The Effects of Chlorpyrifos on Blood Pressure ... - Wiley Online Library

C Basic & Clinical Pharmacology & Toxicology 2005, 96, 503–511. Printed in Denmark . All rights reserved Copyright C ISSN 1742-7835

The Effects of Chlorpyrifos on Blood Pressure and Temperature Regulation in Spontaneously Hypertensive Rats Edward G. Smith1 and Christopher J. Gordon2 1

Livingstone College, Department of Biological Sciences, 701 West Monroe Street, Salisbury, North Carolina 28144, and 2Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, U.S.A. (Received October 20, 2004; Accepted December 27, 2004) Abstract: Using radiotelemetry to monitor blood pressure and core temperature, studies in our laboratory have shown that a prolonged hypertensive response is elicited in rats exposed to chlorpyrifos, an organophosphate-based insecticide. Chlorpyrifos inhibits acetylcholinesterase activity, resulting in central and peripheral stimulation of central cholinergic pathways involved in blood pressure regulation. The spontaneously hypertensive rat has been shown to be more sensitive to central cholinergic stimulation. Therefore, we hypothesized that these rats would be more susceptible and sustain a greater hypertensive response when exposed to chlorpyrifos. Heart rate, cardiac contractility, core temperature, and blood pressure were monitored by radiotelemetry in SHRs and their Wistar Kyoto (WKY) normotensive controls following exposure to chlorpyrifos (10 mg/kg or 25 mg/kg, orally). Baseline blood pressure of SHRs was ∂35 mmHg above that of WKYs prior to dosing. SHRs exhibited a greater and more sustained elevation in diastolic, mean and systolic blood pressure following exposure to 25 mg/kg of chlorpyrifos. The rise in blood pressure lasted for ∂56 hours in SHRs compared to ∂32 hours in WKYs. Chlorpyrifos also led to a prolonged elevation in daytime heart rate in both strains. There was a transient elevation in cardiac contractility in both strains lasting ∂7 hr after exposure to chlorpyrifos. The hypothermic response to chlorpyrifos was similar in magnitude and duration for both strains. Plasma cholinesterase activity measured 4 hr after exposure to 25 mg/kg chlorpyrifos was inhibited to ∂40% of control levels in both strains. Using the SHR strain as a model to study susceptible populations, the data suggest that individuals with a genetic predisposition to hypertension may be more susceptible from exposure to organophosphate-based insecticide, as manifested by an exacerbated hypertensive response.

High blood pressure (hypertension) is a major cause of morbidity and mortality in the United States (Thrilling & Froom 2000; Whelton et al. 2004). A large number of people have a genetic predisposition to hypertension, however, the aetiology has also been found to be environmentally influenced and involves a number of regulatory pathways (Trippodo & Frohlich 1981; Buccafusco 1996; Takahash & Smithies 2004). In this regard, diet, climate, and environmental toxicants have been suggested to have a key role in the etiology of hypertension (Colin et al. 2000; Svetkey et al. 2001; Jehn et al. 2002; Savoca et al. 2004). Therefore, appropriate models must be developed and used to evaluate the impact of these risk factors on the etiology and prognosis of hypertension. Several studies have implicated organophosphate-based insecticide exposure in derangements in blood pressure regulation, thermoregulation, and other autonomic nervous system functions (Vargas & Brezenoff 1998; Gordon 1994; Storm et al. 2000; Saadeh 2001; Asari et al. 2004). Organophosphate-based pesticides inhibit acetylcholinesterase ac-

Author for correspondence: Christopher J. Gordon, NHEERL/ Neurotoxicology Division MD-B105-04, U.S. Environmental Protection Agency, 109 Alexander Drive, Research Triangle Park, NC 27711, USA (fax π1 919 541 4416, e-mail Gordon.christopher/ epa.gov).

tivity resulting in central and peripheral stimulation of cholinergic pathways (Ballentyne & Marrs 1992). Cholinergic pathways in the brainstem that mediate pressor responses are thought to be operative in organophosphateinduced hypertension. Hypertension and other cardiovascular maladies have been reported in humans exposed to organophosphate-based insecticides (Ballantyne & Marrs. 1992; Saadeh 2001). Myocardial damage resulting from sympathetic and parasympathetic overactivity was noted in organophosphate-poisoned victims. In addition, atrial fibrillation, ventricular tachycardia, and diffuse myocarditis are common clinical findings, and in children with severe anticholinesterase insecticide poisoning, the presence of cardiac arrhythmias is associated with a poor prognosis (Saadeh et al. 1997; Verhulst et al. 2002). Many health care providers may not fully appreciate the risk of cardiac toxicity from organophosphate-based insecticide exposure in the normal population, let alone that portion of the population that may be more susceptible due to a genetic predisposition for hypertension or increased blood pressure associated with advancing age. The spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rat have been extensively used as a model for essential hypertension in man (Kiprov 1980; Bianchi et al. 1986; Lawler et al. 1988; Yamori 1991; Pinto et al. 1998). Central cholinergic stimulation of the pressor areas in the brainstem of the SHR has been shown to elicit greater hypertensive responses com-

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EDWARD G. SMITH AND CHRISTOPHER J. GORDON

pared to WKYs (Trippodo & Frohlich 1981; Buccafusco 1996). This may be attributed to higher density of muscarinic receptors in pathways involved in blood pressure regulation (Gattu et al. 1997). Hence, we suspect that the SHR strain could be more sensitive to organophosphate-based insecticides in terms of a greater hypertensive response. It may follow that there is a susceptible human population with increased sensitivity to organophosphate-based pesticides, i.e. those individuals predisposed to essential hypertension and other forms of elevated blood pressure. Radiotelemetry provides an ideal means of detecting subtle effects of a drug or toxicant on the regulation of blood pressure and other autonomic parameters (Kramer & Kinter 2003). The purpose of this study is to determine if the SHR and WKY strains exhibit differential sensitivity to the organophosphate chlorpyrifos in terms of the regulation of blood pressure, heart rate, cardiac contractility, and core temperature monitored by radiotelemetry.

Materials and Methods This protocol used male spontaneously hypertensive rats (SHR) and their Wistar Kyoto (WKY) normotensive controls obtained from Charles River Laboratories (Raleigh, NC, USA). The rats were maintained individually in standard cages with wood shavings and housed in an environmental chamber with an ambient temperature of 22–24 æ and 12:12 hr light: dark cycle. Telemetry implant. Radiotelemetry is an ideal and practical means of monitoring multiple physiological variables in undisturbed rats, 24 hr per day. The implant (Data Sciences Int., St. Paul, MN, USA; model C50-PXT) provides a direct measurement of blood pressure (systolic, mean, and diastolic) and the electrocardiogram (ECG). Heart rate and the QA interval are derived from the recording of blood pressure and the ECG. The QA interval is a parameter of the systolic time interval and is defined as the interval between the Qwave of the ECG and the onset of the aortic blood pressure pulse and has been used as a selective index of cardiac contractility (Cambridge & Whiting 1986). Therefore, a hypodynamic heart would have a longer QA index; conversely, a hyperdynamic heart would have a shorter QA index. The telemetry implant also provides a measure of core body temperature and motor activity. Surgical procedure. The rats were anaesthetized with sodium pentobarbital (50 mg/kg; intraperitoneally). The rats were 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 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 cyanoacrylate-based glue and a biocompatible cellulose patch. Once the ligature was removed, blood flow was restored and the entry 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. Rats were administered penicillin (30,000 units) and an analgesic (buprenorphine; 0.03 mg/kg; subcutaneously) immediately after surgery. The rats were allowed at least two weeks to recover from surgery prior to testing (Gordon & Padnos 2000). The survival rate for this surgery with experienced personnel is generally greater than 95%. Protocol. The telemetry parameters were monitored at 5 min. intervals from rats housed individually in the animal facility. They received food and water ad libitum throughout testing. At least 24 hr of baseline data were collected in undisturbed animals prior to the treatment regiment. The rats were approximately 4 months of age at the time of dosing. In the first phase of the study, SHR (nΩ9) and WKY (nΩ7) rats were administered the corn oil vehicle (0.1 ml/100 g bw) or chlorpyrifos (Chem Services; West Chester, PA, USA) at a dose of 10 or 25 mg/kg. The treatments were administered per os between 11:45 a.m. and 12:00 p.m. Care was taken to dose the rats quickly without excessive handling or stress. The animals were left undisturbed in their cages for 3 days after dosing. The rats were allowed to recover for at least 14 days from this treatment and then they were subjected to a cross-over design in which rats that were initially dosed with corn oil were given chlorpyrifos and conversely, rats that had been dosed with chlorpyrifos were dosed with corn oil. In the second phase of the study a new group of rats without the telemetry implant were exposed to chlorpyrifos or the corn oil vehicle alone. In this protocol an initial group of SHR (nΩ9) and WKY (nΩ9) rats were dosed with 10 mg/kg of chlorpyrifos or corn oil. Subsequently, a second separate group of SHR (nΩ6) and WKY (nΩ6) rats were dosed with 25 mg/kg chlorpyrifos or corn oil. All rats were euthanized 4 hr after dosing by CO2. Immediately after asphyxiation blood was collected in heparinized syringes by cardiac puncture. Plasma was separated (4000 g for 15 min. at 4 æ), aliquotted into 1 ml vials, and stored at ª22 æ. Plasma contains acetylcholinesterase and pseudocholinesterase or butylcholinesterase, both of which are inhibited by organophosphate-based insecticides. Measuring cholinesterase inhibition with the Ellman method provides a measure of the activity of both acetyl- and butylinesterase activity. Hence, the data in this study are denoted as plasma cholinesterase activity. Following the recommended procedures by the manufacturer of the kit (Sigma Diagnostics, procedure 422), cholinesterase activity was determined by measuring the breakdown of propionylthiocholine iodide to propionic acid and thiocholine. Thiocholine complexes with 5,5ø-dithiobis-2-nitrobenzoic acid and is measured spectrophotometrically at 405 nm. Cholinesterase activity was measured by incubating the reagents with the plasma for 30 sec. at 30 æ. Statistical analysis. The 5 min. telemetry data were reviewed for any artifacts that occasionally occur during noise in the telemetry signal. These data were then averaged into 30 min. bins and the standard error was calculated for graphic presentation. The data were further averaged into 12 hr. day and night groups and used for statistical analysis. Repeated measures analysis of variance (ANOVA) with time as a repeated factor was used to test for effects of chlorpyrifos treatment and strain on each telemetry parameter (SASA, Cary, North Carolina, USA). It is important to note in this analysis that the responses of each rat to corn oil and chlorpyrifos were assumed to be independent observations. This assumption is justified because the animals were allowed such a long period of time between administration of the control and test agent. Two-way repeated measures ANOVA’s (strain and chlorpyrifos treatment) with time as a repeated factor were applied to each telemetry parameter to determine if genetic strain had a significant effect on the response to chlorpyrifos. All probabilities for the analysis of the telemetry parameters were adjusted with the Greenhouse-Geisser correction factor. In all cases, the level of significance was set at Pⱕ0.05.

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HYPERTENSIVE RESPONSE TO CHLORPYRIFOS

Results Baseline responses. After a 14-day recovery period from implantation of transmitters, 24 hr recordings from undisturbed rats showed a robust circadian rhythm in all of the recorded variables, i.e., blood pressure, heart rate, QA interval, core temperature, and motor activity (table 1, fig. 1–6). As expected from previous studies in our laboratory (Gordon & Padnos 2000; Smith et al. 2001), there was a nocturnal elevation in core temperature, motor activity, blood pressure and heart rate in both strains. Systolic, mean, diastolic blood pressures of the SHR strain were approximately 36 mmHg above that of the WKY during the day and night. Also, the QA interval of the SHR strain was approximately 5 msec. less than that of the WKY during the day and night. The QA interval abruptly decreased immediately after the lights went off at 6:00 p.m. and remained decreased until the start of the next light phase at 6:00 a.m. On the other hand, heart rate was unaffected by strain. Daytime core temperature of the SHRs was significantly higher than that of the WKYs, whereas nighttime temperature was unaffected by strain. Response to chlorpyrifos. There was an abrupt increase in blood pressure, heart rate, core temperature, motor activity, and reduction in QA-interval following oral gavage of the corn oil vehicle and 25 mg/kg of chlorpyrifos. This transient response was attributable to the effects of handling and dosing (fig. 1–5). A description of the response of each telemetry variable to the administration of 25 mg/kg chlorpyrifos is given below. Both the time course of each variable averaged into 30 min. bins and averaged by day and night are presented. Results of repeated measures ANOVA of the average day and night responses are given in table 2. Blood pressure. Dosing with 25 mg/kg of chlorpyrifos resulted in a sustained elevation in systolic, diastolic, and mean blood pressure in both the SHR and WKY strains. Blood pressure of the SHR strain was markedly more sensitive to chlorpyrifos as indicated by a greater elevation with slower recovery to control levels. Systolic, mean, and diastolic blood pressure of the SHR strain peaked at 206 mmHg, 180 mmHg, and 153 mmHg, respectively. The peak elevation in mean blood pressure (D pressureΩ47 mmHg) of the SHR strain was observed at 4.5 hr after dosing. Following the peak pressure,

there was a gradual decrease over the next several hr but blood pressure remained elevated over the next several days. For example, by 48 hr after dosing, mean blood pressure of SHR rats given chlorpyrifos was ∂16 mmHg above controls. Systolic and diastolic pressures rose and fell in parallel fashion with mean pressure. Blood pressure of the WKYs given 25 mg/kg chlorpyrifos did not increase to the level seen in the SHRs, and the recovery was faster (fig. 1A, B, C). For example, the peak elevation in mean blood pressure (D pressureΩ23 mmHg) of the WKY strain was significantly above that of the corn oil controls during the first day of dosing but this change in pressure was only 50% of that observed in the SHR strain Over the next 60 hr, the analysis failed to detect significant effects of chlorpyrifos on systolic, mean, or diastolic pressure when averaged into day and nigh means. It is nonetheless important to note in the plot of the 30 min. means of blood pressure a trend for a hypertensive response in the WKY strain that appeared to reach a near complete recovery by the third day after dosing. Heart rate. Chlorpyrifos led to tachycardic effects in both strains that were manifested during the light phase and absent during the dark phase. The tachycardia persisted into the second day after treatment with 25 mg/kg of chlorpyrifos (fig. 2). As compared to blood pressure, heart rate of the SHR and WKY strains was equally sensitive to chlorpyrifos. Heart rate of both SHR and WKY strains increased from 280 to ∂340 beats/min. for several hr after chlorpyrifos administration and remained significantly elevated above controls throughout the light phase (fig. 2). During the dark phase, heart rate of the treated and control groups was similar as a result of the nocturnal increase in heart rate of control animals. During the next day (L2), heart rate of controls dropped to daytime levels whilst heart rate of rats dosed with chlorpyrifos remained elevated by 30 to 50 beats/min. With the next dark phase (D2), heart rate was again similar between treated and control groups. By the third day after chlorpyrifos treatment, there was a slight but statistically insignificant elevation in heart rate of both the SHR and WKY rats. QA interval. There were no treatment or treatment*time effects on the QA interval when analyzed over the 72 hr period (fig. 3; table 2). On the other hand, there appeared to be a transient

Table 1. Mean∫S.E. of blood pressure, heart rate, QA interval, core temperature, and motor activity of rats of the SHR and WKY strains when measured over a 24 hr period with a 12:12 light:dark cycle (lights on 6 a.m.). Strain-time

Systolic

Mean

Diastolic

Heart rate, b/min.

QA, msec.

Tc, E æC

SHR-day WKY-day SHR-night WKY-night

160.25∫4.4 122.08∫2.5 155.48∫11.8 125.23∫2.4

139.26∫3.2 104.18∫2.0 144.26∫4.7 106.99∫2.0

117.97∫2.7 87.75∫2.0 123.39∫3.6 90.8∫2.0

288.36∫3.6 285.16∫6.7 327.92∫4.6 329.41∫4.6

38.39∫0.83 42.93∫0.40 36.16∫0.99 41.5∫0.39

37.5∫0.06 37.1∫0.06 38.3∫0.06 38.2∫0.03

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Fig. 1A. Time-course of systolic blood pressure in normotensive (WKY) and hypertensive (SHR) rats when dosed with the corn oil vehicle (CO) or 25 mg/kg chlorpyrifos (CHP). Upper panel represents data averaged at 30 min. intervals. Lower panel represents data averaged during the day and night. Numbers in parentheses indicates sample size. Asterisks in bottom panel represent significant difference between control and treated for a given strain (P⬍0.05). B. Time-course of mean blood pressure in WKY and SHR rats dosed corn oil or 25 mg/kg chlorpyrifos. C. Time-course of diastolic blood pressure in WKY and SHR rats dosed corn oil or 25 mg/kg chlorpyrifos.

reduction in QA interval that persisted for several hr after dosing. Due to the transient reduction in QA interval compared to the other telemetry parameters, a post-hoc analysis of the response was reassessed with a repeated measures ANOVA for 6 hr at 30 min. intervals after dosing. When analyzed over this restricted time interval, there was a significant treatment*time effect for the SHR (PΩ0.006) but not for the WKY strain. Core temperature. Both strains became hypothermic following administration of 25 mg/kg chlorpyrifos (fig. 4). Mean core temperature

decreased from 37.2 æ to 35.8 æ by 4 hr after dosing then rose slowly over the next several hr and reached control levels by the start of the next light phase (L2). There was more variability in the WKY strain during L1 and there was not a statistically significant difference to the controls. Throughout the next day (L2), core temperature of both strains given chlorpyrifos remained elevated above their respective controls. For example, mean core temperature of SHR group during the day (L2) was 38.2 æ as compared to 37.4 æ for SHRs receiving corn oil. At the same time, WKYs receiving chlorpyrifos had a core temperature of 37.6 æ as compared to 37.0 æ for controls. During the next night (D2),


the typical nocturnal rise in core temperature of controls resulted in a near equaling of core temperature in the control and treated groups. During the following day (L3), core temperature of the SHR strain remained elevated above control levels. With the onset of the dark phase, the differences in core temperature between control and treated rats were again negated because of the nocturnal rise in core temperature of the control groups. The following day (L3), core temperature of the SHRs dosed with chlorpyrifos was significantly elevated whilst that of the WKYs had recovered.

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of the SHRs was again suppressed the following night as compared to the SHR control group. Motor activity of the WKY’s was unaffected by chlorpyrifos treatment. Plasma cholinesterase activity.

During the first night after dosing, motor activity of the SHRs given chlorpyrifos was significantly reduced compared to the controls (fig. 5). The following day (L2), motor activity was unaffected by strain and treatment but activity

When measured 4 hr after oral dosing with corn oil, plasma cholinesterase activity of the SHR strain was significantly higher than that of the WKYs (fig. 6). This difference in controls was seen in both the 10 and 25 mg/kg chlorpyrifos cohort studies with cholinesterase activity of the WKY strain 25 to 28% less than that of the SHR strain. The relative reduction in plasma cholinesterase activity following low and high doses of chlorpyrifos was similar between strains (fig. 6). Percent cholinesterase was reduced to 43.8% and 50.7% of control levels following 10 mg/kg and 38.9% to 41.7% of control levels following 25 mg/kg in the SHR and WKY strains, respectively.

Fig. 2. Time-course of heart rate in SHR (upper panel) and WKY (middle panel) strains dosed corn oil or 25 mg/kg chlorpyrifos. Lower panel is meanπS.E. of day and night periods. Abbreviations as in fig. 1.

Fig. 3. Time-course of the QA interval (index of cardiac contractility) and 12 hr day and night analysis in the SHR and WKY strains. Abbreviations as in fig. 1.

Motor activity.

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Fig. 4. Time-course of core temperature and 12 hr day and night analysis in the SHR and WKY strains. Abbreviations as in fig. 1.

Fig. 5. Time-course of motor activity and 12 hr day and night analysis in the SHR and WKY strains Abbreviations as in fig. 1.

Table 2. Summary of one-way repeated measures ANOVA analysis of the day and night time averaging of each telemetry parameter. Probabilities adjusted with Greenhouse-Geiser factor. NSSΩnot statistically significant. Strain

Parameter

Treatment effect

Treatment-time effect

SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY

Systolic pressure Systolic pressure Mean pressure Mean pressure Diastolic pressure Diastolic pressure Heart Rate Heart Rate QA Interval QA Interval Core Temperature Core Temperature Motor Activity Motor Activity

F F F F F F F F F F F F F F

F F F F F F F F F F F F F F

(1, (1, (1, (1, (1, (1, (1, (1, (1, (1, (1, (1, (1, (1,

11)Ω12.1, PΩ0.005 10)Ω7.3, PΩ0.022 11)Ω13.6, PΩ0.004 10)Ω5.8, PΩ0.04 11)Ω10.6, PΩ0.008 10)Ω8.04, PΩ0.02 10)Ω3.4, PΩ0.09 (NSS) 9)Ω2.0, PΩ0.19 (NSS) 10)Ω0.00, PΩ0.97 (NSS) 9)Ω0.29, PΩ0.60 (NSS) 10)Ω3.9, PΩ.08 (NSS) 9)Ω0.04, PΩ0.85 (NSS) 10)Ω4.2, PΩ0.07 (NSS) 9)Ω0.03, PΩ0.86 (NSS)

(5, (5, (5, (5, (5, (5, (5, (5, (5, (5, (5, (5, (5, (5,

55)Ω8.78, P⬍0.0002 55)Ω1.9, PΩ0.16 (NSS) 55)Ω8.7, PΩ0.0003 50)Ω3.0, PΩ0.06 (NSS) 55)Ω13.11, P⬍ 0.0001 50)Ω4.8, PΩ0.01 50)Ω14.1, P⬍0.0001 45)Ω2.7, PΩ0.09 (NSS) 50)Ω9.1, P⬍ 0.0003 50)Ω2.1, PΩ0.18 (NSS) 50)Ω45.3, P⬍ 0.0001 45)Ω6.4, PΩ0.007 50)Ω15.03, P⬍ 0.0001 45)Ω1.19, PΩ0.33 (NSS)


Fig. 6. Plasma cholinesterase (ChE) activity in WKY and SHR rats when measured 4 hr after gavage dosing with the corn oil vehicle, 10, or 25 mg/kg chlorpyrifos. ANOVA analysis: 10 mg/kg, FΩ206.8, P⬍0.0001; 25 mg/kg, F224.6, P⬍. 0001. **P⬍0.01 when compared to control SHR strain. Numbers in boxes represent percent inhibition in ChE activity relative to control. All chlorpyrifos treatment groups are significantly different from respective controls (P⬍0.01).

Ten mg/kg chlorpyrifos. The cardiovascular and thermoregulatory responses to 10 mg/kg chlorpyrifos were meager in both strains in spite of the marked inhibition in plasma cholinesterase activity. Overall, blood pressure of the SHR strain increased by approximately 15 mmHg during the first night after dosing (data not shown). There was little change in blood pressure of the WKY strain. Heart rate, core temperature, QA interval, and motor activity were essentially unaffected by the low dose of chlorpyrifos in both the SHR and WKY strains. Discussion The results of this study suggest that a genetic predisposition to hypertension exacerbates the sensitivity to the hypertensive effects of an organophosphate-based insecticide. Compared to the normotensive WKY strain, the SHR strain showed a marked sensitivity to chlorpyrifos, in terms of an exaggerated elevation in blood pressure. On the other hand, the response of other autonomic parameters, such as heart rate, QA interval, and core temperature, were similar to that of the WKY strain. In both strains, the rise in blood pressure persisted in spite of the recovery from the hypothermic and tachycardic effects of chlorpyrifos. The inhibition in plasma cholinesterase activity was similar in both strains when measured 4 hr after chlorpyrifos administration, suggesting that the stimulation of cholinergic pathways was not augmented in the SHR strain. Hence, the exaggerated hypertensive effects of chlorpyrifos in the SHR strain would not appear to be a result of increased sensitivity to the effects of chlorpyrifos on cholinergic systems per se. Since the hypothermic response to anti-cholinesterase agents in rats is mediated primarily by stimulation of muscarinic pathways in the CNS thermoregulatory centers (Gordon & Padnos 2000), the near identical hypothermic response to chlorpyrifos in the SHR and WKY strains indi-

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cates that these strains possess similar sensitivities to the cholinergic stimulation of an organophosphate-based insecticide. The marked hypertensive response in the SHR strain to chlorpyrifos would appear to be independent of the other cholinergic effects of the organophosphate-based insecticide. The initial effects of anti-cholinesterases such as chlorpyrifos on blood pressure would be expected to be hypertension since cholinergic pathways are well known in this process. Cholinergic neurones in the rostral ventrolateral medulla play a role in the regulation of blood pressure through an essential contribution to efferent sympathetic tone (Kubo et al. 1995). Systemic injection of chlolinergic agonist such as oxotremorine leads to an elevation in blood pressure in the telemetered rat (Smith et al. 2001). Moreover, immunohistochemical and pharmacological studies have implicated the observed increase in acetylcholine in the rostral ventrolateral medulla in the pathogenesis and maintenance of experimental hypertension regardless of its etiology (Kubo et al. 1998; Kubo 1998). Other studies have demonstrated that the hypothalamus is involved in the release of acetylcholine in the rostral ventrolateral medulla in hypertension (Li & Ku 2002; Kubo et al. 2003). The acute hypothermia and delayed hyperthermic response to chlorpyrifos has been reported previously in other strains of the rat (Gordon et al. 1997; Gordon & Padnos 2000). It is of interest to note that the hypothermic response of the SHR and WKY strains was considerably greater when compared to that of the Long-Evans (LE) rat given 25 mg/kg chlorpyrifos (Gordon & Padnos 2000). In the LE rat dosed orally with 25 mg/kg chlorpyrifos, the hypothermia peaked at 1.4 æ below controls and recovered in approximately 12 hr, while the hypertension peaked at 20 mmHg above control levels and persisted for approximately 27 hr. The hypothermic response is a well-characterized response to cholinergic stimulation of muscarinic receptors in the thermoregulatory centers (Gordon 1994). Stimulation of theses pathways leads to a heat loss response, characterized by tail vasodilation and preference for cooler ambient temperatures. The delayed hyperthermia, on the other hand, is not mediated by muscarinic receptor stimulation. The hyperthermia is much like a fever in that rats allowed to behaviourally thermoregulate prefer relatively warm temperatures and the hyperthermia is rapidly blocked by administration of sodium salicylate, an antipyretic agent (Gordon 1997; Gordon et al. 1997). It is possible that the pathways that are activated in the development of the delayed fever are also operative in the mediation of the hypertensive response. The initial burst in pressure following dosing with chlorpyrifos is most likely an acute autonomic response that, along with hypothermia, is likely a result of the marked inhibition in brain Acholinesterase activity and concomitant stimulation of pressor cholinergic pathways. The marked rise in cardiac contractility is most likely an important factor responsible for the initial elevation in blood pressure. Both strains underwent rapid but transient reduc-

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tions in QA interval following dosing with chlorpyrifos. To the best of our knowledge, this is the first report of an effect of an organophosphate-based insecticide on an index of cardiac contractility. It is interesting to note that, while QA interval was reduced during the first day at the same time as a marked elevation in heart rate, there was no change in QA interval the day after dosing in spite of an elevated heart rate. The hypertensive response persisting for several days after a single dose of chlorpyrifos is probably not mediated solely by cholinergic stimulation. Even though brain Acholinesterase activity is inhibited for several days after a single exposure to an organophosphate such as chlorpyrifos, the cholinergic stimulation of the CNS will not persist over the same time-course. That is, the hypothermic effects of chlorpyrifos abated after 4 to 6 hr in spite of the expected inhibition (i.e., ⬎50% reduction) by 24 hr after oral dosing with chlorpyrifos (Gordon et al. 1997; Nostrandt et al. 1997). Cholinergic stimulation could trigger a long-term hypertensive response, with this response being amplified in an SHR strain but little is known on how such a mechanism would operate. Other genetic factors not directly related to cholinergic (Nostrandt et al. 1997; Gordon et al. 1997). Cholinergic stimulation could trigger a long-term hypertensive response, with this response being amplified in an SHR strain but little is known on how such a mechanism would operate. Other genetic factors not directly related to cholinergic stimulation may magnify a hypertensive response. One possibility is PON1 polymorphism. PON1 is a HDL-associated enzyme that exhibits substrate-dependent activity polymorphism, as well as large variability in plasma concentration among organisms studied, i.e., mouse, rabbit, and man (Costa et al. 2003). HDL-associated PON1 is responsible for the metabolizing the chlorpyrifos oxon. It is interesting to note that mice lacking the PON1 gene are more susceptible to organophosphate toxicity (Costa et al. 2003). Therefore, the slow recovery in blood pressure to control levels may be partially explained by a difference in the degree and rate of hydrolysis of the chlorpyrifos oxon based on which isoform of PON1 is present in SHRs versus the WKY and LE rat strains (Costa et al. 2000). Overall, the SHR rat posses a marked sensitivity to chlorpyrifos in terms of a prolonged elevation in blood pressure, but similar sensitivity for other markers of organophosphate-exposure, including hypothermia, tachycardia, increased cardiac contractility, reduced motor activity, and inhibition which isoform of PON1 is present in SHRs versus the WKY and LE rat strains (Costa et al. 2000). and more prolonged hypertensive response to an organophosphate-based insecticide should be considered a possible risk in terms of the classic symptoms of cardiovascular disease. Taking into consideration the necessary caution for extrapolation from animal model to human, the current data suggest that individuals with a genetic predisposition to hypertension may represent a vulnerable group to the toxicological effects of organophosphate-based insecticides.

Acknowledgements We thank Beth Padnos and Peggy Becker for providing technical assistance. We are also most appreciative for technical reviews and analysis provided by Drs. J. Brown, R. Prewitt, and K. Ehman. 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.

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