- Email: [email protected]
Tolerance to the hypothermic and hyperthermic effects of chlorpyrifos1
Toxicology 121 (1997) 215 – 221
Tolerance to the hypothermic and hyperthermic effects of chlorpyrifos1 Pamela Johnson Rowsey a,*, Christopher J. Gordon b a
The Uni6ersity of North Carolina at Chapel Hill, School of Nursing, Carrington Hall, CBc 7460, Chapel Hill, NC 27599 -7460, USA b Neurotoxicology Di6ision, National Health and En6ironmental Effects Research Laboratory, U.S. En6ironmental Protection Agency, Research Triangle Park, NC 27711, USA Received 28 January 1997; accepted 31 March 1997
Abstract Hypothermia is a commonly reported thermoregulatory response in rodents acutely exposed to ogranophosphates (OP); however, our laboratory has recently found a delayed hyperthermic response following the initial hypothermia when exposed to the OP, chlorpyrifos. It is well known that rodents display tolerance to OP-induced hypothermia but little is known about tolerance to OP-induced hyperthermia. Twenty female rats of the Long-Evans strain were made tolerant to chlorpyrifos by administering 0 or 10 mg/g chlorpyrifos by gavage daily for four days. Core temperature (Tc) and motor activity (MA) were monitored continuously by telemetry. Twenty-four hours after the fourth 10 mg/kg injection, the animals were administered a challenge dose of 25 mg/kg chlorpyrifos or corn oil while the telemetry data were monitored for the next 72 h. Non-tolerant rats displayed an initial hypothermic response with reduced MA followed by a delayed increase in Tc 24 h after exposure. The tolerant animals displayed a blunted hypothermic response with virtually no change in MA, but a delayed increase in Tc similar to that of non-tolerant animals. The hyperthermic response of the non-tolerant animals persisted for two days, whereas the tolerant animals recovered by the second day. The data indicate that tolerance to the hypothermic and hyperthermic effects of chlorpyrifos involve separate neurochemical pathways. © 1997 Elsevier Science Ireland Ltd. Keywords: Endotoxin; Fever; Organophosphate; Hyperthermia; Hypothermia; Tolerance
* Corresponding author. Tel: + 1 919 5413019; fax: 919541-4849; e-mail: [email protected] 1 This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
1. Introduction Although hypothermia is a commonly reported thermoregulatory response in rodents exposed to organophosphate (OPs)-based insecticides, our
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laboratory has recently found a delayed hyperthermic response following the initial hypothermia lasting approximately 72 h (Gordon, 1993; Gordon et al., 1997). The hyperthermic response of diisopropyl fluorophosphate (DFP) and chlorpyrifos is, in part, prostaglandin mediated just like the fever of infection because the delayed hyperthermic responses are blocked with sodium salicylate. It is important to note that fever, in some cases lasting for over a week, appears to be a predominant response in humans exposed acutely to OP pesticides (Namba et al., 1971; Hirshberg and Lerman, 1984). It is possible that the delayed hyperthermic response in the rat exposed to DFP and chlorpyrifos represents an important model to study the febrile response of humans exposed to OPs. It is well known that rodents display tolerance to the hypothermia and other acute sequelae of most organophosphates (OP). That is, with repeated injections of an OP, the hypothermic response is generally attenuated or completely blocked which implies that the animal has developed tolerance to the OP (Costa et al., 1982; Russell et al., 1986). Tolerance to OP-induced hypothermia is thought to result from a downregulation of type one muscarinic (M1) cholinergic receptors which appear to activate heat loss processes in rodents (Gordon, 1994). On the other hand, little is known regarding the possible tolerance to the hyperthermic effects of an OP. The hyperthermic effects are less studied but probably involve mechanisms other than stimulation of M1 receptors because: 1. the hyperthermic effects are blocked with sodium salicylate and 2. muscarinic stimulation is normally associated with hypothermic responses in rodents (for review, see Gordon, 1994). One approach to understanding the mechanism of action of OPs such as chlorpyrifos is to assess the pattern of tolerance to the hypothermic and hyperthermic response. For example, if a tolerant animal shows an attenuated hypothermic and hyperthermic response, then it would indicate that the mechanism of action of hyperthermia is similar to the mechanism of action of hypothermia. On the other hand, if hyperthermia persists in an
animal tolerant to the hypothermic effects of the OP, then this would indicate that there are separate pathways of action. The purpose of this study is to identify whether animals become tolerant to the hyperthermic effects of chlorpyrifos.
2. Material and methods Twenty female rats of the Long-Evans strain (Charles River Laboratories, Raleigh, NC) were received at 90 days of age. The animals were housed individually in acrylic cages lined with wood shavings at an ambient temperature of 22°C, 50% relative humidity, and 12:12 light/dark photoperiod (lights on at 06.00 h). All animals were implanted with battery-operated temperature-activity transmitters (Data Sciences International, St. Paul, MN). Details of the telemetry system have been published (Gordon, 1993). Briefly, each animal was anesthetized with sodium pentobarbital and an abdominal incision was made for the implantation of the transmitter into the peritoneal cavity. The abdominal muscle was sutured and the skin was closed with wound clips. Animals were allowed at least 7 days of recovery before testing. Core temperature and motor activity were monitored at 5 min intervals by receivers placed under the floor of each cage. Data were monitored on line as well as stored on computer for later analysis. After recording 24 h of baseline responses, animals were weighed and given either 0 or 10 mg/kg chlorpyrifos (Chemical Services, West Chester, PA) in a volume of 0.1 ml/100 g body weight in corn oil by gavage at 1030 h for four consecutive days and continuously monitored. Twenty-four hours after the fourth 10 mg/kg injection, the animals were administered a challenge dose of 25 mg/kg chlorpyrifos or corn oil (gavage, 0.1 ml/100 g) while the telemetry data were monitored for the next 72 h. Overall, there were four treatment groups: 4 days of corn oil and corn oil challenge (control/control); 4 days of 10 mg/kg chlorpyrifos and corn oil challenge (10/control); 4 days of corn oil and chlorpyrifos challenge (control/25); 4 days of 10 mg/kg chlorpyrifos and chlorpyrifos challenge (10/25).
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Fig. 1. Time course of core temperature and motor activity of female rats administered repeated injections of chlorpyrifos (10 mg/kg). L and D represent light and dark phases, respectively, over a four day test period; NS represents not significant. Repeated measures ANOVA: L1 (treatment, P =0.088; treatment-time, P B 0.0001), D1 (treatment, P= 0.03; treatment-time, P B0.0001), L2 (treatment, NS; treatment-time, P B 0.0001), D2 (treatment, NS; treatment-time, P =0.0015), L3 (treatment, NS; treatment-time, P B0.0001), D3 (treatment, NS; treatment-time, NS), L4 (treatment, NS; treatment-time, P=0.004), D4 (treatment, NS; treatmenttime, NS). Motor Activity was not affected by chlorpyrifos over the four day time course.
2.1. Statistical analysis
3. Results
Data collected at 5 min intervals were collapsed into hourly averages for graphing and statistics. Effects of chlorpyrifos treatment and treatmenttime interaction were analyzed using a two-way repeated measures analysis of variance (ANOVA) in the treated and non-treated groups (GB STAT, Silver Springs, MD).
3.1. Chlorpyrifos repeated injection Oral dosing with 10 mg/kg chlorpyrifos had significant effects on core temperature but not motor activity (Fig. 1). A typical hypothermic response to 10 mg/kg chlorpyrifos was observed on the first day of injection. The hypothermic
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response was characterized by a decrease in core temperature beginning 1 h after exposure with a gradual recovery over the next 12 h. However, on days 2 through 4, animals showed a significant attenuation in the hypothermic response to chlorpyrifos; by the fourth injection, core temperature decreased by just 0.2°C. A hypothermic response of chlorpyrifos persisted during the dark phase after the first injection (Fig. 1). The duration of hypothermia was much shorter following the 2nd, 3rd, and 4th injections. There were no significant treatment effects of chlorpyrifos on core temperature during the dark phases D2, D3 or D4. In view of the attenuated hypothermic response to 10 mg/kg chlorpyrifos by the 4th day, this group of animals was assumed to have developed tolerance to chlorpyrifos and here-in-after referred to as the tolerant group.
3.2. Chlorpyrifos challenge The 25 mg/kg challenge dose of chlorpyrifos led to a rapid drop in core temperature beginning 1 h after administration in both the tolerant and non-tolerant animals (Fig. 2A). However, the nontolerant group displayed a significantly greater hypothermic response (3°C)to the chlorpyrifos challenge when compared to the tolerant group (1.8°C) (Fig. 2A). Twenty-four hours after chlorpyrifos injection, there were significant elevations in the diurnal core temperature in both the tolerant and non-tolerant animals. Forty-eight hours after chlorpyrifos, the diurnal core temperature of the non-tolerant treated animals remained elevated while the tolerant animals showed nearly complete recovery (Fig. 2A). There was a marked reduction of motor activity in the non-tolerant group during the first night after the chlorpyrifos challenge (Fig. 2B). On the other hand, motor activity was unaffected by chlorpyrifos challenge in the tolerant animals. By the second night, the tolerant and non-tolerant animals resumed normal patterns of nocturnal activity.
3.3. Body weight Repeated administration of 10 mg/kg chlorpyrifos led to a significant fall in body weight for 4
days (Table 1). However, by the day of the chlorpyrifos challenge, their body weight had begun to show recovery. The animals administered corn oil for four days displayed a steady increase in body weight. Overall, control animals showed a 3.9-g increase in body weight by day four of injection with an overall increase of 3 g by day five. On the other hand, the chlorpyrifos-treated animals displayed a gradual decrease in body weight of 12.3 g by day four with an overall decrease of 8.3 g on the day of the chlorpyrifos challenge (day five).
4. Discussion Repeated daily administration of a relatively low dose of 10 mg/kg chlorpyrifos over a four day period appeared to induce a state of tolerance in the female Long-Evans rat by the time the 25 mg/kg challenge dose of chlorpyrifos was administered. Following exposure to the challenge dose, the tolerant rats displayed a blunted hypothermic response and their level of motor activity was maintained near that of the controls. Furthermore, by the time of the challenge dose, the body weight loss was starting to recover in the animals repeatedly exposed to 10 mg/kg chlorpyrifos, further suggesting that tolerance to this aspect of OP exposure was developing. On the other hand, the tolerant animal displayed a delayed hyperthermic response on the day after the challenge dose similar to that seen in the non-tolerant group. Overall, the hyperthermic response of the non-tolerant animals persisted for two days, whereas the tolerant animals recovered by the second day. In the tolerant group, the magnitude of the hypothermic response was attenuated but the duration of hypothermia was unaffected. Repeated administration of an OP has been shown to attenuate or completely block the hypothermic response to a challenge dose of the same OP (Overstreet et al., 1979; Russell et al., 1986). On the other hand, the results of the present study show that tolerance to the hyperthermic effects of chlorpyrifos apparently does not develop in this manner as tolerance to the hypothermic effects. This finding may not be unexpected considering that the acute hypothermic and delayed hyperthermic effects of an OP appear
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Fig. 2. (A) Time course of core temperature of female rats administered a chlorpyrifos (25 mg/kg) challenge. Abbreviations same as in Fig. 1. Repeated measures of ANOVA. Non-tolerant animals: L5 (treatment, PB0.001; treatment-time, P B0.0001), D5 (treatment, P= 0.003; treatment-time, P =0.01), L6 (treatment, P =0.013; treatment-time, P B0.0001), D6 (treatment, P= 0.049; treatment-time, NS), L7 (treatment, P =0.0009; treatment-time, P =0.03), D7 (treatment, P=0.015; treatment-time, NS). Tolerant animals: L5 (treatment, P B 0.0113; treatment-time, P B 0.0001), D5 (treatment, NS; treatment-time, NS), L6 (treatment, P= 0.0012; treatment-time, NS), D6 (treatment, NS; treatment-time, NS), L7 (treatment, NS; treatment-time, P \0.0001), D7 (treatment, NS; treatment-time, NS). (B) Time course of motor activity of female rats administered a chlorpyrifos (25 mg/kg) challenge. Abbreviations same as in Fig. 1. Non-tolerant animals: L5 (treatment, NS; treatment-time, NS), D5 (treatment, P =0.0131; treatment-time, NS), L6 –L7 (treatment, NS; treatment-time, NS), D6 – D7 (treatment, NS; treatment-time, NS). Tolerant animals: repeated measures ANOVA: NS.
to operate through separate neurochemical pathways. It appears that tolerance to the hypothermic effects of OPs occurs primarily because of down-regulation of muscarinic receptors which control heat loss thermoeffectors (Bushnell et al.,
1991; Ehlert et al., 1980; Gordon, 1994). The delayed hyperthermic response to OP exposure has only recently been elucidated in this laboratory. Contrary to the acute hypothermic response to OPs which is blocked with muscarinic antago-
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Fig. 2.
nists such as scopolamine and atropine, the delayed hyperthermia is inhibited by administration of sodium salicylate, a commonly used antipyretic (Gordon, 1996; Gordon et al., 1997). Tolerance to OPs is thought to represent a mechanism of adaptation allowing the animal to endure exposure to the OP without sustaining ill effects. Indeed, tolerance to the hypothermic effects of OPs occurs relatively quickly when compared to other autonomic and behavioral functions such as motor activity, food and water consump-
tion, growth, cognition, and others (Costa et al., 1982; Gordon, 1994). The present study suggests that another facet of the thermoregulatory response to OP exposure does not develop tolerance over the same time course as the hypothermia. It will be of interest to understand: 1. if tolerance can develop to the delayed hyperthermic effects of an OP and 2. how the time course of tolerance to the hyperthermic effects of chlorpyrifos compares to the tolerance of other responses as listed above.
P. Johnson Rowsey, C.J. Gordon / Toxicology 121 (1997) 215–221 Table 1 Effect of repeated injection of chlorpyrifos on body weight Day of injection
Non-tolerant (n=10)
Tolerant (n = 10)
1 2 3 4 5
263.90 9 3.95 266.409 4.10 268.80 9 4.07 267.809 4.50 266.909 4.67
267.50 94.80 260.3094.62 256.2094.53 255.2094.30 259.2094.80
Attenuation in the febrile response to bacterial endotoxin is generally seen after the second or third repeated administration (Soszynski et al., 1991). The results of the present study suggest that tolerance to the hyperthermic effects of the chlorpyrifos are not induced in such a manner. Because chlorpyrifos-induced hyperthermia and endotoxin-induced fever are both blocked by sodium salicylate, it was hypothesized that the former would also show a tolerance to repeated exposure. However, because the hyperthermic response persisted in tolerant animals, it would suggest that the chlorpyrifos-induced hyperthermia, although somewhat similar to typical fever, nonetheless involves a unique mechanism of action. Future work in this laboratory is planned to understand the distinction between bacterial endotoxin-mediated fever and delayed OP-mediated fever. Chlorpyrifos-induced hyperthermia could be a factor in explaining the tolerance to the hypothermic effects of repeated exposures to chlorpyrifos. That is, assuming that the hypothermic and hyperthermic effects of chlorpyrifos are mediated through separate neurochemical pathways, if the hyperthermic response is sensitized with repeated dosing, then a net reduction in the hypothermic effects of chlorpyrifos would be expected. This novel model of tolerance to OPs will be evaluated in future studies.
.
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Acknowledgements We thank Drs Virginia Neelon and Diane Miller for their helpful review of the manuscript. We also thank Dr Ying Yang for her technical assistance.
References Bushnell, P.J., Padilla, S., Ward, T., Pope, C.N. and Olszyk, V. (1991). Behavioral and neurochemical changes in rats dosed repeatedly with diisopropylfloourophosphate. J. Pharmacol. Exp. Ther. 256, 741 – 750. Costa, L.G., Schwab, B.W. and Murphy, S.D. (1982). Differential alterations of cholinergic muscarinic receptors during chronic and acute tolerance to organophosphorous insecticides. Biochem. Pharmacol. 31, 3407 – 3413. Ehlert, F.J., Kokka, N. and Fairhurst, A.S. (1980). Altered [3H]quinuclidinyl benzilate binding in the striatum of rats following chronic cholinesterase inhibition with diisopropylfluorophosphate. Mol. Pharmacol. 17, 24 – 30. Gordon, C.J. (1996). Pharmacological analysis of diisopropylfluorophoshate: effects on core temperature, heart rate, and motor activity in the unrestrained rat. Pharm. Biochem. Behav. 55, 185 – 194. Gordon, C.J., Grantham, T. and Yang, Y. (1997). Hypothermia and delayed fever in the male and female rat exposed to chlorpyrifos. Toxicol., in press. Gordon, C.J. (1994). Thermoregulation in laboratory mammals and humans exposed to antichlolinesterase agents. Neurotoxicol. Terat. 16, 427 – 453. Gordon, C.J. (1993). Temperature Regulation in Laboratory Rodents. Cambridge University, New York. 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. Namba, T., Nolte, C.T., Jackrel, J. and Grob, D. (1971). Poisoning due to organophosphate insecticides: acute and chronic manifestations. Am. J. Med. 50, 475 – 492. Overstreet, D.H., Russell, R.W., Helps, S.C., Runge, P., and Prescott, A.M. (1979). Sex differences following pharmacological manipulations of the cholinergic system by DFP and pilocarpine. Psychopharmacology 61, 49 – 58. Russell, R.W., Booth, R.A., Lauretz, S.D., Smith, C.A. and Jenden, D.H. (1986). Behavioral, neurochemical and physiological effects of repeated exposures to subsymptomatic levels of the anticholinesterase, soman. Neurobehav. Toxicol. Terat. 8, 675 – 685. Soszynski, D., Kozak, W. and Scewczenko, M. (1991). Course of fever response to repeated administration of sublethal doses of lipopolysaccharides, ployinosinic:polycytidylic acid and muramyl dipeptide to rabbits. Experientia 47, 43 – 47.
Tolerance to the hypothermic and hyperthermic effects of chlorpyrifos1 Pamela Johnson Rowsey a,*, Christopher J. Gordon b a
The Uni6ersity of North Carolina at Chapel Hill, School of Nursing, Carrington Hall, CBc 7460, Chapel Hill, NC 27599 -7460, USA b Neurotoxicology Di6ision, National Health and En6ironmental Effects Research Laboratory, U.S. En6ironmental Protection Agency, Research Triangle Park, NC 27711, USA Received 28 January 1997; accepted 31 March 1997
Abstract Hypothermia is a commonly reported thermoregulatory response in rodents acutely exposed to ogranophosphates (OP); however, our laboratory has recently found a delayed hyperthermic response following the initial hypothermia when exposed to the OP, chlorpyrifos. It is well known that rodents display tolerance to OP-induced hypothermia but little is known about tolerance to OP-induced hyperthermia. Twenty female rats of the Long-Evans strain were made tolerant to chlorpyrifos by administering 0 or 10 mg/g chlorpyrifos by gavage daily for four days. Core temperature (Tc) and motor activity (MA) were monitored continuously by telemetry. Twenty-four hours after the fourth 10 mg/kg injection, the animals were administered a challenge dose of 25 mg/kg chlorpyrifos or corn oil while the telemetry data were monitored for the next 72 h. Non-tolerant rats displayed an initial hypothermic response with reduced MA followed by a delayed increase in Tc 24 h after exposure. The tolerant animals displayed a blunted hypothermic response with virtually no change in MA, but a delayed increase in Tc similar to that of non-tolerant animals. The hyperthermic response of the non-tolerant animals persisted for two days, whereas the tolerant animals recovered by the second day. The data indicate that tolerance to the hypothermic and hyperthermic effects of chlorpyrifos involve separate neurochemical pathways. © 1997 Elsevier Science Ireland Ltd. Keywords: Endotoxin; Fever; Organophosphate; Hyperthermia; Hypothermia; Tolerance
* Corresponding author. Tel: + 1 919 5413019; fax: 919541-4849; e-mail: [email protected] 1 This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
1. Introduction Although hypothermia is a commonly reported thermoregulatory response in rodents exposed to organophosphate (OPs)-based insecticides, our
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laboratory has recently found a delayed hyperthermic response following the initial hypothermia lasting approximately 72 h (Gordon, 1993; Gordon et al., 1997). The hyperthermic response of diisopropyl fluorophosphate (DFP) and chlorpyrifos is, in part, prostaglandin mediated just like the fever of infection because the delayed hyperthermic responses are blocked with sodium salicylate. It is important to note that fever, in some cases lasting for over a week, appears to be a predominant response in humans exposed acutely to OP pesticides (Namba et al., 1971; Hirshberg and Lerman, 1984). It is possible that the delayed hyperthermic response in the rat exposed to DFP and chlorpyrifos represents an important model to study the febrile response of humans exposed to OPs. It is well known that rodents display tolerance to the hypothermia and other acute sequelae of most organophosphates (OP). That is, with repeated injections of an OP, the hypothermic response is generally attenuated or completely blocked which implies that the animal has developed tolerance to the OP (Costa et al., 1982; Russell et al., 1986). Tolerance to OP-induced hypothermia is thought to result from a downregulation of type one muscarinic (M1) cholinergic receptors which appear to activate heat loss processes in rodents (Gordon, 1994). On the other hand, little is known regarding the possible tolerance to the hyperthermic effects of an OP. The hyperthermic effects are less studied but probably involve mechanisms other than stimulation of M1 receptors because: 1. the hyperthermic effects are blocked with sodium salicylate and 2. muscarinic stimulation is normally associated with hypothermic responses in rodents (for review, see Gordon, 1994). One approach to understanding the mechanism of action of OPs such as chlorpyrifos is to assess the pattern of tolerance to the hypothermic and hyperthermic response. For example, if a tolerant animal shows an attenuated hypothermic and hyperthermic response, then it would indicate that the mechanism of action of hyperthermia is similar to the mechanism of action of hypothermia. On the other hand, if hyperthermia persists in an
animal tolerant to the hypothermic effects of the OP, then this would indicate that there are separate pathways of action. The purpose of this study is to identify whether animals become tolerant to the hyperthermic effects of chlorpyrifos.
2. Material and methods Twenty female rats of the Long-Evans strain (Charles River Laboratories, Raleigh, NC) were received at 90 days of age. The animals were housed individually in acrylic cages lined with wood shavings at an ambient temperature of 22°C, 50% relative humidity, and 12:12 light/dark photoperiod (lights on at 06.00 h). All animals were implanted with battery-operated temperature-activity transmitters (Data Sciences International, St. Paul, MN). Details of the telemetry system have been published (Gordon, 1993). Briefly, each animal was anesthetized with sodium pentobarbital and an abdominal incision was made for the implantation of the transmitter into the peritoneal cavity. The abdominal muscle was sutured and the skin was closed with wound clips. Animals were allowed at least 7 days of recovery before testing. Core temperature and motor activity were monitored at 5 min intervals by receivers placed under the floor of each cage. Data were monitored on line as well as stored on computer for later analysis. After recording 24 h of baseline responses, animals were weighed and given either 0 or 10 mg/kg chlorpyrifos (Chemical Services, West Chester, PA) in a volume of 0.1 ml/100 g body weight in corn oil by gavage at 1030 h for four consecutive days and continuously monitored. Twenty-four hours after the fourth 10 mg/kg injection, the animals were administered a challenge dose of 25 mg/kg chlorpyrifos or corn oil (gavage, 0.1 ml/100 g) while the telemetry data were monitored for the next 72 h. Overall, there were four treatment groups: 4 days of corn oil and corn oil challenge (control/control); 4 days of 10 mg/kg chlorpyrifos and corn oil challenge (10/control); 4 days of corn oil and chlorpyrifos challenge (control/25); 4 days of 10 mg/kg chlorpyrifos and chlorpyrifos challenge (10/25).
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Fig. 1. Time course of core temperature and motor activity of female rats administered repeated injections of chlorpyrifos (10 mg/kg). L and D represent light and dark phases, respectively, over a four day test period; NS represents not significant. Repeated measures ANOVA: L1 (treatment, P =0.088; treatment-time, P B 0.0001), D1 (treatment, P= 0.03; treatment-time, P B0.0001), L2 (treatment, NS; treatment-time, P B 0.0001), D2 (treatment, NS; treatment-time, P =0.0015), L3 (treatment, NS; treatment-time, P B0.0001), D3 (treatment, NS; treatment-time, NS), L4 (treatment, NS; treatment-time, P=0.004), D4 (treatment, NS; treatmenttime, NS). Motor Activity was not affected by chlorpyrifos over the four day time course.
2.1. Statistical analysis
3. Results
Data collected at 5 min intervals were collapsed into hourly averages for graphing and statistics. Effects of chlorpyrifos treatment and treatmenttime interaction were analyzed using a two-way repeated measures analysis of variance (ANOVA) in the treated and non-treated groups (GB STAT, Silver Springs, MD).
3.1. Chlorpyrifos repeated injection Oral dosing with 10 mg/kg chlorpyrifos had significant effects on core temperature but not motor activity (Fig. 1). A typical hypothermic response to 10 mg/kg chlorpyrifos was observed on the first day of injection. The hypothermic
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response was characterized by a decrease in core temperature beginning 1 h after exposure with a gradual recovery over the next 12 h. However, on days 2 through 4, animals showed a significant attenuation in the hypothermic response to chlorpyrifos; by the fourth injection, core temperature decreased by just 0.2°C. A hypothermic response of chlorpyrifos persisted during the dark phase after the first injection (Fig. 1). The duration of hypothermia was much shorter following the 2nd, 3rd, and 4th injections. There were no significant treatment effects of chlorpyrifos on core temperature during the dark phases D2, D3 or D4. In view of the attenuated hypothermic response to 10 mg/kg chlorpyrifos by the 4th day, this group of animals was assumed to have developed tolerance to chlorpyrifos and here-in-after referred to as the tolerant group.
3.2. Chlorpyrifos challenge The 25 mg/kg challenge dose of chlorpyrifos led to a rapid drop in core temperature beginning 1 h after administration in both the tolerant and non-tolerant animals (Fig. 2A). However, the nontolerant group displayed a significantly greater hypothermic response (3°C)to the chlorpyrifos challenge when compared to the tolerant group (1.8°C) (Fig. 2A). Twenty-four hours after chlorpyrifos injection, there were significant elevations in the diurnal core temperature in both the tolerant and non-tolerant animals. Forty-eight hours after chlorpyrifos, the diurnal core temperature of the non-tolerant treated animals remained elevated while the tolerant animals showed nearly complete recovery (Fig. 2A). There was a marked reduction of motor activity in the non-tolerant group during the first night after the chlorpyrifos challenge (Fig. 2B). On the other hand, motor activity was unaffected by chlorpyrifos challenge in the tolerant animals. By the second night, the tolerant and non-tolerant animals resumed normal patterns of nocturnal activity.
3.3. Body weight Repeated administration of 10 mg/kg chlorpyrifos led to a significant fall in body weight for 4
days (Table 1). However, by the day of the chlorpyrifos challenge, their body weight had begun to show recovery. The animals administered corn oil for four days displayed a steady increase in body weight. Overall, control animals showed a 3.9-g increase in body weight by day four of injection with an overall increase of 3 g by day five. On the other hand, the chlorpyrifos-treated animals displayed a gradual decrease in body weight of 12.3 g by day four with an overall decrease of 8.3 g on the day of the chlorpyrifos challenge (day five).
4. Discussion Repeated daily administration of a relatively low dose of 10 mg/kg chlorpyrifos over a four day period appeared to induce a state of tolerance in the female Long-Evans rat by the time the 25 mg/kg challenge dose of chlorpyrifos was administered. Following exposure to the challenge dose, the tolerant rats displayed a blunted hypothermic response and their level of motor activity was maintained near that of the controls. Furthermore, by the time of the challenge dose, the body weight loss was starting to recover in the animals repeatedly exposed to 10 mg/kg chlorpyrifos, further suggesting that tolerance to this aspect of OP exposure was developing. On the other hand, the tolerant animal displayed a delayed hyperthermic response on the day after the challenge dose similar to that seen in the non-tolerant group. Overall, the hyperthermic response of the non-tolerant animals persisted for two days, whereas the tolerant animals recovered by the second day. In the tolerant group, the magnitude of the hypothermic response was attenuated but the duration of hypothermia was unaffected. Repeated administration of an OP has been shown to attenuate or completely block the hypothermic response to a challenge dose of the same OP (Overstreet et al., 1979; Russell et al., 1986). On the other hand, the results of the present study show that tolerance to the hyperthermic effects of chlorpyrifos apparently does not develop in this manner as tolerance to the hypothermic effects. This finding may not be unexpected considering that the acute hypothermic and delayed hyperthermic effects of an OP appear
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Fig. 2. (A) Time course of core temperature of female rats administered a chlorpyrifos (25 mg/kg) challenge. Abbreviations same as in Fig. 1. Repeated measures of ANOVA. Non-tolerant animals: L5 (treatment, PB0.001; treatment-time, P B0.0001), D5 (treatment, P= 0.003; treatment-time, P =0.01), L6 (treatment, P =0.013; treatment-time, P B0.0001), D6 (treatment, P= 0.049; treatment-time, NS), L7 (treatment, P =0.0009; treatment-time, P =0.03), D7 (treatment, P=0.015; treatment-time, NS). Tolerant animals: L5 (treatment, P B 0.0113; treatment-time, P B 0.0001), D5 (treatment, NS; treatment-time, NS), L6 (treatment, P= 0.0012; treatment-time, NS), D6 (treatment, NS; treatment-time, NS), L7 (treatment, NS; treatment-time, P \0.0001), D7 (treatment, NS; treatment-time, NS). (B) Time course of motor activity of female rats administered a chlorpyrifos (25 mg/kg) challenge. Abbreviations same as in Fig. 1. Non-tolerant animals: L5 (treatment, NS; treatment-time, NS), D5 (treatment, P =0.0131; treatment-time, NS), L6 –L7 (treatment, NS; treatment-time, NS), D6 – D7 (treatment, NS; treatment-time, NS). Tolerant animals: repeated measures ANOVA: NS.
to operate through separate neurochemical pathways. It appears that tolerance to the hypothermic effects of OPs occurs primarily because of down-regulation of muscarinic receptors which control heat loss thermoeffectors (Bushnell et al.,
1991; Ehlert et al., 1980; Gordon, 1994). The delayed hyperthermic response to OP exposure has only recently been elucidated in this laboratory. Contrary to the acute hypothermic response to OPs which is blocked with muscarinic antago-
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Fig. 2.
nists such as scopolamine and atropine, the delayed hyperthermia is inhibited by administration of sodium salicylate, a commonly used antipyretic (Gordon, 1996; Gordon et al., 1997). Tolerance to OPs is thought to represent a mechanism of adaptation allowing the animal to endure exposure to the OP without sustaining ill effects. Indeed, tolerance to the hypothermic effects of OPs occurs relatively quickly when compared to other autonomic and behavioral functions such as motor activity, food and water consump-
tion, growth, cognition, and others (Costa et al., 1982; Gordon, 1994). The present study suggests that another facet of the thermoregulatory response to OP exposure does not develop tolerance over the same time course as the hypothermia. It will be of interest to understand: 1. if tolerance can develop to the delayed hyperthermic effects of an OP and 2. how the time course of tolerance to the hyperthermic effects of chlorpyrifos compares to the tolerance of other responses as listed above.
P. Johnson Rowsey, C.J. Gordon / Toxicology 121 (1997) 215–221 Table 1 Effect of repeated injection of chlorpyrifos on body weight Day of injection
Non-tolerant (n=10)
Tolerant (n = 10)
1 2 3 4 5
263.90 9 3.95 266.409 4.10 268.80 9 4.07 267.809 4.50 266.909 4.67
267.50 94.80 260.3094.62 256.2094.53 255.2094.30 259.2094.80
Attenuation in the febrile response to bacterial endotoxin is generally seen after the second or third repeated administration (Soszynski et al., 1991). The results of the present study suggest that tolerance to the hyperthermic effects of the chlorpyrifos are not induced in such a manner. Because chlorpyrifos-induced hyperthermia and endotoxin-induced fever are both blocked by sodium salicylate, it was hypothesized that the former would also show a tolerance to repeated exposure. However, because the hyperthermic response persisted in tolerant animals, it would suggest that the chlorpyrifos-induced hyperthermia, although somewhat similar to typical fever, nonetheless involves a unique mechanism of action. Future work in this laboratory is planned to understand the distinction between bacterial endotoxin-mediated fever and delayed OP-mediated fever. Chlorpyrifos-induced hyperthermia could be a factor in explaining the tolerance to the hypothermic effects of repeated exposures to chlorpyrifos. That is, assuming that the hypothermic and hyperthermic effects of chlorpyrifos are mediated through separate neurochemical pathways, if the hyperthermic response is sensitized with repeated dosing, then a net reduction in the hypothermic effects of chlorpyrifos would be expected. This novel model of tolerance to OPs will be evaluated in future studies.
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Acknowledgements We thank Drs Virginia Neelon and Diane Miller for their helpful review of the manuscript. We also thank Dr Ying Yang for her technical assistance.
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