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Prolonged elevation in blood pressure in the unrestrained rat exposed to chlorpyrifos
Toxicology 146 (2000) 1 – 13 www.elsevier.com/locate/toxicol
Prolonged elevation in blood pressure in the unrestrained rat exposed to chlorpyrifos Christopher J. Gordon *, Beth K. Padnos Neurotoxicology Di6ision, National Health and En6ironmental Effects Research Laboratory, MD-74B, U.S. En6ironmental Protection Agency, Research Triangle Park, NC 27711, USA Received 10 November 1999; accepted 6 January 2000
Abstract Organophosphate (OP) pesticides are likely to alter the regulation of blood pressure (BP) because (i) BP control centers in the brain stem utilize cholinergic synapses and (ii) the irreversible inhibition of acetylcholinesterase activity by OP’s causes cholinergic stimulation in the CNS. This study used radiotelemetric techniques to monitor systolic (S), diastolic (D), mean (M) BP, pulse pressure (systolic–diastolic), heart rate (HR), core temperature (Tc), and motor activity in male Long–Evans rats treated with the OP pesticide chlorpyrifos (CHP) at doses of 0, 5, 10, and 25 mg/kg (p.o.) at 15:00 h 10 and 25 mg/kg CHP led to parallel elevations in S-BP, M-BP, and D-BP within 2 h after dosing. BP increased 15–20 mmHg above controls and increases persisted throughout the night and into the next day. HR decreased slightly in rats administered 25 but not 10 mg/kg CHP. Tc was reduced by treatment with 25 mg/kg CHP and then increased above controls the next day. Motor activity was reduced by treatment with 25 but not 10 mg/kg CHP. Pulse pressure was elevated by 2–4 mmHg for 40 h after exposure to 10 and 25 mg/kg CHP. The increase in BP without an increase in HR suggests that CHP increases total peripheral resistance and may alter the baroreflex control of BP. Cholinergic stimulation of the CNS may explain the initial effects of CHP on BP; however, the persistent elevation suggests an involvement of neurohumoral pressor pathways. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Organophosphate pesticide; Blood pressure; Heart rate; Pulse pressure; Body temperature; Motor activity; Anticholinesterase
1. Introduction
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. * Corresponding author. Tel.: +1-919-5411509; fax + 1919-5414849. E-mail address: [email protected] (C.J. Gordon)
Organophosphate (OP)-based pesticides such as chlorpyrifos, parathion, and methylparathion irreversibly inhibit acetylcholinesterase (AChE) activity, resulting in stimulation of cholinergic synapses. Muscarinic and nicotinic cholinergic synapses in the peripheral and central nervous
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systems are stimulated by OP exposure; this stimulation is responsible for many of the abnormal autonomic effects observed in OP-poisoned animals (Ballantyne and Marrs, 1992). The cardiovascular system utilizes cholinergic pathways in the sensory, integrative, and effector pathways and is thus susceptible to altered function following OP exposure (Ballantyne and Marrs, 1992; Gordon, 1994). Integrative sites in the medulla utilize muscarinic pathways to drive pressor responses (Buccafusco, 1996). This may explain why many studies have shown that treatment with cholinesterase inhibitors or CNS application of cholinomimetics consistently elicits a rise in blood pressure (i.e. pressor response). There are inconsistent effects of OP and carbamate pesticides on blood pressure and heart rate in humans and experimental animals with cases of hypotension and bradycardia as well as hypertension and tachycardia (Saad et al., 1997). The inconsistency in cardiovascular effects in humans exposed to these agents can be attributed to factors such as dose and type of pesticide, age of subject, and environmental conditions. Inconsistency in experimental animal studies may be attributed to the use of anesthetized or restrained preparations, procedures that are likely to alter the cardiovascular responses to cholinergic stimulation. Chlorpyrifos is one of the most heavily used OP pesticides in domestic and industrial applications throughout the world (Aspelin, 1994). However, there is little known regarding the effects of chlorpyrifos on blood pressure in experimental animals or humans. A systematic study of the effects of chlorpyrifos on blood pressure and related autonomic processes would be useful to understanding the toxicology of this pesticide. Radiotelemetry technology is an ideal means for monitoring autonomic processes such as blood pressure and heart rate because the measurements are made continuously in unrestrained and undisturbed animals. By using telemetry, physiological data that are free of artifacts caused by handling can be collected continuously, thus including critical time points that might be missed by conventional techniques. In addition, most experimental animal studies have assessed the effects of OPs on blood pressure with
intravenous injection. There is little information on the effects of oral exposure, a more environmentally relevant mode of exposure. This study was designed to use radiotelemetry to assess the effects of oral exposure to chlorpyrifos on blood pressure, heart rate, core temperature, and motor activity in the unrestrained rat.
2. Materials and methods Animals used in this study were male rats of the Long–Evans strain obtained from Charles River Laboratories (Raleigh, NC) at 90 days of age. The animals were housed individually in acrylic cages lined with wood shavings at an ambient temperature (Ta) of 22°C, relative humidity of 50%, and a 12:12 light:dark photoperiod. Rats were tested at an age of approximately 4 months with a body weight of 500 g.
2.1. Surgery Arterial blood pressure, heart rate, core temperature, and motor activity were monitored using surgically implanted radiotelemetry implants (Data Sciences Int., St Paul, MN; model C50PXT). Details on the surgical implantation are provided by the manufacturer. The radiotelemetry unit consists of a cylinder containing the ancillary electronics including sensors for temperature, pressure, and an electrocardiogram (ECG). Motor activity is measured by the movement of the transmitter in the animal cage relative to the position over the receiver. A gel-filled catheter with anticoagulant properties is connected to the pressure transducer within the body of the transmitter. Also attached to the cylinder are two wire leads for the monitoring of the ECG. Rats were anesthetized with sodium pentobarbital (50 mg/kg; i.p.). The rat was placed supine on a heated table and a 7 cm incision was made along the midline of the abdomen. A similar incision was made into the abdominal muscles. The viscera were pushed aside to expose the descending aorta. The aorta was isolated between the bifurcations of the renal and inguinal arteries and cleansed of connective tissue. To insert the
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catheter into the aorta, a ligature was placed around the aorta and lifted with slight tension to temporarily stop blood flow. The wall of the artery was pierced below the ligature with the tip of a 21-g needle. The gel-filled catheter tip was pushed 5 mm into the lumen of the aorta. The site of entry into the aorta was then sealed with a cyanoacrylic-based glue and biocompatible cellulose patch. Once the ligature was removed, blood flow was restored and the entry site of the catheter was checked for leakage. The entire procedure to insert the catheter was limited to less than 2 min to assure no permanent tissue damage from anoxia. The body of the radio transmitter was positioned along the midline of the abdominal cavity and sutured in place to the abdominal wall with 4-0 silk. The abdominal muscle incision was closed with 4-0 silk and the skin was closed with wound clips. The two ECG leads were tunneled under the skin and sutured to the left and right sides of the thorax. The ECG leads were only used in four of the surgeries. In the surgeries in which the ECG leads were not utilized, the leads were wound into a tight coil and left attached to the radio transmitter. Rats were administered penicillin (30 000 units) and an analgesic (buprenorphine; 0.03 mg/kg; s.c.) immediately after surgery. The rats were allowed at least two weeks to recover from the surgery prior to testing.
2.2. Protocol In most of the experiments the rats were placed in an environmental chamber maintained at a Ta of 22–24°C and a 12:12 light:dark cycle while housed individually in their standard cages with wood shavings. Food and water were provided ad libitum. In one experiment the rats were studied while housed in the animal room. The telemetry parameters were monitored at 5-min intervals and stored for later analysis using the LabPro® software system (Data Sciences). The rats were allowed to adapt to the environmental chamber for one night prior to testing. Chlorpyrifos (Chem Services; West Chester, PA) was dissolved in corn oil and administered by oral gavage (0.1 ml/100 g) at doses of 0, 5, 10, or
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25 mg/kg. These doses were selected such that rats would be exposed to a dose that caused little or no behavioral alterations and to a higher dose that would cause marked behavioral and autonomic effects. An earlier study has shown that oral chlorpyrifos at a dose of 10 mg/kg in the male Long–Evans rat has no effect on motor activity or other behavioral measures but inhibits brain AChE activity by 40%; 30 mg/kg reduces motor activity, disrupts other behavioral and autonomic endpoints, and inhibits brain AChE activity by 70% (Nostrandt et al. 1997). We expected 25 mg/kg chlorpyrifos to cause a significant but not severe autonomic and behavioral response. Chlorpyrifos was administered between 14:30 and 15:30 h. This laboratory has found that rats are more sensitive to the thermoregulatory effects of chlorpyrifos when it is administered in the afternoon rather than the morning (Gordon and Rowsey, 1998). Hence, it was expected that effects on blood pressure would be more apparent when chlorpyrifos was administered in the afternoon. The telemetry parameters were monitored for the next 40 h after exposure. Each rat was retested with corn oil or chlorpyrifos using a cross-over design. Animals were allowed to recover from chlorpyrifos administration for at least 10 days before being retested. Overt signs of cholinesterase inhibition, including salivation, ataxia, reduced activity, lacrimation, and tremor, show recovery by 72 h after oral exposure to 80 mg/kg chlorpyrifos (Moser and Padilla, 1998). Recovery of brain and blood AChE activity is slower but is nearly complete 2 weeks after exposure.
2.3. Analysis The telemetry system computed the systolic, diastolic, and mean arterial pressure by sampling the aortic pressure waveform for 10 s (cf. Fig. 1). Heart rate was derived from the pulsatile pressure wave. Core temperature and motor activity were recorded along with blood pressure and heart rate at 5-min intervals. These parameters were then averaged into 1-h bins for statistical analysis. Pulse pressure, the difference between systolic and diastolic pressure, was calculated after averaging
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the data. The data were analyzed with repeated measures analysis of variance by assessing the effects of treatment and time on each telemetry variable during the light and dark phases of the circadian cycle. Mean, systolic, diastolic, and pulse pressure were converted to change in pressure prior to statistical analysis. This was necessary because the variability of blood pressure in individual rats obscured the effects of chlorpyrifos. The change in pressure was calculated by subtracting the average pressure measured between 6:00 and 14:00 h on the day of administration from the pressure measured after corn oil and chlorpyrifos treatments.
3. Results
3.1. Baseline response In general, the telemetry system provided a clear recording of the aortic pressure waveform (Fig. 1). This waveform was typical in most of the rats; however, a few animals had a very damped waveform and abnormally low pressure which was likely a result of a clot in the lumen of the catheter. The pressure readings from these animals was excluded from the data analysis. A 24-h recording in six undisturbed animals shows a clear circadian rhythm for all variables (Fig. 2). The nocturnal elevation in blood pressure was relatively small compared to the elevations in core temperature, heart rate, and motor activity.
3.2. Chlorpyrifos effects
Fig. 1. Example of the trace of the aortic pressure and electrocardiogram waveforms. Large deflection in ECG represents depolarization of ventricles (i.e. QRS complex). P wave of ECG not apparent due to position of electrodes.
An example of the time course of raw data illustrates the typical dynamic characteristics of systolic, diastolic, mean, and pulse blood pressure, heart rate, and core temperature before and after administration of 25 mg/kg chlorpyrifos (Fig. 3). The hourly averaging of the data smooths the responses and facilitates comparison among doses (Fig. 4A). Administration of either the corn oil vehicle or chlorpyrifos led to an abrupt elevation in blood pressure, heart rate, core temperature, and motor activity that was attributed to the stress of handling and gavage procedure (Fig. 4a and b). There was quick recovery in blood pressure after corn oil administration followed by an abrupt elevation coinciding with the onset of the dark phase. Blood pressure increased by approximately 5 mmHg throughout much of the dark phase (D1) in the control group. There was a transient elevation in pressure at the end of the dark phase and then a rapid reduction at the onset of the light phase (L2). Heart rate, core temperature, and motor activity also increased at the onset of the dark phase and remained elevated throughout this period (Fig. 4b). Rats dosed with chlorpyrifos at doses of 10 and 25 mg/kg underwent marked elevations in blood pressure throughout the first dark phase (Fig. 4a; see Appendix A for statistical analysis of data).
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Fig. 2. Twenty-four hour pattern of blood pressure (systolic, mean, and diastolic), heart rate, core temperature, and motor activity of six rats maintained at an ambient temperature of 22°C. Horizontal bars indicate dark phase.
The effects of chlorpyrifos on blood pressure were apparent 2 h after dosing as indicated by a recovery in blood pressure of control animals while that of the treated rats remained elevated and then increased further with the onset of the dark phase. Mean arterial pressure of the 25 mg/kg dose group increased by 20 mmHg over baseline by 3 h into the dark phase. Mean blood pressure of the 10 mg/kg dose group increased by 15 mmHg and remained elevated throughout the dark phase. Mean pressure returned towards baseline but remained elevated in the 25 mg/kg dose group during the next light phase (L2). A slight elevation in mean pressure above controls was seen during the first hours of the next dark phase (D2) (Fig. 4a). Diastolic and systolic pressure showed similar responses to that of mean pressure. There was a notable elevation in systolic pressure in the 10 and 25 mg/kg groups during the second night after treatment (D2). Core temperature of rats administered 25 mg/ kg chlorpyrifos underwent a significant decrease from 37 to 35°C within 4 h after administration. There was no hypothermic response following 10 mg/kg chlopyrifos (Fig. 4b). At the onset of the
next light phase (L2), core temperature of the 10 and 25 mg/kg treatment groups was significantly elevated above control levels. Core temperature of the 25 mg/kg group remained elevated above controls throughout most of the second dark phase (D2). Heart rate decreased significantly during the first half of the dark phase following 25 mg/kg chlorpyrifos; however, there was no change in heart rate in rats dosed with 10 mg/kg. There was a slight but significant elevation in heart rate during the next light phase (L2). Motor activity was not markedly affected by the 10 or 25 mg/kg treatments. There was a slight but significant reduction in motor activity during the first half of the second dark phase (D2) after exposure to 25 mg/kg (Fig. 4b). Chlorpyrifos at a dose of 5 mg/kg had little effect on either the cardiovascular, thermoregulatory, or behavioral parameters (data not shown). There was a significant elevation in pulse pressure in both the 10 and 25 mg/kg treatment groups that persisted throughout the 40 h monitoring period after treatment (Fig. 5). Overall, pulse pressure increased by 2–4 mmHg relative to rats receiving corn oil.
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Fig. 3. Example of the time course of unaveraged data of blood pressure, heart rate, and core temperature collected at 5 min intervals in a rat administered 25 mg/kg chlorpyrifos (p.o.) Arrows denotes time of chlorpyrifos administration. Other symbols same as Fig. 2.
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Fig. 4. Time-course of hourly averages of change in blood pressure, heart rate, core temperature, and motor activity of rats gavaged with the chlorpyrifos at doses of 0, 10, and 25 mg/kg (p.o.). Numbers in parenthesis indicate sample size. Abbreviations same as Fig. 2.
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Fig. 4. (Continued)
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4. Discussion Continuous monitoring of blood pressure and other physiological parameters in undisturbed rats by radiotelemetry has demonstrated that exposure to the OP pesticide chlorpyrifos elicits a prolonged hypertensive response that appears to be independent of the toxic effects on core temperature, heart rate, and motor activity. The elevation in blood pressure was evident within 2 h and persisted for approximately 24 h after exposure to chlorpyrifos. The hypertensive response appears to be a sensitive indicator of exposure to this OP because it occurred following exposure to 10 mg/ kg chlorpyrifos, a dose that had little effect on core temperature (i.e. no hypothermia), heart rate, and motor activity. It has been well documented that areas in the medulla involved in the regulation of sympathetic tone and blood pressure utilize cholinergic pathways and respond to cholinomimetics and anticholinesterase agents with a pressor response (Buccafusco, 1996). In general, unanesthetized species (excluding the cat) administered carbamates undergo a hypertensive response (Philippu, 1981). For example, the hypertensive effects of physostigmine, a reversible cholinesterase inhibitor, have been well documented in experimental animals and humans (Janowsky et al., 1986; Varagic et al., 1991; Lazartigues et al., 1998). The peripheral effects of cholinergic stimulation would be expected to counter the central influences. That is, the pacemaker of the heart is innervated by
Fig. 5. Time-course of change in pulse pressure ( = systolic– diastolic) of rats gavaged with chlorpyrifos at doses of 0, 10, and 25 mg/kg (p.o.). Abbreviations same as Fig. 2.
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vagal efferents which elicit a bradycardic response when stimulated with cholinomimetics (Guyton, 1986). Hence, the inhibition of AChE activity in the peripheral tissues following exposure to an OP would be expected to result in a decrease in heart rate. However, the feedback interactions between blood pressure regulation and heart rate appears to offset a direct effect of an OP on heart rate. There was a reduction in heart rate after exposure to the highest dose of chlorpyrifos; however, the decrease may be a regulatory, negative feedback response to the chlorpyrifos-induced rise in blood pressure (cf. Figs. 3 and 4). Studies on the cardiovascular effects of OP’s and other AChE inhibitors have generally shown a hypertensive response but inconsistent responses are also reported. Intravenous administration of the OP’s sarin and paraoxon to the awake rat lead to an immediate elevation in blood pressure and decrease in heart rate that persists for 8 h after injection (Bataillard et al., 1990). Intravenous physostigmine administered to the awake rat causes a peak hypertensive response within 5 min of injection concomitant with bradycardia (Lazartigues et al., 1998). Intravenous soman administration elicits the most rapid increase in blood pressure when compared to intramuscular, subcutaneous, and intraperitoneal routes in the urethane-anesthetized rat (Brezenoff et al., 1984). The urethane-anesthetized rat given a lethal dose of OP pesticides (diazinon or fenthion) becomes bradycardic and develops hypotension, a response which may be a peripheral, noncholinergic mechanism (Kojima et al., 1992). The anesthetized rabbit displays a consistent hypotensive response and vasodilation to the OP’s soman, sarin, and DFP (Preston and Heath, 1972). It is important to note that these studies utilized anesthetized or tethered subjects and the cardiovascular measurements were made over a relatively short time frame. We contend that a more complete picture of the cardiovascular effects on an OP can be assessed with telemetry. Changes in blood pressure lasting over 24 h elicited by exposure to an OP can be readily detected using the telemetric techniques. The hypertensive response to chlorpyrifos and other OP’s is remarkable when one considers the toxicological effects of these agents on heart rate, skin blood flow, and body temperature:
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1. Hypertension from chlorpyrifos developed with relatively little change in heart rate. Indeed, the higher dose of 25 mg/kg initially led to a reduction in heart rate. This would suggest that the elevation in blood pressure following chlorpyrifos exposure is attributed to an increase in peripheral resistance of the systemic circulation rather than to an increase in cardiac output. That is, if cardiac output is constant, then pressure can only increase by increasing the resistance to blood flow as would occur with vasoconstriction (Guyton, 1986). Interestingly, several studies have shown that peripheral vasodilation of the tail occurs during the first hours after exposure to OP’s such as soman (Meeter, 1971), DFP (Gordon et al., 1991), and chlopryifos (Gordon and Yang, 2000). Vasodilation of the tail would undoubtedly place a greater challenge to the cardiovascular system to raise blood pressure during OP exposure because such a response lowers resistance to blood flow in the tail. Increased resistance is likely to occur in other tissues and/or organs during chlorpyrifos exposure. The failure to vasodilate the tail could exacerbate the hypertensive effect of chlorpyrifos. 2. The greater hypertensive response observed in the 25 mg/kg dose group concomitant with a marked period of hypothermia was unexpected. Hypothermia can alter the sensitivity of the cardiovascular system to various cardiac drugs (Vidrio and Garcia-Marquez, 1987; Kim et al., 1997). The hypertensive responses to 10 mg/kg chlorpyrifos, which did not cause hypothermia, and to 25 mg/kg chlorpyrifos were similar in magnitude during the first night after exposure. This would suggest that the pressor response to chlorpyrifos is a relatively strong toxicological effect that is not attenuated by the OP’s hypothermic effects. It is important to note that the maximal inhibition in AChE in the CNS occurs 4 h after oral exposure to chlorpyrifos (Nostrandt et al., 1997). The male Long–Evans rat administered 10 mg/kg chlorpyrifos undergoes a 50% inhibition in AChE activity in the pons/medulla and recovers to 25% inhibition by 24 h postexposure. Chlorpyri-
fos (30 mg/kg) leads to a 75% inhibition in AChE activity at 3.5 h and 50% inhibition by 24 h (Nostrandt et al., 1997). The hypertensive and thermoregulatory effects of chlorpyrifos persisted into the second night after exposure in spite of the recovery in AChE activity. Hence, it is unlikely that inhibition in AChE is solely responsible for the hypertensive response to chlorpyrifos. The chlorpyrifos-induced rise in pulse pressure was statistically significant for both 10 and 25 mg/kg chlorpyrifos; however, the pathophysiological significance is uncertain because the rise in pulse pressure was only 2–4 mmHg above controls. Nonetheless, the persistent rise in pulse pressure at the low and high doses of chlorpyrifos could represent an important indicator of toxicity of this OP. It is of interest to note the lack of dose-related effect of chlorpyrifos on pulse pressure; the 10 mg/kg dose was as effective as the 25 mg/kg treatment dose on pulse pressure. Two critical factors affect the pulse pressure: the stroke volume of the heart (i.e. blood ejected with each contraction) and the compliance or distensibility of the arterial tree (Guyton, 1986). The cholinergic stimulation resulting from AChE inhibition is likely to affect these factors. Stroke volume could also be affected by chlorpyrifos exposure; however, an increase in stroke volume is associated with a reduced heart rate. We suspect that chlorpyrifos reduces the compliance of the arterial tree since there is little change in heart rate after chlopyrifos exposure. Other than the reduced heart rate during the first night after 25 mg/kg chlorpyrifos, mean heart rate was similar to controls during the next day and night after chlorpyrifos. It is important to consider if the rise in blood pressure represents a possible hazard to humans exposed to chlorpyrifos and other OP pesticides. Clinical studies have shown that physostigmine causes marked elevations in blood pressure (Janowsky et al., 1986). Physostigmine in humans stimulated the adrenal release of epinephrine which can explain the hypertension and tachycardia of this agent (Kennedy et al., 1984). On the other hand, clinical case reports of humans exposed to anticholinesterase pesticides indicate hypotension or hypertension as well as bradycardia
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and tachycardia (Tafuri and Roberts, 1987; Saadeh et al., 1996, 1997). Of course, the clinical case reports have to viewed with caution because of the innumerable uncontrolled variables such as dose, compound purity, age of subject, drug intervention, and environmental conditions. The marked increase in blood pressure without a compensatory change in heart rate following chlorpyrifos exposure suggests that this OP alters the sensitivity of the control of blood pressure. The sensing and central processing of blood pressure by the baroreceptors in the carotid sinus and elsewhere in the circulatory system may be altered by chlorpyrifos. If this were not the case, then one would expect a reduction in heart rate and a concomitant reduction in cardiac output to reduce blood pressure. A resetting of the baroreflex could be mediated by a neurohormonal system such as vasopressin or angiotensin II. Vasopressin administered centrally can attenuate the baroreceptor reflex (Lebrun et al., 1987). Cholinergic stimulation of the CNS with physostigmine has been
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shown to stimulate vasopressin release (Lazartigues et al., 1998, 1999). The renin-angiotensin pathway is also activated by central cholinergic stimulation (Saad et al., 1997). There is apparently little information on how OP exposure can affect the activation of these neurohumoral pathways. Such a response could pose a risk to populations susceptible to heart disease, particularly those with high blood pressure. Hence, it will be important to discern the mechanism of action of OP-induced hypertension and to study the potential hypertensive responses in experimental models of hypertension such as the spontaneously hypertensive rat.
Acknowledgements We thank Drs W.P. Watkinson and A.H. Rezvani for their review of the manuscript. We also thank P. Becker for providing technical assistance.
Appendix A. Repeated measures ANOVA results of the effects of chlorpyrifos on the telemetry variables
Data set
Period
Treatment
Treatment-time
Mean pressure ‘‘ ’’ ‘‘ ’’ Systolic pressure ‘‘ ’’ ‘‘ ’’ Diastolic pressure ‘‘ ’’ ‘‘ ’’ Pulse pressure ‘‘ ’’ ‘‘ ’’ Heart rate ‘‘ ’’ ‘‘ ’’
D1 L2 D2 D1 L2 D2 D1 L2 D2 D1 L2 D2 D1 L2 D2
F(2,42) = 34.9, PB0.0001 F(2,42) = 11.8, PB0.0001 Not significant F(2,42) = 30.5, PB0.0001 Not significant Not significant F(2,42)= 34.4, PB0.0001 F(2,42) = 9.5, P= 0.0004 Not significant F(2,42) = 3.9, P= 0.03 F(2,42) = 17.5, PB0.0001 F(2,42) = 15.2, PB0.0001 F(2,45) = 8.5, P= 0.0007 Not significant Not significant
F(22,462)= 4.2, PB0.0001 F(22,462)= 2.4, P= .0004 F(22,462)= 3.5, PB0.0001 F(22,462= 3.7, PB0.0001 F(22,462)= 2.1, P= 0.002 F(22,462)= 3.6, PB0.0001 F(22,462)= 4.1, PB0.0001 F(22,462)= 2.7, PB0.0001 F(22,463)= 3.3, PB0.001 F(22,462)= 5.4, PB0.0001 F(22,462)= 3.1, PB0.0001 F(22,462)= 3.2, PB0.0001 F(22,495)= 4.7, PB0.0001 F(22,495)= 2.5, P= 0.0002 F(22,495)= 3.6, PB0.0001
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Core temperature ‘‘ ’’ ‘‘ ’’ Motor activity ‘‘ ’’ ‘‘ ’’
D1 L2 D2 D1 L2 D2
F(2,45) =18.9, PB0.0001 F(2,45) =23.7, PB0.0001 F(2,45) = 1.5, P= 0.024 Not significant Not significant F(2,45) =6.1, P= 0.005
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.
F(22,495)= 8.5, F(22,495)= 6.3, F(22,495)=3.6, F(22,495)= 4.1, F(22,495)= 5.1, F(22,495)=4.3,
PB0.0001 PB0.0001 PB0.0001 PB0.0001 PB0.0001 PB0.0001
cardia induced by mivazerol, a selective alpha 2-adrenoceptor agonist, is amplified during mild hypothermia in the pentobarbital-anesthetized rat. Cardiovasc. Drugs Ther. 11, 751 – 755. Kojima, T., Tsuda, S., Shirasu, Y., 1992. Non-cholinergic mechanisms underlying the acute lethal effects of P =S type organophosphorus insecticides in rats. J. Vet. Med. Sci. 54, 529 – 533. Lazartigues, E, Freslon, J.L., Tellioglu, T., Brefel-Courbon, C., Pelat, M., Tran, M.A., Montastruc, J.L., Rascol, O., 1998. Pressor and bradycardic effects of tacrine and other acetylcholinesterase inhibitors in the rat. Eur. J. Pharmacol. 361, 61 – 71. Lazartigues, E., Brefel-Courbon, C., Tran, M.A., Montastruc, J.L., Rascol, O., 1999. Spontaneously hypertensive rats cholinergic hyper-responsiveness: central and peripheral pharmacological mechanisms. Br. J. Pharmacol. 127, 1657 – 1665. Lebrun, C., Rohmeiss, P., Demmert, G., Unger, T., 1987. Central cardiovascular actions of vasopressin in the rat. Can. J. Physiol. Pharmacol. 65, 1712 – 1716. Moser, V.C., Padilla, S., 1998. Age- and gender-related differences in the time-course of behavioral and biochemical effects produced by oral chlorpyrifos in rats. Toxicol. Appl. Pharmacol. 149, 107 – 119. Meeter, E., 1971. The effect of atropine on the hypothermia and the shift in set-point for heat release evoked by a cholinesterase inhibitor in the rat. Proc. Royal Neth. Acad. Sci. Series C 74, 105 – 112. Nostrandt, A.C., Padilla, S., Moser, V.C., 1997. The relationship of oral chlorpyrifos effects on behavior, cholinesterase inhibition, and muscarinic receptor density in rat. Pharm. Biochem. Behav. 58, 15 – 23. Philippu, A., 1981. Involvement of cholinergic system of the brain in the central regulation of cardiovascular functions. J. Auton. Pharmac. 1, 321 – 330. Preston, E., Heath, C., 1972. Atropine-insensitive vasodilation and hypotension in the organophosphate-poisoned rabbit. Arch. Int. Pharmacodyn. 200, 231 – 244. Saad, W.A., Luiz, A.C., Camargo, L.A., Silveira, J.E., Foglia, S., Menani, J.V., Saad, W.A., 1997. Functional evidence that the central renin-angiotensin system plays a role in the pressor response induced by central injection of carbachol. Braz. J. Med. Biol. Res. 30, 493 – 496.
C.J. Gordon, B.K. Padnos / Toxicology 146 (2000) 1–13 Saadeh, A.M., Al-Ali, M.K., Farsakh, N.A., Ghani, M., 1996. Clinical and sociodemographic features of acute carbamate and organophosphate poisoning: a study of 70 adult patients in North Jordan. Clin. Toxicol. 34, 45–51. Saadeh, A.M., Farsakh, N.A., al-Ali, M.K., 1997. Cardiac manifestations of acute carbamate and organophosphate poisoning. Heart 77, 461–464. Tafuri, J., Roberts, J., 1987. Organophosphate poisoning.
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Ann. Emerg. Med. 16, 193 – 202. Varagic, V.M., Prostran, M.S., Stepanovic, S., Savic, J., Vujnov, S., 1991. Transmitter interactions in the central cholinergic control of blood pressure regulation. Drug Metab. Interact. 9, 49 – 76. Vidrio, H., Garcia-Marquez, F., 1987. Reversal by hypothermia of vasodilator-induced tachycardia in anesthetized rats. Arch. Int. Pharmacodyn. Ther. 288, 217 – 228.
Prolonged elevation in blood pressure in the unrestrained rat exposed to chlorpyrifos Christopher J. Gordon *, Beth K. Padnos Neurotoxicology Di6ision, National Health and En6ironmental Effects Research Laboratory, MD-74B, U.S. En6ironmental Protection Agency, Research Triangle Park, NC 27711, USA Received 10 November 1999; accepted 6 January 2000
Abstract Organophosphate (OP) pesticides are likely to alter the regulation of blood pressure (BP) because (i) BP control centers in the brain stem utilize cholinergic synapses and (ii) the irreversible inhibition of acetylcholinesterase activity by OP’s causes cholinergic stimulation in the CNS. This study used radiotelemetric techniques to monitor systolic (S), diastolic (D), mean (M) BP, pulse pressure (systolic–diastolic), heart rate (HR), core temperature (Tc), and motor activity in male Long–Evans rats treated with the OP pesticide chlorpyrifos (CHP) at doses of 0, 5, 10, and 25 mg/kg (p.o.) at 15:00 h 10 and 25 mg/kg CHP led to parallel elevations in S-BP, M-BP, and D-BP within 2 h after dosing. BP increased 15–20 mmHg above controls and increases persisted throughout the night and into the next day. HR decreased slightly in rats administered 25 but not 10 mg/kg CHP. Tc was reduced by treatment with 25 mg/kg CHP and then increased above controls the next day. Motor activity was reduced by treatment with 25 but not 10 mg/kg CHP. Pulse pressure was elevated by 2–4 mmHg for 40 h after exposure to 10 and 25 mg/kg CHP. The increase in BP without an increase in HR suggests that CHP increases total peripheral resistance and may alter the baroreflex control of BP. Cholinergic stimulation of the CNS may explain the initial effects of CHP on BP; however, the persistent elevation suggests an involvement of neurohumoral pressor pathways. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Organophosphate pesticide; Blood pressure; Heart rate; Pulse pressure; Body temperature; Motor activity; Anticholinesterase
1. Introduction
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. * Corresponding author. Tel.: +1-919-5411509; fax + 1919-5414849. E-mail address: [email protected] (C.J. Gordon)
Organophosphate (OP)-based pesticides such as chlorpyrifos, parathion, and methylparathion irreversibly inhibit acetylcholinesterase (AChE) activity, resulting in stimulation of cholinergic synapses. Muscarinic and nicotinic cholinergic synapses in the peripheral and central nervous
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 1 5 8 - X
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systems are stimulated by OP exposure; this stimulation is responsible for many of the abnormal autonomic effects observed in OP-poisoned animals (Ballantyne and Marrs, 1992). The cardiovascular system utilizes cholinergic pathways in the sensory, integrative, and effector pathways and is thus susceptible to altered function following OP exposure (Ballantyne and Marrs, 1992; Gordon, 1994). Integrative sites in the medulla utilize muscarinic pathways to drive pressor responses (Buccafusco, 1996). This may explain why many studies have shown that treatment with cholinesterase inhibitors or CNS application of cholinomimetics consistently elicits a rise in blood pressure (i.e. pressor response). There are inconsistent effects of OP and carbamate pesticides on blood pressure and heart rate in humans and experimental animals with cases of hypotension and bradycardia as well as hypertension and tachycardia (Saad et al., 1997). The inconsistency in cardiovascular effects in humans exposed to these agents can be attributed to factors such as dose and type of pesticide, age of subject, and environmental conditions. Inconsistency in experimental animal studies may be attributed to the use of anesthetized or restrained preparations, procedures that are likely to alter the cardiovascular responses to cholinergic stimulation. Chlorpyrifos is one of the most heavily used OP pesticides in domestic and industrial applications throughout the world (Aspelin, 1994). However, there is little known regarding the effects of chlorpyrifos on blood pressure in experimental animals or humans. A systematic study of the effects of chlorpyrifos on blood pressure and related autonomic processes would be useful to understanding the toxicology of this pesticide. Radiotelemetry technology is an ideal means for monitoring autonomic processes such as blood pressure and heart rate because the measurements are made continuously in unrestrained and undisturbed animals. By using telemetry, physiological data that are free of artifacts caused by handling can be collected continuously, thus including critical time points that might be missed by conventional techniques. In addition, most experimental animal studies have assessed the effects of OPs on blood pressure with
intravenous injection. There is little information on the effects of oral exposure, a more environmentally relevant mode of exposure. This study was designed to use radiotelemetry to assess the effects of oral exposure to chlorpyrifos on blood pressure, heart rate, core temperature, and motor activity in the unrestrained rat.
2. Materials and methods Animals used in this study were male rats of the Long–Evans strain obtained from Charles River Laboratories (Raleigh, NC) at 90 days of age. The animals were housed individually in acrylic cages lined with wood shavings at an ambient temperature (Ta) of 22°C, relative humidity of 50%, and a 12:12 light:dark photoperiod. Rats were tested at an age of approximately 4 months with a body weight of 500 g.
2.1. Surgery Arterial blood pressure, heart rate, core temperature, and motor activity were monitored using surgically implanted radiotelemetry implants (Data Sciences Int., St Paul, MN; model C50PXT). Details on the surgical implantation are provided by the manufacturer. The radiotelemetry unit consists of a cylinder containing the ancillary electronics including sensors for temperature, pressure, and an electrocardiogram (ECG). Motor activity is measured by the movement of the transmitter in the animal cage relative to the position over the receiver. A gel-filled catheter with anticoagulant properties is connected to the pressure transducer within the body of the transmitter. Also attached to the cylinder are two wire leads for the monitoring of the ECG. Rats were anesthetized with sodium pentobarbital (50 mg/kg; i.p.). The rat was placed supine on a heated table and a 7 cm incision was made along the midline of the abdomen. A similar incision was made into the abdominal muscles. The viscera were pushed aside to expose the descending aorta. The aorta was isolated between the bifurcations of the renal and inguinal arteries and cleansed of connective tissue. To insert the
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catheter into the aorta, a ligature was placed around the aorta and lifted with slight tension to temporarily stop blood flow. The wall of the artery was pierced below the ligature with the tip of a 21-g needle. The gel-filled catheter tip was pushed 5 mm into the lumen of the aorta. The site of entry into the aorta was then sealed with a cyanoacrylic-based glue and biocompatible cellulose patch. Once the ligature was removed, blood flow was restored and the entry site of the catheter was checked for leakage. The entire procedure to insert the catheter was limited to less than 2 min to assure no permanent tissue damage from anoxia. The body of the radio transmitter was positioned along the midline of the abdominal cavity and sutured in place to the abdominal wall with 4-0 silk. The abdominal muscle incision was closed with 4-0 silk and the skin was closed with wound clips. The two ECG leads were tunneled under the skin and sutured to the left and right sides of the thorax. The ECG leads were only used in four of the surgeries. In the surgeries in which the ECG leads were not utilized, the leads were wound into a tight coil and left attached to the radio transmitter. Rats were administered penicillin (30 000 units) and an analgesic (buprenorphine; 0.03 mg/kg; s.c.) immediately after surgery. The rats were allowed at least two weeks to recover from the surgery prior to testing.
2.2. Protocol In most of the experiments the rats were placed in an environmental chamber maintained at a Ta of 22–24°C and a 12:12 light:dark cycle while housed individually in their standard cages with wood shavings. Food and water were provided ad libitum. In one experiment the rats were studied while housed in the animal room. The telemetry parameters were monitored at 5-min intervals and stored for later analysis using the LabPro® software system (Data Sciences). The rats were allowed to adapt to the environmental chamber for one night prior to testing. Chlorpyrifos (Chem Services; West Chester, PA) was dissolved in corn oil and administered by oral gavage (0.1 ml/100 g) at doses of 0, 5, 10, or
3
25 mg/kg. These doses were selected such that rats would be exposed to a dose that caused little or no behavioral alterations and to a higher dose that would cause marked behavioral and autonomic effects. An earlier study has shown that oral chlorpyrifos at a dose of 10 mg/kg in the male Long–Evans rat has no effect on motor activity or other behavioral measures but inhibits brain AChE activity by 40%; 30 mg/kg reduces motor activity, disrupts other behavioral and autonomic endpoints, and inhibits brain AChE activity by 70% (Nostrandt et al. 1997). We expected 25 mg/kg chlorpyrifos to cause a significant but not severe autonomic and behavioral response. Chlorpyrifos was administered between 14:30 and 15:30 h. This laboratory has found that rats are more sensitive to the thermoregulatory effects of chlorpyrifos when it is administered in the afternoon rather than the morning (Gordon and Rowsey, 1998). Hence, it was expected that effects on blood pressure would be more apparent when chlorpyrifos was administered in the afternoon. The telemetry parameters were monitored for the next 40 h after exposure. Each rat was retested with corn oil or chlorpyrifos using a cross-over design. Animals were allowed to recover from chlorpyrifos administration for at least 10 days before being retested. Overt signs of cholinesterase inhibition, including salivation, ataxia, reduced activity, lacrimation, and tremor, show recovery by 72 h after oral exposure to 80 mg/kg chlorpyrifos (Moser and Padilla, 1998). Recovery of brain and blood AChE activity is slower but is nearly complete 2 weeks after exposure.
2.3. Analysis The telemetry system computed the systolic, diastolic, and mean arterial pressure by sampling the aortic pressure waveform for 10 s (cf. Fig. 1). Heart rate was derived from the pulsatile pressure wave. Core temperature and motor activity were recorded along with blood pressure and heart rate at 5-min intervals. These parameters were then averaged into 1-h bins for statistical analysis. Pulse pressure, the difference between systolic and diastolic pressure, was calculated after averaging
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the data. The data were analyzed with repeated measures analysis of variance by assessing the effects of treatment and time on each telemetry variable during the light and dark phases of the circadian cycle. Mean, systolic, diastolic, and pulse pressure were converted to change in pressure prior to statistical analysis. This was necessary because the variability of blood pressure in individual rats obscured the effects of chlorpyrifos. The change in pressure was calculated by subtracting the average pressure measured between 6:00 and 14:00 h on the day of administration from the pressure measured after corn oil and chlorpyrifos treatments.
3. Results
3.1. Baseline response In general, the telemetry system provided a clear recording of the aortic pressure waveform (Fig. 1). This waveform was typical in most of the rats; however, a few animals had a very damped waveform and abnormally low pressure which was likely a result of a clot in the lumen of the catheter. The pressure readings from these animals was excluded from the data analysis. A 24-h recording in six undisturbed animals shows a clear circadian rhythm for all variables (Fig. 2). The nocturnal elevation in blood pressure was relatively small compared to the elevations in core temperature, heart rate, and motor activity.
3.2. Chlorpyrifos effects
Fig. 1. Example of the trace of the aortic pressure and electrocardiogram waveforms. Large deflection in ECG represents depolarization of ventricles (i.e. QRS complex). P wave of ECG not apparent due to position of electrodes.
An example of the time course of raw data illustrates the typical dynamic characteristics of systolic, diastolic, mean, and pulse blood pressure, heart rate, and core temperature before and after administration of 25 mg/kg chlorpyrifos (Fig. 3). The hourly averaging of the data smooths the responses and facilitates comparison among doses (Fig. 4A). Administration of either the corn oil vehicle or chlorpyrifos led to an abrupt elevation in blood pressure, heart rate, core temperature, and motor activity that was attributed to the stress of handling and gavage procedure (Fig. 4a and b). There was quick recovery in blood pressure after corn oil administration followed by an abrupt elevation coinciding with the onset of the dark phase. Blood pressure increased by approximately 5 mmHg throughout much of the dark phase (D1) in the control group. There was a transient elevation in pressure at the end of the dark phase and then a rapid reduction at the onset of the light phase (L2). Heart rate, core temperature, and motor activity also increased at the onset of the dark phase and remained elevated throughout this period (Fig. 4b). Rats dosed with chlorpyrifos at doses of 10 and 25 mg/kg underwent marked elevations in blood pressure throughout the first dark phase (Fig. 4a; see Appendix A for statistical analysis of data).
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Fig. 2. Twenty-four hour pattern of blood pressure (systolic, mean, and diastolic), heart rate, core temperature, and motor activity of six rats maintained at an ambient temperature of 22°C. Horizontal bars indicate dark phase.
The effects of chlorpyrifos on blood pressure were apparent 2 h after dosing as indicated by a recovery in blood pressure of control animals while that of the treated rats remained elevated and then increased further with the onset of the dark phase. Mean arterial pressure of the 25 mg/kg dose group increased by 20 mmHg over baseline by 3 h into the dark phase. Mean blood pressure of the 10 mg/kg dose group increased by 15 mmHg and remained elevated throughout the dark phase. Mean pressure returned towards baseline but remained elevated in the 25 mg/kg dose group during the next light phase (L2). A slight elevation in mean pressure above controls was seen during the first hours of the next dark phase (D2) (Fig. 4a). Diastolic and systolic pressure showed similar responses to that of mean pressure. There was a notable elevation in systolic pressure in the 10 and 25 mg/kg groups during the second night after treatment (D2). Core temperature of rats administered 25 mg/ kg chlorpyrifos underwent a significant decrease from 37 to 35°C within 4 h after administration. There was no hypothermic response following 10 mg/kg chlopyrifos (Fig. 4b). At the onset of the
next light phase (L2), core temperature of the 10 and 25 mg/kg treatment groups was significantly elevated above control levels. Core temperature of the 25 mg/kg group remained elevated above controls throughout most of the second dark phase (D2). Heart rate decreased significantly during the first half of the dark phase following 25 mg/kg chlorpyrifos; however, there was no change in heart rate in rats dosed with 10 mg/kg. There was a slight but significant elevation in heart rate during the next light phase (L2). Motor activity was not markedly affected by the 10 or 25 mg/kg treatments. There was a slight but significant reduction in motor activity during the first half of the second dark phase (D2) after exposure to 25 mg/kg (Fig. 4b). Chlorpyrifos at a dose of 5 mg/kg had little effect on either the cardiovascular, thermoregulatory, or behavioral parameters (data not shown). There was a significant elevation in pulse pressure in both the 10 and 25 mg/kg treatment groups that persisted throughout the 40 h monitoring period after treatment (Fig. 5). Overall, pulse pressure increased by 2–4 mmHg relative to rats receiving corn oil.
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Fig. 3. Example of the time course of unaveraged data of blood pressure, heart rate, and core temperature collected at 5 min intervals in a rat administered 25 mg/kg chlorpyrifos (p.o.) Arrows denotes time of chlorpyrifos administration. Other symbols same as Fig. 2.
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Fig. 4. Time-course of hourly averages of change in blood pressure, heart rate, core temperature, and motor activity of rats gavaged with the chlorpyrifos at doses of 0, 10, and 25 mg/kg (p.o.). Numbers in parenthesis indicate sample size. Abbreviations same as Fig. 2.
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Fig. 4. (Continued)
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4. Discussion Continuous monitoring of blood pressure and other physiological parameters in undisturbed rats by radiotelemetry has demonstrated that exposure to the OP pesticide chlorpyrifos elicits a prolonged hypertensive response that appears to be independent of the toxic effects on core temperature, heart rate, and motor activity. The elevation in blood pressure was evident within 2 h and persisted for approximately 24 h after exposure to chlorpyrifos. The hypertensive response appears to be a sensitive indicator of exposure to this OP because it occurred following exposure to 10 mg/ kg chlorpyrifos, a dose that had little effect on core temperature (i.e. no hypothermia), heart rate, and motor activity. It has been well documented that areas in the medulla involved in the regulation of sympathetic tone and blood pressure utilize cholinergic pathways and respond to cholinomimetics and anticholinesterase agents with a pressor response (Buccafusco, 1996). In general, unanesthetized species (excluding the cat) administered carbamates undergo a hypertensive response (Philippu, 1981). For example, the hypertensive effects of physostigmine, a reversible cholinesterase inhibitor, have been well documented in experimental animals and humans (Janowsky et al., 1986; Varagic et al., 1991; Lazartigues et al., 1998). The peripheral effects of cholinergic stimulation would be expected to counter the central influences. That is, the pacemaker of the heart is innervated by
Fig. 5. Time-course of change in pulse pressure ( = systolic– diastolic) of rats gavaged with chlorpyrifos at doses of 0, 10, and 25 mg/kg (p.o.). Abbreviations same as Fig. 2.
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vagal efferents which elicit a bradycardic response when stimulated with cholinomimetics (Guyton, 1986). Hence, the inhibition of AChE activity in the peripheral tissues following exposure to an OP would be expected to result in a decrease in heart rate. However, the feedback interactions between blood pressure regulation and heart rate appears to offset a direct effect of an OP on heart rate. There was a reduction in heart rate after exposure to the highest dose of chlorpyrifos; however, the decrease may be a regulatory, negative feedback response to the chlorpyrifos-induced rise in blood pressure (cf. Figs. 3 and 4). Studies on the cardiovascular effects of OP’s and other AChE inhibitors have generally shown a hypertensive response but inconsistent responses are also reported. Intravenous administration of the OP’s sarin and paraoxon to the awake rat lead to an immediate elevation in blood pressure and decrease in heart rate that persists for 8 h after injection (Bataillard et al., 1990). Intravenous physostigmine administered to the awake rat causes a peak hypertensive response within 5 min of injection concomitant with bradycardia (Lazartigues et al., 1998). Intravenous soman administration elicits the most rapid increase in blood pressure when compared to intramuscular, subcutaneous, and intraperitoneal routes in the urethane-anesthetized rat (Brezenoff et al., 1984). The urethane-anesthetized rat given a lethal dose of OP pesticides (diazinon or fenthion) becomes bradycardic and develops hypotension, a response which may be a peripheral, noncholinergic mechanism (Kojima et al., 1992). The anesthetized rabbit displays a consistent hypotensive response and vasodilation to the OP’s soman, sarin, and DFP (Preston and Heath, 1972). It is important to note that these studies utilized anesthetized or tethered subjects and the cardiovascular measurements were made over a relatively short time frame. We contend that a more complete picture of the cardiovascular effects on an OP can be assessed with telemetry. Changes in blood pressure lasting over 24 h elicited by exposure to an OP can be readily detected using the telemetric techniques. The hypertensive response to chlorpyrifos and other OP’s is remarkable when one considers the toxicological effects of these agents on heart rate, skin blood flow, and body temperature:
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1. Hypertension from chlorpyrifos developed with relatively little change in heart rate. Indeed, the higher dose of 25 mg/kg initially led to a reduction in heart rate. This would suggest that the elevation in blood pressure following chlorpyrifos exposure is attributed to an increase in peripheral resistance of the systemic circulation rather than to an increase in cardiac output. That is, if cardiac output is constant, then pressure can only increase by increasing the resistance to blood flow as would occur with vasoconstriction (Guyton, 1986). Interestingly, several studies have shown that peripheral vasodilation of the tail occurs during the first hours after exposure to OP’s such as soman (Meeter, 1971), DFP (Gordon et al., 1991), and chlopryifos (Gordon and Yang, 2000). Vasodilation of the tail would undoubtedly place a greater challenge to the cardiovascular system to raise blood pressure during OP exposure because such a response lowers resistance to blood flow in the tail. Increased resistance is likely to occur in other tissues and/or organs during chlorpyrifos exposure. The failure to vasodilate the tail could exacerbate the hypertensive effect of chlorpyrifos. 2. The greater hypertensive response observed in the 25 mg/kg dose group concomitant with a marked period of hypothermia was unexpected. Hypothermia can alter the sensitivity of the cardiovascular system to various cardiac drugs (Vidrio and Garcia-Marquez, 1987; Kim et al., 1997). The hypertensive responses to 10 mg/kg chlorpyrifos, which did not cause hypothermia, and to 25 mg/kg chlorpyrifos were similar in magnitude during the first night after exposure. This would suggest that the pressor response to chlorpyrifos is a relatively strong toxicological effect that is not attenuated by the OP’s hypothermic effects. It is important to note that the maximal inhibition in AChE in the CNS occurs 4 h after oral exposure to chlorpyrifos (Nostrandt et al., 1997). The male Long–Evans rat administered 10 mg/kg chlorpyrifos undergoes a 50% inhibition in AChE activity in the pons/medulla and recovers to 25% inhibition by 24 h postexposure. Chlorpyri-
fos (30 mg/kg) leads to a 75% inhibition in AChE activity at 3.5 h and 50% inhibition by 24 h (Nostrandt et al., 1997). The hypertensive and thermoregulatory effects of chlorpyrifos persisted into the second night after exposure in spite of the recovery in AChE activity. Hence, it is unlikely that inhibition in AChE is solely responsible for the hypertensive response to chlorpyrifos. The chlorpyrifos-induced rise in pulse pressure was statistically significant for both 10 and 25 mg/kg chlorpyrifos; however, the pathophysiological significance is uncertain because the rise in pulse pressure was only 2–4 mmHg above controls. Nonetheless, the persistent rise in pulse pressure at the low and high doses of chlorpyrifos could represent an important indicator of toxicity of this OP. It is of interest to note the lack of dose-related effect of chlorpyrifos on pulse pressure; the 10 mg/kg dose was as effective as the 25 mg/kg treatment dose on pulse pressure. Two critical factors affect the pulse pressure: the stroke volume of the heart (i.e. blood ejected with each contraction) and the compliance or distensibility of the arterial tree (Guyton, 1986). The cholinergic stimulation resulting from AChE inhibition is likely to affect these factors. Stroke volume could also be affected by chlorpyrifos exposure; however, an increase in stroke volume is associated with a reduced heart rate. We suspect that chlorpyrifos reduces the compliance of the arterial tree since there is little change in heart rate after chlopyrifos exposure. Other than the reduced heart rate during the first night after 25 mg/kg chlorpyrifos, mean heart rate was similar to controls during the next day and night after chlorpyrifos. It is important to consider if the rise in blood pressure represents a possible hazard to humans exposed to chlorpyrifos and other OP pesticides. Clinical studies have shown that physostigmine causes marked elevations in blood pressure (Janowsky et al., 1986). Physostigmine in humans stimulated the adrenal release of epinephrine which can explain the hypertension and tachycardia of this agent (Kennedy et al., 1984). On the other hand, clinical case reports of humans exposed to anticholinesterase pesticides indicate hypotension or hypertension as well as bradycardia
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and tachycardia (Tafuri and Roberts, 1987; Saadeh et al., 1996, 1997). Of course, the clinical case reports have to viewed with caution because of the innumerable uncontrolled variables such as dose, compound purity, age of subject, drug intervention, and environmental conditions. The marked increase in blood pressure without a compensatory change in heart rate following chlorpyrifos exposure suggests that this OP alters the sensitivity of the control of blood pressure. The sensing and central processing of blood pressure by the baroreceptors in the carotid sinus and elsewhere in the circulatory system may be altered by chlorpyrifos. If this were not the case, then one would expect a reduction in heart rate and a concomitant reduction in cardiac output to reduce blood pressure. A resetting of the baroreflex could be mediated by a neurohormonal system such as vasopressin or angiotensin II. Vasopressin administered centrally can attenuate the baroreceptor reflex (Lebrun et al., 1987). Cholinergic stimulation of the CNS with physostigmine has been
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shown to stimulate vasopressin release (Lazartigues et al., 1998, 1999). The renin-angiotensin pathway is also activated by central cholinergic stimulation (Saad et al., 1997). There is apparently little information on how OP exposure can affect the activation of these neurohumoral pathways. Such a response could pose a risk to populations susceptible to heart disease, particularly those with high blood pressure. Hence, it will be important to discern the mechanism of action of OP-induced hypertension and to study the potential hypertensive responses in experimental models of hypertension such as the spontaneously hypertensive rat.
Acknowledgements We thank Drs W.P. Watkinson and A.H. Rezvani for their review of the manuscript. We also thank P. Becker for providing technical assistance.
Appendix A. Repeated measures ANOVA results of the effects of chlorpyrifos on the telemetry variables
Data set
Period
Treatment
Treatment-time
Mean pressure ‘‘ ’’ ‘‘ ’’ Systolic pressure ‘‘ ’’ ‘‘ ’’ Diastolic pressure ‘‘ ’’ ‘‘ ’’ Pulse pressure ‘‘ ’’ ‘‘ ’’ Heart rate ‘‘ ’’ ‘‘ ’’
D1 L2 D2 D1 L2 D2 D1 L2 D2 D1 L2 D2 D1 L2 D2
F(2,42) = 34.9, PB0.0001 F(2,42) = 11.8, PB0.0001 Not significant F(2,42) = 30.5, PB0.0001 Not significant Not significant F(2,42)= 34.4, PB0.0001 F(2,42) = 9.5, P= 0.0004 Not significant F(2,42) = 3.9, P= 0.03 F(2,42) = 17.5, PB0.0001 F(2,42) = 15.2, PB0.0001 F(2,45) = 8.5, P= 0.0007 Not significant Not significant
F(22,462)= 4.2, PB0.0001 F(22,462)= 2.4, P= .0004 F(22,462)= 3.5, PB0.0001 F(22,462= 3.7, PB0.0001 F(22,462)= 2.1, P= 0.002 F(22,462)= 3.6, PB0.0001 F(22,462)= 4.1, PB0.0001 F(22,462)= 2.7, PB0.0001 F(22,463)= 3.3, PB0.001 F(22,462)= 5.4, PB0.0001 F(22,462)= 3.1, PB0.0001 F(22,462)= 3.2, PB0.0001 F(22,495)= 4.7, PB0.0001 F(22,495)= 2.5, P= 0.0002 F(22,495)= 3.6, PB0.0001
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Core temperature ‘‘ ’’ ‘‘ ’’ Motor activity ‘‘ ’’ ‘‘ ’’
D1 L2 D2 D1 L2 D2
F(2,45) =18.9, PB0.0001 F(2,45) =23.7, PB0.0001 F(2,45) = 1.5, P= 0.024 Not significant Not significant F(2,45) =6.1, P= 0.005
References Aspelin, A. 1994. Pesticides industry sales and usage-1992 and 1993 market estimates. U.S. Environmental Protection Agency, report no. 733-K-94-001, June. Ballantyne, B., Marrs, T.C., 1992. Overview of the biological and clinical aspects of organophosphates and carbamates. In: Ballantyne, B., Marrs, T.C. (Eds.), Clinical and Experimental Toxicology of Organophosphates and Carbamates. Butterworth – Heinemann, Oxford, pp. 3–14. Bataillard, A., Sannajust, F., Yoccoz, D., Blanchet, G., Sentenac-Roumanou, H., Sassard, J., 1990. Cardiovascular consequences of organophosphorus poisoning and of antidotes in conscious unrestrained rats. Pharmacol. Toxicol. 67, 27– 35. Brezenoff, H.E., McGee, J., Knight, V., 1984. The hypertensive response to soman and its relation to brain acetylcholinesterase inhibition. Acta Pharmacol. Toxicol. 55, 270 – 277. Buccafusco, J.J., 1996. The role of central cholinergic neurons in the regulation of blood pressure and in experimental hypertension. Pharmacol. Rev. 48, 179–211. Gordon, C.J., Fogelson, L., Lee, L., Highfill, J., 1991. Acute effects of diisopropyl fluorophosphate (DFP) on autonomic and behavioral thermoregulatory responses in the Long-Evans rat. Toxicology 67, 1–14. Gordon, C.J., 1994. Thermoregulation in laboratory mammals and humans exposed to anticholinesterase agents. Neurotoxicol. Terat. 16, 427–453. Gordon, C.J., Rowsey, P.J., 1998. Poisons and fever. Clin. Exp. Pharm. Physiol. 25, 145–149. Gordon, C.J., Y.-L. Yang. 2000. Chlorpyrifos-induced hypothermia and vasodilation in the tail of the rat: blockade by scopolamine. Pharmacol. Toxicol. (in press). Guyton, A.C., 1986. Textbook of Medical Physiology, seventh edition. W.B. Saunders, Philadelphia. Janowsky, D.S., Rish, S.C., Kennedy, B., Ziegler, M., Huey, L., 1986. Central muscarinic effects of physostigmine on mood, cardiovascular function, pituitary and adrenal neuroendocrine release. Psychopharmacol. 89, 150–154. Kennedy, B., Janowsky, D.S., Risch, S.C., Ziegler, M.G., 1984. Central cholinergic stimulation causes adrenal epinephrine release. J. Clin. Invest. 74, 972–975. Kim, S.Y., Raikoff, K., Wulfert, E., Hanin, I., 1997. Brady-
.
F(22,495)= 8.5, F(22,495)= 6.3, F(22,495)=3.6, F(22,495)= 4.1, F(22,495)= 5.1, F(22,495)=4.3,
PB0.0001 PB0.0001 PB0.0001 PB0.0001 PB0.0001 PB0.0001
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