition and during recovery of activity. Thus, we compared the ChE inhibition profile produced by chlorpyrifos, an organophosphate that requires hepatic activation and produces an extremely long-lasting inhibition of ChE activity (recovery of activity does not begin for weeks), to the pat- tern of ChE inhibition produced by paraoxon, an organophosphate that does not require liver activation, and produces short-lived inhibition of ChE activity (recovery usually commences within hours). Our results show that, under certain conditions, blood ChE levels may be useful in predicting ChE inhibition in target tissues.

2. Materials and methods

2.1. Materials Chlorpyrifos (Dursban®; diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate),

99% pure, was obtained from Chem Service (West Chester, PA). Paraoxon (O,O'- diethyl-p-nitrophenyl phosphate) obtained from Sigma Chemical Co. (St. Louis, MO)(95% pure) was further purified by washing with bicarbonate (Johnson, 1977).

2.2. Animals and treatment Long-Evans male rats, 60-90 days old (Crl:(LE)BR, Charles River Laboratories,

Raleigh, NC) were singly housed on heat-treated pine bedding and allowed free ac- cess to water. For the chlorpyrifos experiment, food (Purina rat chow) was limited to maintain body weights at 350 ± 10 g. For the paraoxon experiment, the animals were approximately 70 days old and weighed between 325 and 350 g at the time of dosing. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee of the Health Effects Research Laboratory of the US Environmental Protection Agency.

Chlorpyrifos was dissolved in peanut oil at concentrations of 15, 30 or 62.5 mg/ml; the injection volume was 2 ml/kg; i.e. dosages were 30, 60 or 125 mg/kg. Each rat was injected subcutaneously with the appropriate solution between 9 and I 1:30 a.m. on Day 0. Paraoxon was dissolved in corn oil at concentrations of 0.17 or 0.34 mg/ml; the injection volume was 1 ml/kg; i.e., final dosage was 0.17 or 0.34 mg/kg. Each rat was injected subcutaneously with the appropriate solution between 9 and 11:00 a.m.

2.3. Tissue collection and handling In the chlorpyrifos experiment the animals were given mild methoxyflurane

14 S. Padilla et al. /Toxicology 92 (1994) 11-25

(Pittman-Moore, Mundelein, IL) anesthesia by inhalation and then killed by decapitation. Trunk blood was collected in heparinized microfuge tubes, an aliquot of whole blood was taken and the remainder separated into plasma and erythrocytes by centrifugation. The brain was dissected into the following areas: hypothalamus (a cube, approximately 2 mm on a side, was removed from the ventral surface of the brain between the optic chiasm and the mammillary bodies), striatum (caudate putamen, rostral to the optic chiasm), forebrain (the remaining cortical tissue rostral to the optic chiasm) and the hippocampus; all were dissected free-hand using Pax- inos and Watson (1982) as a guide.

In the paraoxon experiment, the animals were deeply anesthetized with pentobar- bital, the abdominal cavity opened, and blood from the dorsal aorta was collected in heparinized tubes. An aliquot of whole blood was taken and the remainder cen- trifuged to separate plasma from the erythrocytes. The brain tissue taken in this experiment was the entire cerebrum (i.e., everything rostral to the cerebellum).

The tissue samples were homogenized using a Polytron ® homogenizer (Brinkmann Instruments, Westbury, NY; power setting 6, for 15 s, on ice) in 0.t M Na phosphate buffer (pH 8). The samples were again diluted with 0.1 M phosphate buffer containing 1% Triton X-100 and frozen at -80°C. This final tissue homoge- nate (in at least 0.5% Triton) remained frozen until the ChE activity was determined. Final dilutions for the brain areas were typically 1:100, and 5-20 ~1 were used in the ChE assay; final dilution for the diaphragm tissue was 1:70 and 20 tA were used for the assay; final dilution for whole blood and erythrocytes was 1:25 and 5 tzl were used in the assay and 5 #1 of plasma were assayed undiluted.

2.4. Cholinesterase assays The ChE assay was essentially as described by Ellman and co-workers (Ellman et

al., 1961). This ChE assay was performed on each tissue sample in a single experiment (i.e., all times and dosages were assayed together, n = 100). The assay was adapted for use with a microtiter plate reader (ThermoMax Microtiter Plate Reader, Molecular Devices, Menlo Park, CA); total volume was 200 ~1. The samples were placed in a microtiter plate (approximately four plates/tissue group) in triplicate and brought up to 195 ~1 with 0.1 M Na phosphate buffer (pH 8) containing the chromogen dithiobisnitrobenzoic acid. Each plate was run with control and treated samples, substrate blank and tissue blanks. The plates were preincubated for t0 min; during the preincubation, the samples were shaken and warmed. After preincubation, the substrate, acetylthiocholine, was added (final concentration = 0.5 mM). The samples were read kinetically for 5 min, during which time, 31 separate absor- bance readings were taken for calculating the mean change in mOD/min. The temperature of the buffer and incubation chamber were adjusted so that the assay temperature was approximately 26°C. Protein levels were determined in the hypothalamus homogenates using the Bio-Rad DC protein assay kit (Bio-Rad, Rich- mond, CA, based on Lowry et al., 1951). ChE activity was calculated in nmol acetylthiocholine hydrolysed/min by dividing the readings from the assay which were in units of mOD/min by the slope of a glutathione standard curve (39.9 mOD/nmol SH).


2.5. Statistics All raw data for the ChE levels (i.e., nmol acetylthiocholine hydrolysed/min/vol

or mg protein or wet weight) were converted to percent of control activity using the mean of the control values from the corresponding tissue from the vehicle-treated controls at each time after dosing. This percent control activity is referred to as 'cholinesterase activity' or 'residual cholinesterase activity'. Least-squares linear regressions were then carried out across all dose groups to calculate the correlations (Pearson's r) between ChE activity in various tissues at each time point. The linear functions relating ChE activity in target tissue as a function of blood ChE activity were also calculated when appropriate.

3. Results

3.1. Chlorpyrifos experiment No overt signs of a cholinergic crisis (i.e., lacrimation, diarrhea, salivation,

tremor) as assessed by periodic observation were noted in the chlorpyrifos-treated animals.

The amount of ChE activity remaining after an acute dose of chlorpyrifos in the frontal cortex, plasma, whole blood and erythrocytes was clearly dose-dependent and changed with time after dosing (Fig. 1). The ChE activity in the other tissues (i.e., striatum, hippocampus, hypothalamus and diaphragm) exhibited the same general, qualitative trends as depicted here for frontal cortex: (1) at 1 day post- dosing, ChE activity in all three blood components was lower than the frontal cortex; (2) by 7 days after dosing, both the frontal cortex and blood components were maximally inhibited with the degree of inhibition dependent upon the chlorpyrifos dosage; (3) by 21 days after dosing, some recovery of ChE activity was usually apparent in both the frontal cortex and in two of the blood components (whole blood and plasma); (4) ChE activity in the plasma recovered most rapidly, while the erythrocyte ChE recovered most slowly.

If the residual ChE activity levels for each tissue are compared to the residual activity in the blood components in each animal across all doses two general relationships emerge (see Table 1). First, ChE activity in the CNS structures and the diaphragm was more tightly correlated with the activity in all the blood components at the time of maximal ChE inhibition, (i.e., from 7 to 21 days post dosing) than either before or after this period. Second, correlation coefficients were higher for the erythrocytes and whole blood than they were for plasma, especially at times when the activity levels were changing, i.e., 1-4 days or 35 days after dosing. In addition, correlations among the CNS structures (i.e., activity in the frontal cortex, striatum and hippocampus) were very high at all time points (data not shown). Because of these high intra-cerebrai correlations, those three CNS structures tended to follow the same trends with regard to correlation with blood components. In contrast, the correlation of the activity in the blood with the activity in the hypothalamus was very poor except at 7 days after dosing. This may be due to the lesser degree and more variable inhibition of hypothalamic ChE activity at each dose compared to the three other CNS structures (data not shown).

16 S. Padilla et al. / Toxicology 92 (1994) l l - 2 5

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S. Padilla et aL /Toxicology 92 (1994) 11-25 17

Table 1 Correlation of cholinesterase inhibition by tissue and time-after-dosing in animals treated with chlorpyrifos

Wholeblood Erythrocytes Plasma

1 Day after dosing Forebrain 0.68 *a 0.65* 0.58* Hippocampus 0.64" 0.71" 0.53 Striatum 0.77* 0.75* 0.58* Hypothalamus -0.21 0.06 -0.16 Diaphragm 0.35 0.39 0.02

4 Days after dosing Forebrain 0.85* 0.81 * 0.69* Hippocampus 0.82* 0.82* 0.70* Striatum 0.90* 0.82* 0.76* Hypothalamus 0.38 0.46 0.31 Diaphragm 0.61" 0.50 0.47

7 Days after dosing Forebrain 0.89* 0.89* 0.89* Hippocampus 0.90* 0.86* 0.81 * Striatum 0.87* 0.88* 0.81" Hypothalamus 0.87* 0.88* 0.77* Diaphragm 0.53 0.40 0.35

21 Days after dosing Forebrain 0.91' 0.84* 0.75* Hippocampus 0.90* 0.75* 0.72* Striatum 0.91" 0.88* 0.63" Hypothalamus 0.27 0.27 0.13 Diaphragm 0.85* 0.68* 0.58*

35 Days after dosing Forebrain 0.76* 0.67* 0.28 Hippocampus 0.75" 0.65" 0.39 Striatum 0.73* 0.71 * 0.23 Hypothalamus 0.52 0.79* 0.02 Diaphragm -0.09 0.27 -0.22

aCorrelation coefficients (r values) for the comparison of each blood component compared to each target tissue in each animal sampled at various times after chlorpyrifos administration. An r value more than or equal to 0.561 is considered significant at the 0.01 level.

Fig. 1. Time course of frontal cortical, plasma, erythrocyte, and whole blood cholinesterase activity after a single dose of chlorpyrifos. Animals received one injection (s.c.) of either 0, 30 (A), 60 (B) or 125 (C) mg/kg cblorpyrifos in corn oil. At the indicated times after dosing, the animals were sacrificed and cholinesterase activity was determined.


Residual Activity In Strlatum vs. Residual Activity in the Whole Blood

Residual Activity in the Diaphragm vs. Residual Activity in the Whole Blood

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Fig. 3. Cholinesterase activity at 4 or 24 h after a single, s.c. dose of paraoxon. All tissues except the erythrocytes were analysed (at 37°C) using the Ellman method (see Methods for details), whereas the radiometric method (Johnson and Russell, 1975) was used to analyse the erythrocyte ChE activity. The bar heights represent the actual activity per wet weight or volume; the numbers within the bars indicate the percentage inhibition.


Table 2 Correlation of cholinesterase inhibition by tissue and time-after-dosing in animals treated with paraoxon

Wholeblood Erythrocyte Plasma

4 Hours after dosing Diaphragm 0.75 *a 0.89* 0.74* Brain 0.82* 0.92* 0.81"

24 Hours after dosing Diaphragm 0.79* 0.80* 0.29 Brain 0.83* 0.85* 0.46

aCorrelation coefficients (r values) for the comparison of each blood component compared to each target tissue in each animal sampled at 4 and 24 hours after paraoxon administration. An r value more than or equal to 0.623 is considered significant at the 0.01 level.

S~'atterplots of ChE activity in whole blood versus striatum and diaphragm for each animal at 1, 21 and 35 days after dosing revealed several important points (Fig. 2). First, activity in the whole blood correlated better with the activity in the striatum than with the activity in the diaphragm. Second, as previously noted, the correlation of activity in both tissues was better at 21 days after dosing than at either 1 or 35 days after dosing. This dependence upon time after dosing resulted from the faster onset of, and recovery from, inhibition of whole blood ChE activity as compared to brain or muscle ChE activity (Fig. 1), and is evident in the steepening of the slope of the function relating whole blood ChE activity to striatal ChE activity across days (Fig. 2A-C). Third, the functions relating ChE activity in whole blood to those in the target tissues do not have a slope of 1 and an intercept of 0. Thus, residual ChE activity in the target tissues does not necessarily equal the residual ChE activity in the whole blood, even at the time of maximum correlation between the two.

3.2. P a r a o x o n e x p e r i m e n t

Four hours after administration of 0.34 mg/kg paraoxon, the animals showed a few cholinergic signs such as gnawing and tremor. There were no cholinergic signs in the low dosage group at 4 h and none in either dosage group by 24 h after dosing.

Paraoxon administration caused a dose-dependent inhibition of ChE activity in each tissue (Fig. 3). By 24 h after dosing the ChE activity, especially in the animals given 0.34 mg/kg paraoxon, had begun to recover. As with chlorpyrifos (Fig. 1), activity in plasma tended to be higher (i.e., less inhibited) than in the erythrocytes or whole blood (Fig. 3C).

Generally, the correlation of brain or diaphragm ChE activity with the residual activity in the blood components was stronger 4 h after dosing as compared to 24 h after dosing (Table 2). Moreover, as with chlorpyrifos, the correlation coefficients were weaker and less stable for the plasma: at 4 h the correlation coefficients were significant (Table 2), but by 24 h, at the time when the plasma activity had almost

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S. Padilla et al. / Toxicology 92 (1994) 11-25

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Fig. 4. Panel A shows the scattergram of the data for brain ChE activity compared to the erythrocyte ChE activity in paraoxon dosed animals at 4 h (r = 0.92; P ~ 0.01), whereas panel B shows the scattergram of the brain activity for each animal vs. its plasma activity at 24 h after paraoxon dosing (r = 0.46; P ~ 0.05). Note that the correlation is much higher in panel A, illustrating that one must be careful when choosing the time after dosing and the blood component for prediction of 'target tissue' cholinesterase activity.


returned to control values (Fig. 3B), the correlation coefficients decreased below sig- nificance.

Fig. 4 illustrates the point that it is important to choose carefully the blood component and time after dosing when attempting to draw conclusions about the degree of brain ChE activity using the activity levels in a blood component. The correlation between erythrocyte and brain ChE activity (Fig. 4A; r = 0.92; P _< 0.01) is extremely strong at 4 h after dosing, while there is no significant correlation between plasma and brain ChE activity 24 h after dosing (Fig. 4B; r = 0.46; P _> 0.05).

4. Discussion

These studies were undertaken to determine the utility of employing blood ChE data for estimating ChE inhibition in other, less accessible tissues. The studies were designed using two different, environmentally-relevant, inhibitors of ChE activity. The first, chlorpyrifos, an organophosphate insecticide activated by the liver, requires days to produce maximal depression of ChE activity in subcutaneously dosed animals (Fig. 1). The second, paraoxon, is the active metabolite of the insecticide parathion and does not require hepatic activation, and therefore maximal inhibition of ChE activity occurs just hours after dosing. Moreover, even though both are organophosphate compounds, paraoxon is approximately two orders of magnitude more potent than chlorpyrifos: chlorpyrifos oral LDs0 in rats is 1500 vs. 2.5 mg/kg for paraoxon (Eto, 1974). Despite these differences, some important similarities were evident in the neurochemical effects of these two compounds.

Because ChE activity in plasma decreased and recovered more rapidly than it did in the target tissues after dosing with either organophosphate, the activity of plasma ChE may be a poor predictor of activity in the target tissues, unless the comparison is made at the height of inhibition (see Tables 1 and 2). This propensity for rapid recovery of plasma ChE activity has been noted by many other investigators using a variety of ChE inhibitors in many different species (Yaksh et al., 1975; Fleming and Grue, 1981; Holmes and Boag, 1990; Pope et al., 1991). It is thought that the plasma compartment of activity regenerates quickly because it is continually resyn- thesized by the liver, whereas the turnover of the acetylcholinesterase tethered to the erythrocyte is dependent upon the life-time of the erythrocyte. The ChE activity in whole blood and in erythrocytes seems to track the activity in target tissues more accurately than does the plasma compartment (Figs. 1 and 3; see also Frawley et al., 1952). To a first approximation, the relationship between the erythrocyte or whole blood activity and activity in other tissues is linear at the time of maximal inhibition. The investigator must keep in mind that this correlation does not imply a one-to-one relationship, i.e., blood ChE residual activity does not equal numerically the residual activity in the other tissues. Nevertheless, a good correlation just signifies that there is a mathematically predictable relationship between the values, presenting a possibility that a conversion factor may be determined to calculate target tissue inhibition given the blood inhibition values. This conversion factor would likely take the form of the formula for the line which would be formed by plotting the % activity of the chosen blood component (x axis) versus the % activity of the chosen 'target' tissue (y axis).


Not only does the correspondence between blood component and target tissue seem to be dependent on the choice of the blood component for the correlation, but it also appears that some target tissues correlate with the blood components better than others as has been reported by other investigators (Jimmerson et al., 1989). Three of the brain regions, hippocampus, striatum and frontal cortex, were highly correlated with one another and also with the blood components at 7 and 21 days after dosing with chlorpyrifos. On the other hand, the hypothalamus and diaphragm ChE activity correlated less well with the blood components (see Table I). Even in the paraoxon experiment, the correlation between the brain and the blood components was always higher than the correlations between diaphragm and the blood components. These results are too preliminary to tell if these tissue-specific differences in correlation are due to choice of test compounds or to intrinsic differences in the tissues.

Because of the above caveats, we would recommend the following when attempting to predict ChE activity in target tissue using residual ChE activity in the blood:

(1) in most cases, it would probably be better to use whole blood or erythrocyte values for the correlation because plasma activity changes faster than that of the other tissues;

(2) the time of peak inhibition in the target tissues probably yields the highest correlation;

(3) to determine the actual numerical relationship between the blood component and the target tissues, blood and target ChE activity should be determined over a wide range of activity (i.e., dose-response study) during the period of maximal, stable inhibition. Then, in subsequent experiments, the values for the target tissues may be estimated from the formula for the line relating the blood values to the target tissues calculated in the first experiment.

These studies did not address the issue of the relationships among blood and target tissue ChE activity in situations of chronic or repeated exposure to ChE inhibitors. If it is generally true that the steady-state conditions of ChE activity enhance the correlations between ChE activity in the blood and target tissues, then predictable relationships between the endpoints ought to be obtained when repeated or long-term exposure produces stable levels of ChE inhibition.

Extrapolation to human field or clinical studies will require further research into the ability of rat data to predict the relationships between blood and target tissue ChE activity in human tissues. For instance, rat plasma ChE activity is a mixture of approximately 50% butyrylcholinesterase and 50% acetylcht nesterase activity (Traina and Serpietri, 1984, and unpublished data), whereas human plasma ChE activity is entirely butyrylcholinesterase activity, so the profile of plasma inhibition is likely to differ between the species. Certainly, it would be wise to exert caution at this attempt.

In summary, these studies show that a recognized and accessible biomarker of exposure, blood ChE activity, can, under certain circumstances, be used to predict ChE inhibition in target tissues such as brain and muscle.


Acknowledgements

The au tho r s w o u l d like to thank Dr. Carey Pope o f N o r t h e a s t Lou i s i ana Univer -

sity ( M o n r o e , LA) for his gift o f the p a r a o x o n ; Jack ie F a r m e r for dos ing the para-

oxon animals ; J o h n L e h m a n for co l lec t ing b l o o d samples f rom the p a r a o x o n - t r e a t e d

animals ; Lan T r a n and Jer ry Highfi l l for the i r s tat is t ical expert ise; and T o m

Heidersche i t and D o t Benne t t for their g raphica l expert ise. The research descr ibed

in this ar t icle has been rev iewed by the Hea l th Effects Resea rch L a b o r a t o r y , US

E n v i r o n m e n t a l P ro t ec t i on Agency , and a p p r o v e d for publ ica t ion . A p p r o v a l does

no t signify that the con ten t s necessar i ly ref lect the views and policies o f the Agency

no r does men t ion o f t rade names and c o m m e r c i a l p roduc t s cons t i tu te e n d o r s e m e n t

o r r e c o m m e n d a t i o n for use.

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studies on the correlation between blood cholinesterase inhibition and ‘target tissue’...

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