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Relationship between cholinesterase inhibition and thermoregulation following exposure to diisopropyl fluorophosphate in the rat
Toxicology Letters, 59 (1991) 161-168 0 1991 Elsevier Science Publishers B.V. 0378-4274/91/$ 3.50
161
TOXLET 02664
Relationship between cholinesterase inhibition and thermoregulation following exposure to diisopropyl fluorophosphate in the rat
Christopher J. Gordon’, Lela Fogelson’, Judy Richards2 and Jerry Highfill ‘Neurotoxicology
Division,
U.S. Environmental
‘Special Support Services Division, Health Effects Research Laboratory,
Protection
Agency,
Research
Triangle Park, NC and ‘NSI Technology Services, Inc.,
Research Triangle Park, NC (U.S.A.)
(Received 6 May 1991) (Accepted 13 September 1991) Key worcis: Hypothermia;
Hyperthermia; Colonic temperature; Skin temperature; Acetylcholinesterase
SUMMARY This study examined the relationship between inhibition of cholinesterase activity (CA) and thermoregulatory response in the rat following exposure to the organophosphate (OP), diisopropyl fluorophosphate (DFP). Male Long-Evans rats were injected with DFP dissolved in peanut oil in doses ranging from 0 to 1.5 mg/kg (s.c.). Colonic (T,,,) and tail skin temperature (T,,,) were recorded at 0, 1, 2 and 3 h post-injection, At 3 h post-injection the rat was sacrificed and a blood sample was taken by cardiac puncture and analyzed for CA. There was a biphasic dose effect of DFP on T,,, with slight but significant elevation in T,,, in the dose range of 0.0145 mg/kg and a significant depression in T,,, at doses of 1.Oand 1.5 mg/kg. There was a dose-dependent fall in CA with DFP administration in the erythrocyte, plasma, and whole blood fractions. Hypothermia was associated with 80-87% inhibition in CA, whereas the elevation in T,,, was associated with 2&70% inhibition in CA. DFP also elicited significant elevations in Tti,. Overall, the data fail to demonstrate any clear relationship between inhibition of blood CA and thermoregulatory response following exposure to DFP. However, the elevation in T,, following relatively low doses of DFP may be of relevance to the frequently reported symptom of fever in humans exposed to OP agents.
This paper has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and accepted for publication. Mention of trade names does not constitute endorsement or recommendation for use. Address for correspondence:
NC 27711, U.S.A.
Dr. C.J. Gordon, MD-74B, NTD/HERL, U.S. EPA, Research Triangle Park,
162
INTRODUCTION
The inhibition of acetylcholinesterase (AChE) and the subsequent accumulation of acetylcholine at synaptic junctions is considered to be the primary mechanism of toxicity in laboratory rodents and other species exposed to organophosphate (OP)and carbamate-based pesticides. Clinical and experimental animal studies rely heavily on the degree of inhibition of AChE activity in the erythrocyte and plasma fractions of whole blood and in the central nervous system as an indication of exposure to OPs and carbamates [l-4]. However, the degree of correlation between AChE inhibition in blood and the development of other toxic effects is not clear. Russell et al. [5] concluded that the critical level of AChE activity was 4&45% of normal. Autonomic and behavioral dysfunction in clinical and experimental studies is generally not observed until AChE activity in the blood, brain, and other sites is approximately less than 50% of the control levels [ 1,4,5]. A reduction in body temperature of the laboratory rodent is a commonly reported effect following exposure to a variety of OPs and carbamate agents [1,7,8]. Peripheral vasodilation and the subsequent increase in heat loss appear to be a primary mechanism to explain the hypothermia in rat following acute exposure to OPs [8]. However, our laboratory has recently found that thermoregulatory effecters other than peripheral vasomotor tone may be operative during exposure to DFP [9]. There is little information on the relationship between the OP-induced hypothermia and the degree of AChE inhibition following exposure to a wide dose range of an OP. Hence, the purpose of this study was to correlate the changes in skin and core temperature as a function of inhibition of AChE activity in blood of the rat exposed to varying doses of the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate (DFP). MATERIALS AND METHODS
Animals used in this study were 70 adult, male rats of the Long-Evans strain obtained from Charles River Laboratories (Raleigh, NC). The rats were housed in groups of 2-3 in cages lined with wood shavings and were maintained at an ~bient temperature of 22°C with a 50% relative humidity and a 12:12 L:D cycle. Average body mass of the rats was 347.8 -t 4 (SE) g. At weekly intervals DFP (Sigma) was diluted in peanut oil to dosages of 0, 0.01, 0.05, 0.12, 0.25, 0.5, 1.0 and 1.5 mglkg. The volume of the injection (s.c.) was 0.1 ml/100 g body wt. The DFP solutions were kept at 4°C. The rats were brought to the laboratory and kept in their home cages. The animals were allowed at least 60 min of adjustment to the laboratory conditions before testing. Baseline colonic (TcOJ and tail skin temperatures (T,,ii) were taken prior to DFP administration. Tco, was measured by inserting a thermocouple probe (Physitemp, RET-3) 5 cm past the anal sphincter. Ttail was measured by securing a 26 ga. thermocouple lightly to the tail with a small piece of gum rubber tubing. The ther-
163
mocouple was held in place for approximately 30 s until a stable Ttailreading could be made. After the baseline data were taken the rats were injected with one of the aforementioned doses of DFP and were then returned to their home cage. TcOiand T,, data were taken at 1,2 and 3 h post-injection. Following the last data collection period the rats were asphyxiated with CO2 and 34 ml of blood was quickly withdrawn by cardiac puncture. The blood was collected in an EGTA-coated vacutainer tube. Cholinesterase levels in the erythrocyte, plasma, and whole blood fractions were determined sp~trophotometrically using a commercially prepared kit (Boehringer Mannheim Diagnostics, Indianapolis, IN; No. 124117). Multivariate repeated measured tests were done for each DFP dose group for Tcol and Ttail taken at 0, I,2 and 3 h post-injection. Ranks were substituted for measured temperature data when the variance of a dose group was not homogeneous. Dunnett’s test was used to compare control levels with each dose group if the univariate test was significant. RESULTS
There were significant dose by time interactions for both TcO,(P < 0.001) and Ttai, (P < 0.004). T_, was initially elevated when first measured and then gradually decreased over the 3-h post-injection period. There was a biphasic effect of DFP on TcO,relative to the control animals (Fig. 1). At doses of 0.01-0.5 mg/kg a significant elevation in Tco, was observed at 1 and/or 2 h post-injection, whereas a significant decrease in TeO,was observed at 3 h post-injection in rats given 1.Oand 1.5 m&kg. T,,, was significantly elevated at 2 h post-injection in rats given 0.01,0.05 and 1.5 mg/kg, otherwise there were no significant effects of DFP on this parameter (Fig. 2). Cholinesterase activity (CA) in the plasma and erythrocyte fractions was significantly affected by all doses of DFP (Table I). CA of the plasma and erythrocyte fractions was significantly inhibited even at the lowest dose of DFP studied (Table I). Using the CA of the erythrocyte fraction as an independent variable and mean T,,, over the 3-h post-DFP treatment period as the dependent variable, a clear bimodal effect of inhibition in CA and thermoregulation is apparent (F = 7.6, P < 0.001; Fig. 3). The peak rise in TcOiwas associated with a CA that is 47.1% of control, whereas the peak decrease in TcDlwas associated with a CA that is 19.3% of control. Note that decrease in Tco, at the highest inhibition of CA was not statistically significant in this analysis. This is due to the averaging of T,, over the 3-h period; there was significant hypothe~ia at 3 but not 1 and 2 h post-inj~tion in this treatment group (cf. Fig. 1). DISCUSSION
The apparent biphasic dose-response relationship between DFP dose and T,,, found in the present study further clouds the relationship between cholinesterase inhibition and the~ore~lation following OP exposure. The lower range of doses of
164 38.8 .
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Time,hour Fig. 1. Effect of DFP dose on the colonic t~~mture (T& of tbe Long-Evans rat at 0, 1, 2 and 3 h post-injection. Each panel represents the response of rats to a given dose of DFP compared with the rats given peanut oil (controls). Data plotted as mean f SE; n = 10 per group. Asterisks indicate significant difference between treated and control group at a given time point.
DFP used in this study (0.0145 mglkg) resulted in shght, albeit significant elevations in T-r above the controls while the higher doses of 1.O and 1.5 m&g led to hypothermia. The DFP-induced hypothermia does not occur until CA was inhibited by at least 81% relative to the control animals. On the other hand, DFP-induced elevations in Tcol and Ttail occurred when CA was inhibited by as little as 16%.
165 28.0..
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Time,hour Fig. 2. Effect of DFP dose on the tail skin temperature (T,,,) of the Long-Evans rat at 0, 1, 2 and 3 h post-injection. Abbreviations same as in Fig. 1.
Because an increase in blood flow to the tail is considered to be a primary mechanism of DFP-induced hypothermia in the rat [8], it was decided to measure Ttairin the present study. A sudden rise in Ttai, at a constant ambient temperature is indicative of an increase in blood flow and, hence, an elevation in heat loss [lo]. However, in this study the measure of Ttailwas erratic at times with either slight increases or no change following DFP administration. Ttailcan change rapidly in response to stress and other environmental perturbations and thus may not be an accurate index of OP exposure unless the experimental conditions are more rigorously controlled. The significant
166 38.25
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.
.
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1 30
I 40
0 50
8 60
0 70
8
80
u
90
1 100
110
RBC CHOLlNESTEFiASE ACTM-W, % OF CONTROL
Fig. 3. Relationship between percent inhibition of RBC cholinesterase activity relative to control and mean colonic temperature (T,,,) of the rat recorded over the 3 h post-injection of DFP. Blood sample was taken immediately after the determination of T,, at 3 h post-injection. Asterisks indicate significant difference of T,,, from controls using Tukey’s t-test.
elevation in Ttail noted in rats given 1.5 mg/kg DFP supports the reported mechanism of DFP-mediated hypothermia in the rat [8]. On the other hand, the significant elevations in T,,, in rats given 0.01 and 0.05 mg/kg indicate the presence of thermal stress and the possible attempt to increase heat dissipation. The statistically significant Ttail responses were so slight that a definitive conclusion on their biological significance is not warranted at this time. Taken together, the experimental evidence of the present and past studies strongly suggests a biphasic dose effect of AChE inhibition on temperature regulation in the
TABLE I CHOLINESTERASE ACTIVITY (CA) OF THE ERYTHROCYTE, PLASMA, AND WHOLE BLOOD OF THE LONG-EVANS RAT MEASURED AT 3 h POST-INJECTION OF DFP AT DOSES RANGING FROM 0 TO 1.5 mg/kg Dose (mg/kg)
0
0.01 0.05 0.12 0.25 0.50 1.0 1.5
Cholinesterase activity (mu/ml) Erythrocytes
Plasma
482.9 f 31 (100) 386.6 ?r 25 (80.1) 358.9 + 12 (74.3) 275.1 f 17 (57) 214.3 f 17 (44.4) 146.6 + 7 (30.4) 100.1 + 6 (20.7) 60.1 f 3 (12.4)
455.2 f 360.8 + 327.7 f 247.8 ? 181.5 f 117.8 f 65.1 f 38.3 f
31 (100) 25 (79.3) 13 (72) 17 (54.4) 16 (39.9) 6 (25.9) 7 (14.3) 3 (8.4)
n = 10 per dose group. Significant CA inhibition occurred at all DFP doses (P < 0.05). Data plotted as mean f SE. Numbers in parentheses indicate inhibition of CA as percent of control.
167
rat. Studies with laboratory rodents have repeatedly demonstrated the hypothermic effect of acute administration of OP and carbamates (cf. Introduction). The present study found that DFP at doses of 1.O and 1.5 mgkg resulted in a significant decrease in body temperature; however, there is little evidence in the literature of an elevation in body temperature following exposure to relatively low doses of DFP. That DFP and other OPs led to hypothermia in the rat was generally explained by the mechanism that the accumulation of acetylcholine in the CNS activated heat-loss pathways resulted in the lowering of body temperature [8]. This hypothesis was supported by the frequent observation that direct administration of cholinergic agonists into the anterior hypothalamic area led to autonomic and behavioral thermoregulatory heatloss reponses [ 1l-l 31. Interestingly, fever is an often-reported symptom in humans acutely exposed to OPs and carbamates [4,14]. The fever is occasionally observed at least 24 h after exposure. The study by Namba et al. [4] suggested that the occurrence of fever following OP poisoning was associated with ‘moderate’ to ‘severe’ inhibition of AChE (i.e., 80-100% inhibition). It is premature to suggest any relationship between the DFP-induced elevation in TcO,found in this study and the reports of fever in OP-exposed humans. The elevation in temperature in the present study could be attributed to a slower recovery of Tcolto baseline following the injection of DFP. The temperature elevations were relatively small (5 0.5”C) and cannot be considered hazardous; however, the OP-induced hyperthermia could represent an index of thermoregulatory response to low levels of AChE inhibition. Temperature elevations at low degrees of AChE inhibition and temperature depressions at high degrees of AChE inhibition may be explained by one proposed mechanism of cholinergic control of thermoregulation in the rat and other mammals [ 15,161; namely, the frequently observed hypothermia following cholinergic stimulation, either through massive inhibition of AChE or through administration of cholinomimetic agents, may be a result of saturation of cholinergic CNS receptors and a subsequent impairment of neural transmission. Administering relatively small quantities of cholinomimetics in the CNS of the rat causes hyperthermia [ 17,181. Also, the divergent thermoregulatory effects of cholinergic stimulation may also be attributed to the degree of stimulation of muscarinic and nicotinic receptors in the CNS [ 161.Clearly, it is important in future studies of OPs to evaluate the effect of relatively low doses of the agents on thermoregulatory tone. REFERENCES 1 Coudray-Lucas, C., Prioux-Guyonneau, M., Tassel, A., Coq, H.M. and Wepierre, J. (1981) Influence of intoxication by anticholinesterase agents on core temperature in rats: relationships between hypothermia and acetylcholinesterase inhibition in different brain areas. Acta Pharmacol. Toxicol. 49, 215-222. 2 Harris, L.W., Anderson, D.R., Lennox, W.J. and Solana, R.P. (1989) Effects of subacute administration of physostigmine on blood acetylcholinesterase activity, motor performance, and soman intoxication. Toxicol. Appl. Pharmacol. 97, 267-271.
168 3 Ahdaya, SM., Shaw, P.V. and Guthrie, F.E. (1976) Th~o~~ation in mice treated with parathion, carbaryl, or DDT. Toxic&. Appt. Pharmacol. 35575-580. 4 Namba, T., Nolte, C.T., Jackrel, J. and Grob, D. (1971) Poisoning due to organophosphate insecticides. Am. J. Med. 50,475-492. 5 Russell, R.W., Booth, R.A., Lauretz, SD., Smith, CA. and Jenden, D.J. (1986) Behavioral, neurochemical and physiological effects of repeated exposures to subsymptomatic levels of the antichoiinesterase, soman. Neurobehav. Toxicol. Teratog. 8675-685. 6 Maickel, R.P., Kinney, D.R., Ryker, N.D. and Nichols, M.B. (1990) Effects of environments temperature on hypothermia and neuroendocrine changes induced by soman. Fund. Appl. Toxicol. 14,696 705. 7 Meeter, E. and Wolthuis, O.L. (1968) The effects of cholinesterase inhibitors on the body temperature of the rat. Eur. J. Pharmacol. 4, 18-24. 8 Meeter, E. (1969) The mode of action of cholinesterase inhibitors on the temperature regulation of the rat. Arch. Int. Pha~a~~~. 182,416-419. 9 Gordon, C.J., Fogelson, L., Lee, L. and Highfill, J. (1991) Acute effects of diisopropyl fluorophosphate (DFP) on autonomic and behavioral thermoregulatory responses in the Long-Evans rat. Toxicology 67, l-14. 10 Gordon, C.J. (1990) Thermal biology of the laboratory rat. Physiol. Behav. 47,963-991. 11 Crawshaw, L.I. (1973) Effect of intracranial acetylcholine injection on thermoregulatory responses in the rat. J. Comp. Physiol. Psychol. 83,32-35. 12 Lin, M.T., Chen, F.F., Chern, Y.F. and Fung, T.C. (1979) The role of the cholinergic systems in the central control of thermoregulation in rats. Can. J. Physiol. Pharmacol. 57, 1205-1212. 13 Lomax, P. and Jenden, D.J. (1966) Hypothermia following systematic and intracerebral injection of oxotremorine in the rat. Int. J. Neuropharmacol. 5, 353-359. 14 Hirshberg, A. and Lerman, Y. (1984) Clinical problems in organophosphate insecticide poisoning: the use of a computerized information system. Fund. Appl. Toxicol. 4, S209-S214. 15 Myers, R.D. (1987) Cholinergic systems in the central control of body temperature. In: N.J. Dun and R.L. Perhnan (Ed%), Neurobiology of Acetylcholine. Plenum Publishing Corp., New York, pp. 391402. 16 Myers, R.D. and Lee, T.F. (1989) Neurochemical aspects of thermoregulation. In: L.C.H. Wang (Ed.), Advances in Comparative Environmental Physiology, Springer-Verlag, Berlin, pp. 161-203. 17 Myers, R.D. and Yaksh, T.L. (1968) Feeding and temperature responses in the unrestrained rat after injections of cholinergic and amine@ substances into the cerebral ventricles. Physiol. Behav. 3,917928. 18 Cox, B. and Lomax, P. (1977) Pharmacologic control of temperature regulation. Annu. Rev. Pharmacol. Toxicol. 17, 341-353.
161
TOXLET 02664
Relationship between cholinesterase inhibition and thermoregulation following exposure to diisopropyl fluorophosphate in the rat
Christopher J. Gordon’, Lela Fogelson’, Judy Richards2 and Jerry Highfill ‘Neurotoxicology
Division,
U.S. Environmental
‘Special Support Services Division, Health Effects Research Laboratory,
Protection
Agency,
Research
Triangle Park, NC and ‘NSI Technology Services, Inc.,
Research Triangle Park, NC (U.S.A.)
(Received 6 May 1991) (Accepted 13 September 1991) Key worcis: Hypothermia;
Hyperthermia; Colonic temperature; Skin temperature; Acetylcholinesterase
SUMMARY This study examined the relationship between inhibition of cholinesterase activity (CA) and thermoregulatory response in the rat following exposure to the organophosphate (OP), diisopropyl fluorophosphate (DFP). Male Long-Evans rats were injected with DFP dissolved in peanut oil in doses ranging from 0 to 1.5 mg/kg (s.c.). Colonic (T,,,) and tail skin temperature (T,,,) were recorded at 0, 1, 2 and 3 h post-injection, At 3 h post-injection the rat was sacrificed and a blood sample was taken by cardiac puncture and analyzed for CA. There was a biphasic dose effect of DFP on T,,, with slight but significant elevation in T,,, in the dose range of 0.0145 mg/kg and a significant depression in T,,, at doses of 1.Oand 1.5 mg/kg. There was a dose-dependent fall in CA with DFP administration in the erythrocyte, plasma, and whole blood fractions. Hypothermia was associated with 80-87% inhibition in CA, whereas the elevation in T,,, was associated with 2&70% inhibition in CA. DFP also elicited significant elevations in Tti,. Overall, the data fail to demonstrate any clear relationship between inhibition of blood CA and thermoregulatory response following exposure to DFP. However, the elevation in T,, following relatively low doses of DFP may be of relevance to the frequently reported symptom of fever in humans exposed to OP agents.
This paper has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and accepted for publication. Mention of trade names does not constitute endorsement or recommendation for use. Address for correspondence:
NC 27711, U.S.A.
Dr. C.J. Gordon, MD-74B, NTD/HERL, U.S. EPA, Research Triangle Park,
162
INTRODUCTION
The inhibition of acetylcholinesterase (AChE) and the subsequent accumulation of acetylcholine at synaptic junctions is considered to be the primary mechanism of toxicity in laboratory rodents and other species exposed to organophosphate (OP)and carbamate-based pesticides. Clinical and experimental animal studies rely heavily on the degree of inhibition of AChE activity in the erythrocyte and plasma fractions of whole blood and in the central nervous system as an indication of exposure to OPs and carbamates [l-4]. However, the degree of correlation between AChE inhibition in blood and the development of other toxic effects is not clear. Russell et al. [5] concluded that the critical level of AChE activity was 4&45% of normal. Autonomic and behavioral dysfunction in clinical and experimental studies is generally not observed until AChE activity in the blood, brain, and other sites is approximately less than 50% of the control levels [ 1,4,5]. A reduction in body temperature of the laboratory rodent is a commonly reported effect following exposure to a variety of OPs and carbamate agents [1,7,8]. Peripheral vasodilation and the subsequent increase in heat loss appear to be a primary mechanism to explain the hypothermia in rat following acute exposure to OPs [8]. However, our laboratory has recently found that thermoregulatory effecters other than peripheral vasomotor tone may be operative during exposure to DFP [9]. There is little information on the relationship between the OP-induced hypothermia and the degree of AChE inhibition following exposure to a wide dose range of an OP. Hence, the purpose of this study was to correlate the changes in skin and core temperature as a function of inhibition of AChE activity in blood of the rat exposed to varying doses of the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate (DFP). MATERIALS AND METHODS
Animals used in this study were 70 adult, male rats of the Long-Evans strain obtained from Charles River Laboratories (Raleigh, NC). The rats were housed in groups of 2-3 in cages lined with wood shavings and were maintained at an ~bient temperature of 22°C with a 50% relative humidity and a 12:12 L:D cycle. Average body mass of the rats was 347.8 -t 4 (SE) g. At weekly intervals DFP (Sigma) was diluted in peanut oil to dosages of 0, 0.01, 0.05, 0.12, 0.25, 0.5, 1.0 and 1.5 mglkg. The volume of the injection (s.c.) was 0.1 ml/100 g body wt. The DFP solutions were kept at 4°C. The rats were brought to the laboratory and kept in their home cages. The animals were allowed at least 60 min of adjustment to the laboratory conditions before testing. Baseline colonic (TcOJ and tail skin temperatures (T,,ii) were taken prior to DFP administration. Tco, was measured by inserting a thermocouple probe (Physitemp, RET-3) 5 cm past the anal sphincter. Ttail was measured by securing a 26 ga. thermocouple lightly to the tail with a small piece of gum rubber tubing. The ther-
163
mocouple was held in place for approximately 30 s until a stable Ttailreading could be made. After the baseline data were taken the rats were injected with one of the aforementioned doses of DFP and were then returned to their home cage. TcOiand T,, data were taken at 1,2 and 3 h post-injection. Following the last data collection period the rats were asphyxiated with CO2 and 34 ml of blood was quickly withdrawn by cardiac puncture. The blood was collected in an EGTA-coated vacutainer tube. Cholinesterase levels in the erythrocyte, plasma, and whole blood fractions were determined sp~trophotometrically using a commercially prepared kit (Boehringer Mannheim Diagnostics, Indianapolis, IN; No. 124117). Multivariate repeated measured tests were done for each DFP dose group for Tcol and Ttail taken at 0, I,2 and 3 h post-injection. Ranks were substituted for measured temperature data when the variance of a dose group was not homogeneous. Dunnett’s test was used to compare control levels with each dose group if the univariate test was significant. RESULTS
There were significant dose by time interactions for both TcO,(P < 0.001) and Ttai, (P < 0.004). T_, was initially elevated when first measured and then gradually decreased over the 3-h post-injection period. There was a biphasic effect of DFP on TcO,relative to the control animals (Fig. 1). At doses of 0.01-0.5 mg/kg a significant elevation in Tco, was observed at 1 and/or 2 h post-injection, whereas a significant decrease in TeO,was observed at 3 h post-injection in rats given 1.Oand 1.5 m&kg. T,,, was significantly elevated at 2 h post-injection in rats given 0.01,0.05 and 1.5 mg/kg, otherwise there were no significant effects of DFP on this parameter (Fig. 2). Cholinesterase activity (CA) in the plasma and erythrocyte fractions was significantly affected by all doses of DFP (Table I). CA of the plasma and erythrocyte fractions was significantly inhibited even at the lowest dose of DFP studied (Table I). Using the CA of the erythrocyte fraction as an independent variable and mean T,,, over the 3-h post-DFP treatment period as the dependent variable, a clear bimodal effect of inhibition in CA and thermoregulation is apparent (F = 7.6, P < 0.001; Fig. 3). The peak rise in TcOiwas associated with a CA that is 47.1% of control, whereas the peak decrease in TcDlwas associated with a CA that is 19.3% of control. Note that decrease in Tco, at the highest inhibition of CA was not statistically significant in this analysis. This is due to the averaging of T,, over the 3-h period; there was significant hypothe~ia at 3 but not 1 and 2 h post-inj~tion in this treatment group (cf. Fig. 1). DISCUSSION
The apparent biphasic dose-response relationship between DFP dose and T,,, found in the present study further clouds the relationship between cholinesterase inhibition and the~ore~lation following OP exposure. The lower range of doses of
164 38.8 .
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.
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0 36.6 36.2 37.8
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Time,hour Fig. 1. Effect of DFP dose on the colonic t~~mture (T& of tbe Long-Evans rat at 0, 1, 2 and 3 h post-injection. Each panel represents the response of rats to a given dose of DFP compared with the rats given peanut oil (controls). Data plotted as mean f SE; n = 10 per group. Asterisks indicate significant difference between treated and control group at a given time point.
DFP used in this study (0.0145 mglkg) resulted in shght, albeit significant elevations in T-r above the controls while the higher doses of 1.O and 1.5 m&g led to hypothermia. The DFP-induced hypothermia does not occur until CA was inhibited by at least 81% relative to the control animals. On the other hand, DFP-induced elevations in Tcol and Ttail occurred when CA was inhibited by as little as 16%.
165 28.0..
25.0 1 . 0
1 .o
I 3.0
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28.0..
-Ome/kQ --- .5mg/kQ
-0mQlkQ
..~________~_____---i..~~~~
::::;:pq
--Omg/ko
---.lPmg/kQ
28.0
0
1 :o
210
28.0
25.0 1 . 0
3:o -Omg/kQ --..25mg/kg
1.0
2.0
I 3.0
Time,hour Fig. 2. Effect of DFP dose on the tail skin temperature (T,,,) of the Long-Evans rat at 0, 1, 2 and 3 h post-injection. Abbreviations same as in Fig. 1.
Because an increase in blood flow to the tail is considered to be a primary mechanism of DFP-induced hypothermia in the rat [8], it was decided to measure Ttairin the present study. A sudden rise in Ttai, at a constant ambient temperature is indicative of an increase in blood flow and, hence, an elevation in heat loss [lo]. However, in this study the measure of Ttailwas erratic at times with either slight increases or no change following DFP administration. Ttailcan change rapidly in response to stress and other environmental perturbations and thus may not be an accurate index of OP exposure unless the experimental conditions are more rigorously controlled. The significant
166 38.25
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0
.
.
10
r 20
1 30
I 40
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8 60
0 70
8
80
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90
1 100
110
RBC CHOLlNESTEFiASE ACTM-W, % OF CONTROL
Fig. 3. Relationship between percent inhibition of RBC cholinesterase activity relative to control and mean colonic temperature (T,,,) of the rat recorded over the 3 h post-injection of DFP. Blood sample was taken immediately after the determination of T,, at 3 h post-injection. Asterisks indicate significant difference of T,,, from controls using Tukey’s t-test.
elevation in Ttail noted in rats given 1.5 mg/kg DFP supports the reported mechanism of DFP-mediated hypothermia in the rat [8]. On the other hand, the significant elevations in T,,, in rats given 0.01 and 0.05 mg/kg indicate the presence of thermal stress and the possible attempt to increase heat dissipation. The statistically significant Ttail responses were so slight that a definitive conclusion on their biological significance is not warranted at this time. Taken together, the experimental evidence of the present and past studies strongly suggests a biphasic dose effect of AChE inhibition on temperature regulation in the
TABLE I CHOLINESTERASE ACTIVITY (CA) OF THE ERYTHROCYTE, PLASMA, AND WHOLE BLOOD OF THE LONG-EVANS RAT MEASURED AT 3 h POST-INJECTION OF DFP AT DOSES RANGING FROM 0 TO 1.5 mg/kg Dose (mg/kg)
0
0.01 0.05 0.12 0.25 0.50 1.0 1.5
Cholinesterase activity (mu/ml) Erythrocytes
Plasma
482.9 f 31 (100) 386.6 ?r 25 (80.1) 358.9 + 12 (74.3) 275.1 f 17 (57) 214.3 f 17 (44.4) 146.6 + 7 (30.4) 100.1 + 6 (20.7) 60.1 f 3 (12.4)
455.2 f 360.8 + 327.7 f 247.8 ? 181.5 f 117.8 f 65.1 f 38.3 f
31 (100) 25 (79.3) 13 (72) 17 (54.4) 16 (39.9) 6 (25.9) 7 (14.3) 3 (8.4)
n = 10 per dose group. Significant CA inhibition occurred at all DFP doses (P < 0.05). Data plotted as mean f SE. Numbers in parentheses indicate inhibition of CA as percent of control.
167
rat. Studies with laboratory rodents have repeatedly demonstrated the hypothermic effect of acute administration of OP and carbamates (cf. Introduction). The present study found that DFP at doses of 1.O and 1.5 mgkg resulted in a significant decrease in body temperature; however, there is little evidence in the literature of an elevation in body temperature following exposure to relatively low doses of DFP. That DFP and other OPs led to hypothermia in the rat was generally explained by the mechanism that the accumulation of acetylcholine in the CNS activated heat-loss pathways resulted in the lowering of body temperature [8]. This hypothesis was supported by the frequent observation that direct administration of cholinergic agonists into the anterior hypothalamic area led to autonomic and behavioral thermoregulatory heatloss reponses [ 1l-l 31. Interestingly, fever is an often-reported symptom in humans acutely exposed to OPs and carbamates [4,14]. The fever is occasionally observed at least 24 h after exposure. The study by Namba et al. [4] suggested that the occurrence of fever following OP poisoning was associated with ‘moderate’ to ‘severe’ inhibition of AChE (i.e., 80-100% inhibition). It is premature to suggest any relationship between the DFP-induced elevation in TcO,found in this study and the reports of fever in OP-exposed humans. The elevation in temperature in the present study could be attributed to a slower recovery of Tcolto baseline following the injection of DFP. The temperature elevations were relatively small (5 0.5”C) and cannot be considered hazardous; however, the OP-induced hyperthermia could represent an index of thermoregulatory response to low levels of AChE inhibition. Temperature elevations at low degrees of AChE inhibition and temperature depressions at high degrees of AChE inhibition may be explained by one proposed mechanism of cholinergic control of thermoregulation in the rat and other mammals [ 15,161; namely, the frequently observed hypothermia following cholinergic stimulation, either through massive inhibition of AChE or through administration of cholinomimetic agents, may be a result of saturation of cholinergic CNS receptors and a subsequent impairment of neural transmission. Administering relatively small quantities of cholinomimetics in the CNS of the rat causes hyperthermia [ 17,181. Also, the divergent thermoregulatory effects of cholinergic stimulation may also be attributed to the degree of stimulation of muscarinic and nicotinic receptors in the CNS [ 161.Clearly, it is important in future studies of OPs to evaluate the effect of relatively low doses of the agents on thermoregulatory tone. REFERENCES 1 Coudray-Lucas, C., Prioux-Guyonneau, M., Tassel, A., Coq, H.M. and Wepierre, J. (1981) Influence of intoxication by anticholinesterase agents on core temperature in rats: relationships between hypothermia and acetylcholinesterase inhibition in different brain areas. Acta Pharmacol. Toxicol. 49, 215-222. 2 Harris, L.W., Anderson, D.R., Lennox, W.J. and Solana, R.P. (1989) Effects of subacute administration of physostigmine on blood acetylcholinesterase activity, motor performance, and soman intoxication. Toxicol. Appl. Pharmacol. 97, 267-271.
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