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Cardiovascular effects of oral toluene exposure in the rat monitored by radiotelemetry
Neurotoxicology and Teratology 29 (2007) 228 – 235 www.elsevier.com/locate/neutera
Cardiovascular effects of oral toluene exposure in the rat monitored by radiotelemetry☆ Christopher J. Gordon ⁎, Tracey E. Samsam, Wendy M. Oshiro, Philip J. Bushnell Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, United States Received 10 July 2006; received in revised form 12 September 2006; accepted 16 October 2006 Available online 24 October 2006
Abstract Toluene is a hazardous air pollutant that can be toxic to the nervous and cardiovascular systems. The cardiotoxicity data for toluene come from acute studies in anesthetized animals and from clinical observations made on toluene abusers and there is little known on the response of the cardiovascular and other autonomic processes to graded doses of toluene. This study assessed the effects of toluene (0.4, 0.8, and 1.2 g/kg; gavage) on heart rate (HR), blood pressure, core temperature (Tc), and motor activity (MA) in unrestrained, male Long-Evans rats monitored by telemetry. Toluene doses of 0.8 and 1.2 g/kg elicited significant elevations in HR, characterized by a transient 100 beats/min increase in HR lasting 1 h followed with a steady state tachycardia lasting N 6 h. Overall, HR increased by 25 and 50 beats/min in the 0.8 and 1.2 g/kg groups, respectively. MA increased markedly in the 0.8 and 1.2 g/kg groups but the tachycardia persisted in spite of recovery of MA in the 0.8 g/kg group. There was a small (b 0.5 °C) increase in Tc above controls in rats dosed with 0.8 g/kg toluene, whereas 1.2 g/kg toluene elicited a transient reduction in Tc followed by a small elevation lasting several hours. In a second study, rats were implanted with transmitters to monitor blood pressure (BP), and were administered with toluene as in the first study. HR, Tc, and MA were also monitored. The tachycardic effects of toluene at 0.8 and 1.2 g/kg were associated with a rise in blood pressure. Doses of 0.8 and 1.2 g/kg elicited a mean BP elevation of 6 and 16 mm Hg, respectively, for 7-hour post-dosing. The biphasic tachycardia to toluene suggests multiple sites for eliciting the cardiotoxic effects of this toxicant. © 2006 Elsevier Inc. All rights reserved. Keywords: Radiotelemetry; Heart rate; Tachycardia; Volatile organic compounds
1. Introduction Toluene is a volatile organic compound (VOC) widely used in industrial settings nd has been shown to have a variety of adverse health effects. VOC's are prevalent in urban air and are generated by several types of regulated sources. In addition, many commercial products contain VOC's in the form of organic solvents, including paints, waxes, inks, cleaning solutions, and pesticides. The nervous and cardiovascular systems have been shown to be ☆
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. B105-04, U.S. EPA, 109 S. T.W. Alexander Drive, Research Triangle Park, NC 27711, United States. Tel.: +1 919 541 1509; fax: +1 919 541 4416. E-mail address: [email protected] (C.J. Gordon). 0892-0362/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2006.10.004
markedly susceptible to acute exposure to toluene vapors and other VOC's [2]. Furthermore, recent research suggests that relatively low doses of inhaled toluene can have adverse effects on brain neurochemistry [16]. There are renewed efforts to understand the toxicity of relatively low concentrations of toluene. The clinical reports on the treatment of abusers of toluenebased products (i.e., “glue sniffers”) provides a considerable data base on the hazardous effects of acute toluene on the cardiovascular system. The heart appears to be a sensitive target to toluene and other volatile organic compounds; there are several clinical case reports of ventricular sensitization and arrhythmic effects along with tachycardia or bradycardia in cases of acute toluene abuse [1,5,14]. There are surprisingly few studies on the cardiotoxic effects of toluene in experimental animals. The few studies that have been performed utilized anesthetized preparations subjected to
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acute doses of inhaled or parenterally administered toluene [12,19]. For example, a tachycardic response was seen in anesthetized rats exposed to a nearly saturated atmosphere of toluene or benzene [19]. Morvai et al. [12] showed an appreciable fall in blood pressure of anesthetized rats administered toluene intravenously. On the other hand, anesthetized mice exposed to high doses of inhaled toluene develop bradycardia and atrialventricular block [18]. The depressant action of anesthetics on the CNS and cardiovascular system deems the use of anesthetized rodents as unacceptable models to study the toxicity of VOC's in most cases. Radiotelemetry technology that utilizes surgically implanted radiotransmitters is an ideal means of monitoring cardiovascular and other autonomic processes in awake, undisturbed rodents. The unrestrained, awake rodent is probably the most sensitive model to study the autonomic effects of a toxicant [8,9]. The purpose of this study is to assess the effects of toluene exposure on blood pressure, heart rate, core temperature, and motor activity in the unrestrained, awake rat. It is important to comment on our selection of an oral route of exposure. Inhalation exposure would be most relevant to study the cardiotoxic effects of an air pollutant. Oral route of exposure has often been used in acute toluene studies because it is less costly and time-consuming than inhalation systems. Inhalation “nose-only” systems involve prolonged restraint of the test subject. The stress of restraint is clearly not conducive for collecting physiological data that are free of stress artifacts. A single oral dose of toluene elicits a prolonged elevation in blood toluene that can be used to model the acute effects of inhalation exposure [10,17]. Blood toluene levels can be manipulated in rats dosed orally to match that when they are exposed to given air concentrations [10,17] (see Discussion). In the current study, telemetry is used to monitor the physiological response to an acute, oral dose of toluene that can be used to predict physiological responses to equivalent inhaled doses. 2. Materials and methods Animals used in these studies were male rats of the LongEvans strain acquired from Charles River Laboratory (Raleigh, NC) at approximately 2 months of age. The rats were housed individually in plastic cages with pine wood shavings at an ambient temperature of 22 °C and 50% relative humidity with a 12:12 light:dark photoperiod with lights on at 0600 h. 2.1. Surgery In the first study, heart rate, core temperature, and motor activity were monitored in seven unrestrained rats using radiotelemetry (model TA11CTA-F40; Data Sciences Int., St. Paul, MN). Details of the surgical procedure have been published [8]. Briefly, rats were anesthetized with Nembutal (50 mg/kg; IP). A midline incision was made in the abdominal musculature and a transmitter with electrocardiogram leads was secured inside the abdominal cavity and sutured to the midline. The ECG leads were tunneled under the skin and positioned on the left and right sides of the thorax allowing for the best detection of the ECG
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signal. The skin was closed with wound clips and the rat was administered an analgesic (buprenorphine; 0.03 mg/kg; SC) and an antibiotic (penicillin; ∼ 1000 units; IM) and allowed to recover for at least 10 days prior to study. In a second study, five rats were implanted with radiotransmitters (model TL11M2-C50-PXT; Data Sciences) to monitor blood pressure, ECG, heart rate, core temperature, and motor activity. Details on the surgical procedure have been published [15]. The transmitters were implanted by the staff of Charles River Laboratories in Raleigh, NC. Once the rats had recovered from the surgery for at least 7 days, they were shipped to our facility for study. These transmitters have a catheter that is placed into the lumen of the descending aorta to measure aortic blood pressure. A gel in the tip of the catheter inhibits clot formation in most cases. The transmitter provides a continuous measure of systolic, diastolic, and mean blood pressure. Heart rate is derived from the pressure pulse. Core temperature and motor activity are also monitored. The QA interval, a measure of cardiac contractility, was also monitored at 5 min intervals. The QA interval is defined as the interval between the depolarization of the ECG and the upswing of aortic pressure and is calculated automatically by the telemetry software [15]. 2.2. Protocol Rats were dosed with toluene diluted in corn oil (dosing volume = 4 mL/kg). All rats were given corn oil, 0.4, 0.8, and 1.2 g/kg toluene (99.5% spectrophotometric grade, SigmaAldrich Chemical Co., St. Louis, MO). Each rat received the control and three toluene treatments in a different, randomlyselected sequence. The telemetry parameters were monitored at 5 min intervals in rats left undisturbed in the animal facility. The day before dosing, the rats were weighed and placed into a clean cage of wood shaving bedding. Animals were allowed food and water ad libitum. On the day of dosing, all rats were administered with toluene within 10 min of 1100 h and were then left undisturbed for 72 h. Toluene administered by oral gavage is completely cleared to nearly undetectable levels in the circulation within 24 h after dosing [10,17]. Moreover, the telemetric parameters appeared to be near fully recovered within 48 h after dosing. Hence, it was decided that a recovery interval of at least 3 days was adequate. Rotating the animals through each control and toluene treatment allowed for the experiment to be performed with a minimum number of animals. Mean body weight of rats from the start to the end of each study was 433 to 490 g in Study 1 and 341 to 402 g in Study 2. 2.3. Statistical analysis The average response for each telemetry parameter, beginning 30 min after dosing (1130 h) until the onset of the dark phase (6 pm), was averaged for each animal. These mean responses over the 6.5-hour interval were analyzed for statistical significance using repeated measures, one-way analysis of variance (RMANOVA) (Sigma Stat® 3.1). If there was a significant treatment effect for each telemetry parameter, then a
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comparison of each toluene treatment group to the control was performed using the Hold–Sidak method (Sigma Stat) with an overall significance level set a p = 0.05.
Table 1 Analysis of the overall effects of toluene on each telemetry parameter when compared to the change from control values Treatment
Time
Heart rate, beats/min
Core temperature, °C
Motor activity, counts/min
Control 0.4 g/kg 0.8 g/kg 1.2 g/kg Control 0.4 g/kg 0.8 g/kg 1.2 g/kg
Day Day Day Day Night Night Night Night
338.18 ± 17.62 346.44 ± 6.18 363.05 ± 20.51⁎ 378.81 ± 13.49⁎ 390.65 ± 18.08 375.46 ± 22.45 368.91 ± 13.50⁎ 361.46 ± 21.68⁎
37.34 ± 0.098 37.47 ± 0.154 37.47 ± 0.154 37.44 ± 0.290 38.09 ± 0.076 37.94 ± 0.10 37.95 ± 0.10⁎ 37.89 ± 0.18⁎
1.91 ± 0.67 2.29 ± 0.75 3.42 ± 1.12 6.01 ± 2.57⁎ 5.45 ± 2.48 4.90 ± 1.73 4.97 ± 1.89 4.49 ± 2.07
3. Results 3.1. Study 1 (heart rate, core temperature, and activity) Long-Evans rats displayed distinct circadian rhythms of heart rate, blood pressure, core temperature, and motor activity when left undisturbed in the animal facility (Fig. 1). Prior to dosing, heart rate was at minimal daytime levels of 300– 325 beats/min; core temperature was 37.0 to 37.2 °C and motor activity was minimal (Fig. 1B, C). Administering the corn oil vehicle and toluene led to abrupt increases in all telemetry variables, a response attributed to the stress of handling and dosing. In the first 30 min after dosing, the heart rate of control rats increased to over 400 beats/min then recovered rapidly to a level of ∼ 325 beats/min for the remainder of the light phase. The heart rate of rats dosed with 0.4, 0.8, and 1.2 g/kg peaked at 400 to 425 beats/min during the first 30 min after dosing. These acute tachycardic responses were transient, but heart rate remained elevated above control levels throughout the light phase in rats dosed with 0.8 and 1.2 g/kg. Overall, heart rate during the light phase was significantly elevated by toluene, increasing by 25 and 50 beats/min over the 6.5-hour period at doses of 0.8 and 1.2 g/kg, respectively (Table 1). During the dark phase, heart rate showed a notable reduction in the toluene groups with overall significant reductions at doses of 0.8 and 1.2 g/kg. Heart rate was not significantly affected by 0.4 g/kg. However, this reduction in nocturnal heart rate was not
Data expressed as mean ± S.D. Asterisks indicate significant difference from control ( p b 0.05).
replicated in Study 2 (see below). The electrodes implanted in one rat did not provide a clear ECG signal; hence, the heart rate data for this animal were excluded from the analyses, leaving an N of 6 for the heart rate and N of 7 for temperature and activity. Core temperature increased transiently following dosing with corn oil, 0.4, and 0.8 g/kg toluene (Fig. 1B). Temperature of the 0.4 g/kg group was similar to that of controls whereas the 0.8 g/ kg exhibited an elevated temperature that persisted through much of the light phase. Rats dosed with 1.2 g/kg toluene underwent a transient decrease in temperature and did not exhibit the stress-induced rise in core temperature that was seen in the controls and the 0.4 and 0.8 g/kg treatment groups. At approximately 2.5 h after dosing, core temperature of the 1.2 g/ kg group increased above control levels, reaching 38 °C by 5 h after dosing. Overall, there were no significant effects of toluene on core temperature when it was assessed over the 6.5-hour
Fig. 1. Time-course of heart rate (A), core temperature (B), and motor activity (C) in rats dosed by oral gavage with the corn oil vehicle, 0.4, 0.8, and 1.2 g/kg toluene. Each plot represents the response of a given dose of toluene compared to that of the controls. N = 7 for core temperature and activity; N = 6 for heart rate.
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period after dosing. Nevertheless, exploratory analysis of the core temperature during the periods of maximum hypothermia and hyperthermia after dosing suggested an attenuation of the normal circadian temperature rhythm. There was a significant reduction in core temperature in the highest dose groups during the night after toluene exposure. Motor activity increased markedly during the first hour after dosing with the corn oil vehicle and toluene treatments (Fig. 1C). The peak rise in activity was notably higher in rats dosed with 0.8
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and 1.2 g/kg. Motor activity in the 0.8 g/kg group remained elevated for about an hour after dosing and then returned to control levels. On the other hand, motor activity of rats dosed with 1.2 g/kg was elevated for 2 h after dosing, decreased to near control levels, then increased again for another 3 h. Overall, motor activity in the 1.2 g/kg dose group increased by 4.1 counts/min over the 6.5-hour period after dosing (Table 1). At night, there was a slight reduction in motor activity in rats dosed with toluene but the reduction was not significant (Table 1).
Fig. 2. Time-course of blood pressure heart rate (A), systolic (B), mean (C), and diastolic blood pressure (D), QA interval (E), core temperature (F), and motor activity (G) of rats administered the corn oil vehicle or toluene at doses of 0.4, 0.8, or 1.2 g/kg. N = 5 for all parameters except blood pressure and QA interval where N = 4 for the 1.2 g/kg treatment.
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after dosing. In the 0.8 g/kg group, the elevated blood pressure was maintained for about 3 h and then equaled control levels at the onset of the dark phase. Following administration of the 1.2 g/kg dose, the recording of blood pressure gave spurious readings in one rat and those data were excluded from analysis. In the other four rats administered 1.2 g/kg, the transient increase in blood pressure observed immediately after dosing was higher than the control response. Blood pressure was maintained above control values after dosing and the hypertension persisted for several hours into the dark phase (Fig. 2B, C, D). QA interval was unaffected by toluene (Fig. 2E). Although there were no significant treatment effects on QA interval, there was a trend in the high dose group for a reduction in QA interval that was associated with time of maximal increase in blood pressure. Core temperature of rats dosed with 1.2 g/kg decreased transiently then rebounded abruptly beginning 2.5 h after dosing and increased by nearly 1.0 °C over the next 90 min (Fig. 2F). The time course of core temperature of the 0.4 and 0.8 g/kg groups was similar to that of the control group. 3.3. Heart rate vs. motor activity Fig. 2 (continued ).
3.2. Study 2 (blood pressure, heart rate, core temperature, QA, and activity) The acute effects of toluene on heart rate and motor activity observed in Study 1 were replicated using the blood pressure transmitter system. One exception was a higher mean heart rate in the control group which was ∼20 b/min higher over the 6.5-hour period as compared to the controls in the first study (Fig. 2A). Nonetheless, the significant effects of toluene doses of 0.8 and 1.2 g/kg were quite similar as in Study 1 with heart rate over the 6.5-hour period increasing an average of 38 and 54 b/min above controls, respectively (Table 2). Motor activity was significantly elevated by the 0.8 and 1.2 g/kg toluene treatments. The 0.4 g/kg dose had no significant effects on heart rate or motor activity. The tachycardic effects of toluene were associated with significant elevations in blood pressure (Fig. 2B, C, D; Table 2). Systolic, mean, and diastolic pressure changed in parallel fashion. Toluene doses of 0.8 and 1.2 g/kg elicited elevations in blood pressure that were maintained throughout the light phase
An interesting aside in both studies is the comparison of heart rate and motor activity in the rats dosed with 0.8 g/kg. The time scales have been expanded around the time of dosing to view the dynamics of heart and motor activity (Fig. 3A, B). The time course of heart rate following 0.8 g/kg was remarkably similar in both studies. Heart rate increased rapidly after toluene and remained at a steady level of 425 beats/min (Study 1) and 450 beats/min (Study 2) for 1 h. Heart rate then dropped precipitously, reaching a new steady state level of 40–50 beats/min above controls that persisted for most of the light phase. The abrupt decrease in heart rate in the first hour coincided with a marked drop in motor activity (denoted by dashed arrows in Fig. 3). 4. Discussion Exposure to toluene via oral gavage in the awake, unrestrained rat elicited a marked tachycardia and hyperactivity with relatively little change in body temperature. We suspected that the tachycardic effects of toluene would be more easily detectable when the rat was studied in a resting state. The rat is nocturnal and exhibits a circadian rhythm of heart rate, core temperature, and motor activity. During the daytime, heart rate approaches basal
Table 2 Analysis of effects of toluene on blood pressure, heart rate, QA interval, core temperature, and motor activity Parameter
Control
0.4 g/kg
0.8 g/kg
1.2 g/kg
Mean pressure, mm Hg Diastolic pressure, mm Hg Systolic pressure, mm Hg Heart rate, beats/min QA interval, ms Core temperature, °C Motor activity, counts/min
103.87 ± 4.23 90.66 ± 3.676 119.18 ± 6.35 361.08 ± 23.61 40.27 ± 2.73 37.61 ± 0.121 0.78 ± 0.53
108.08 ± 6.35 95.67 ± 6.94 122.34 ± 7.76 370.64 ± 34.35 40.37 ± 2.61 37.58 ± 0.199 1.06 ± 0.67
110.30 ± 6.86 96.99 ± 5.12 125.06 ± 8.92 399.92 ± 21.74⁎ 39.90 ± 2.91 37.63 ± 0.217 3.52 ± 2.50⁎
119.05 ± 9.82⁎ 104.41 ± 8.77⁎ 134.95 ± 11.21⁎ 415.76 ± 22.19⁎ 39.47 ± 3.78 37.60 ± 0.264 6.58 ± 2.13⁎
Data averaged over 7-hour period from time of dosing until onset of dark phase. N = 5. Asterisks indicate significant difference from control ( p b 0.05).
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Fig. 3. Expanded view of the time-course of heart rate and motor activity in rats dosed with 0.8 g/kg toluene in Study 1 (A) and Study 2 (B). Note the abrupt decrease in heart rate coinciding with secondary peak in motor activity (denoted by dashed arrows) that occurs approximately 1 h after dosing.
levels of approximately 325 beats/min when the rat is housed at an ambient temperature of 22 °C on a wire-screen floor [22]. At night, heart rate increases to over 400 beats/min. Hence, by exposing resting rats to toluene during the daytime when housed on pine shaving bedding that affords insulation and more favorable thermal environment, we contend that the cardiovascular system is close to basal, resting conditions and dosedependent tachycardia and hypertension should be detected at lower doses of toluene. The tachycardic effects of toluene at doses of 0.8 and 1.2 g/ kg were apparent within 15 min after dosing as evidenced by the recovery of the telemetric parameters in rats given corn oil (i.e., stress response) but a persistent elevation in heart rate of the toluene-treated animals. One novel finding in this study is the biphasic heart rate response characterized by an initial tachycardia followed by the establishment of a lower, steady state heart rate that was significantly elevated above controls for at least 6 h after dosing. Another interesting observation was the inability to observe recovery of heart rate. That is, the recovery from tachycardia could not be seen because, at the onset of the dark phase, heart rate of controls and toluene-treated animals increased together, thus eliminating the ability to view a recovery from toluene. Finally, when plotted side-by-side, blood pressure and heart rate essentially rise and fall simultaneously following dosing with toluene (Fig. 4). This would suggest that the hypertensive response to toluene is driven primarily by the tachycardia but this will require further
investigation. Core temperature was mildly affected by toluene with a transient hypothermia that was seen only after exposure to the highest dose, and was followed by a delayed hyperthermia that may be associated with the period of hypertension. There have been a few studies on the effects of toluene and other VOC's on heart rate and blood pressure but the
Fig. 4. Expanded view of the time-course of mean heart rate and blood pressure of rats dosed with 1.2 g/kg toluene. Note how blood pressure tracks closely with the changes in heart rate. N = 5 for heart rate; N = 4 for pressure.
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measurements were made in anesthetized rats [12,19]. These studies reported on tachycardic, bradycardic, and arrhythmogenic effects following acute exposure to VOC's but the results should be viewed with caution in view of the use of anesthesia. Lethal effects of acute toluene in anesthetized dogs can be a result of fatal arrhythmia as a result of direct effects of the VOC on the myocardium [6]. Exposure to near lethal quantities of toluene and benzene in chloralose-anesthetized rats leads to tachycardia [19]. No evidence of hypertension was observed in anesthetized rats administered acute doses of toluene intravenously [12]. To the best of our knowledge, this is the first report of using radiotelemetry to assess the effects of graded doses of toluene on cardiovascular and thermoregulatory parameters in resting, undisturbed rats. It is clear that the cardiovascular responses observed under the conditions of the present study are not parallel with those seen in anesthetized animals. In our opinion, telemetry is the best approach to collect data on the physiological effects of VOC's in experimental animals that can be extrapolated to humans. There has been one study of toluene-induced tachycardia in awake rats monitored by radiotelemetry. A preliminary report by Oshiro et al. [13] used radiotelemetry to determine the effects of inhaled toluene on heart rate and body temperature of LongEvans rats. Heart rate and core temperature were monitored by radiotelemetry in rats trained to perform an operant signal detection task. Rats inhaled toluene vapor for 1 h either while sedentary or while performing the behavioral task. In sedentary rats, 2000 ppm toluene increased heart rate from about 250 to ∼ 350 b/min by 1 h of exposure. Toluene concentrations in the brain under these conditions increased to ∼70 μg/mL over the course of the hour of exposure (unpublished observations). In rats actively performing the task, heart rate in rats breathing clean air was ∼370 beats/min, and toluene increased it by only 20 to about 390 beats/min. Brain toluene concentrations in these active rats reached about 100 μg/mL. Thus, the magnitude of the toluene-induced tachycardia depended more on the control baseline heart rate than upon the concentration of toluene in the brain. In contrast to the present evidence for toluene-induced hyperthermia, inhaled toluene induced a hypothermic response in these trained rats, observed against a ∼ 1 °C increase in temperature associated with performing the task. This difference could reflect the route of exposure or the temperature of the animals in the absence of toluene. How would the no observed adverse effects level (NOAEL) and lowest observed adverse effects level (LOAEL) doses for oral toluene exposure in the current study (i.e., 0.4 and 0.8 g/kg, respectively) compare to the estimated concentrations of inhaled toluene? Two studies provide estimates that used toluene dissolved in corn oil. Sullivan and Conolly [17] compared blood toluene levels in Sprague-Dawley rats exposed to toluene via oral gavage to that of a 6-hour inhalation. Toluene doses of 0.433 g/kg and 0.867 g/kg elicited elevations in blood toluene that reached an asymptote at approximately 2 h after dosing. Blood toluene levels in rats given the lower dose of toluene fell slowly after reaching the asymptote over the next 4 h, whereas rats given the higher dose of toluene exhibited near steady state blood toluene levels for several hours after reaching the
asymptote. A correlation between oral and inhalation routes shows that a concentration of 607 ppm is approximately equal to an oral dose of 0.4 g/kg; 1274 ppm is equivalent to 0.8 g/kg [17]. Gospe and Al-Bayati [10] found that the asymptote for blood toluene in the rat dosed orally with 0.741 and 0.911 g/kg was 4.33 and 6.3 h, respectively. These investigators used a 3-hour exposure as compared to the 6-hour inhalation exposure described above [17]. Blood toluene levels reached their asymptote within 2 h of exposure to ambient concentrations of 99 to 1145 ppm [10]. The analysis of the relationship between oral and inhaled toluene differed substantially from Sullivan and Conolly [17]; 2500 ppm was equivalent to an oral dose of 0.4 g/ kg and 5200 ppm was equivalent to 0.8 g/kg. It is interesting to note that the pattern of tachycardia does not correlate with the predicted elevations in blood and brain toluene concentrations reported by others [10,17]. For example, we found in both studies that an oral dose of 0.8 g/kg toluene resulted in a 100 beats/min increase in heart rate that was maintained for the first hour after dosing. This was followed with a drop in heart rate and maintenance at a steady level above controls for several hours. Hence, the tachycardic response subsides to a steady state level prior to blood and brain toluene reaching their peak concentrations. This would suggest that the cardiac responses to ingested toluene involves multiple points of sensory and effector response to the toxicant. Clinical case reports on accidental and occupational exposure to toluene point to tachycardic and ventricular arrhythmias. Japanese painters exposed to toluene and other VOC's exhibit a variety of CNS maladies [11]. Two symptoms suggestive of cardiovascular dysfunction are cold hands and feet as well as dizziness. Chronic toluene abuse (i.e., “glue-sniffing”) in humans is associated with cardiac sensitization, which generally results in tachyarrhythmias, but some bradycardic events have also been noted [7]. Rats exposed chronically to relatively low concentrations of toluene (40 to 80 ppm) underwent changes in CNS neurochemistry (dopaminergic and serotonergic pathways) as well as behavioral alterations [3,16,20]. Brain regions showing marked changes in catecholamine biosynthesis are involved in neurovegetative and cardiorespiratory regulations. Overall, the use of radiotelemetry allows for assessment of cardiovascular and other autonomic effects of toluene in the undisturbed rat. There is a dose-related tachycardia and hypertensive response that persists for at least 7 h after dosing. It is not clear how long the tachycardia effects persist because the night time elevation in heart rate of control rats obviates any effects of the toxicant on baseline heart rate The literature on cardiotoxic effects of toluene and other VOC's implies that the tachycardic and arrythmogenic effects of these compounds arise from their direct effects on the cardiac tissue. On the other hand, the well known effects of acute toluene on catecholamine pathways in the CNS would suggest that the cardiovascular effects arise from a central origin. Several studies suggest central activation of serotonergic pathways in rats exposed acutely to toluene [4,21]. Such activation of serotonergic pathways could be responsible for the tachcardic effects of this VOC. The unusual pattern of tachycardia that was most
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pronounced in rats dosed with 0.8 g/kg toluene illustrates the peculiar sensory-effector response to toluene. Future studies will discern the mechanisms of action and site of origin (i.e., central and/or peripheral nervous system) for mediation of toluene-induced tachycardia and hypertension. Acknowledgement We thank Drs. L. Leon and V. Benignus for their review of the manuscript. We also thank P. Becker for providing technical assistance. References [1] M. Bass, Sudden sniffing death, J. Am. Med. Assoc. 212 (1970) 2075–2079. [2] V.A. Benignus, Health effects of toluene: a review, Neurotoxicology 2 (1981) 567–588. [3] P. Berenguer, C. Soulage, D. Perrin, J.-M. Pequignot, J.H. Abraini, Behavioral and neurochemical effects induced by subchronic exposure to 40 ppm toluene in rats, Pharmacol. Biochem. Behav. 74 (2003) 997–1003. [4] L. Castilla-Serna, M.G. Barragan-Mejia, R.A. Rodriguez-Perez, A. Carcia Rillo, C. Reyes-Vazquez, Effects of acute and chronic toluene inhalation on behavior, monoamine metabolisms and specific binding (3H-serotonin and 3H-norepinephrine) of rat brain, Arch. Med. Res. 24 (1993) 169–176. [5] S. Einav, Y. Amitai, J. Reichman, D. Geber, Bradycardia in toluene poisoning, J. Toxicol. Clin. Toxicol. 35 (1997) 295–299. [6] N. Ideda, H. Takahashi, K. Umetsu, T. Suzuki, The course of respiration and circulation in “toluene-sniffing”, Forensic Sci. Int. 44 (1990) 151–158. [7] R.D. Gerkin Jr., F. LoVecchio, Rapid reversal of life-threatening tolueneinduced hypokalemia with hemodialysis, J. Emerg. Med. 16 (1998) 723–725. [8] C.J. Gordon, Acute and delayed effects of diisopropyl fluorophosphate (DFP) on body temperature, heart rate, and motor activity in awake, unrestrained rats, J. Toxicol. Environ. Health 39 (1993) 247–260. [9] C.J. Gordon, Temperature and Toxicology: An Integrative, Comparative, and Environmental Approach, CRC Press, Boca Raton, FL, 2005.
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[10] Gospe, M.A.S. Al-Bayati, Comparison of oral and inhalation exposures to toluene, J. Am. Coll. Toxicol. 13 (1994) 21–32. [11] R. Ishi, I. Harabuchi, Y. Katakura, T. Ikeda, H. Miyake, Neurobehavioral effects of chronic occupational exposure to organic solvents among Japanese industrial painters, Environ. Res. 62 (1993) 303–313. [12] V. Morvai, A. Hudak, G. Ungvary, B. Varga, ECG changes in benzene, toluene and xylene poisoned rats, Acta med. Acad. Sci. Hung. 33 (1976) 275–286. [13] W.M. Oshiro, T.E. Samsam, Q.T. Krantz, W.P. Watkinson, P.J. Bushnell, Age-related Differences in Heart Rate, But Not Body Temperature, in Rats Performing Operant Tasks at Equivalent Trial Rates in Air and While Inhaling Toluene, Society of Toxicology, Salt Lake City, UT, March 2003. [14] C.F. Reinhardt, A. Azar, M.E Maxfield, P.E. Smith Jr., L.S. Mullin, Cardiac arrhythmias and aerosol “sniffing, Arch. Environ. Health 22 (1971) 265–279. [15] E.G. Smith, C.J. Gordon, The effects of chlorpyrifos on blood pressure and temperature regulation in spontaneously hypertensive rats, Basic Clin. Pharmacol. Toxicol. 96 (2005) 503–511. [16] C. Soulage, D. Perrin, P. Berenguer, J.M. Pequignot, Sub-chronic exposure to toluene at 40 ppm alters the monamine biosynthesis rate in discrete brain areas, Toxicology 196 (2004) 21–30. [17] M.J. Sullivan, R.B. Conolly, Comparison of blood toluene levels after inhalation and oral administration, Environ. Res. 45 (1988) 64–70. [18] G.J. Taylor, W.S. Harris, Glue sniffing causes heart block in mice, Science 170 (1970) 866–877. [19] H. Vidrio, G.A. Magos, M. Lorenzana-Jimenez, Electrocardiographic effects of toluene in the anesthetized rat, Arch. Int. Pharmacodyn. 279 (1986) 121–129. [20] M. Von Euler, T.M. Pham, M. Hillefors, B. Bjelke, B. Henriksson, G. von Euler, Inhalation of low concentrations of toluene induces persistent effects on a learning retention task, beam-walk performance, and cerebrocortical size in the rat, Exp. Neurol. 163 (2000) 1–8. [21] S. Yamawaki, T. Segawa, K. Sarai, Effects of acute and chronic toluene inhalation on behavior and (3H)-serotonin binding in rat, Life Sci. 30 (1982) 1997–2002. [22] Y. Yang, C.J. Gordon, Ambient temperature limits and stability of temperature regulation in telemetered male and female rats, J. Therm. Biol. 21 (1996) 353–363.
Cardiovascular effects of oral toluene exposure in the rat monitored by radiotelemetry☆ Christopher J. Gordon ⁎, Tracey E. Samsam, Wendy M. Oshiro, Philip J. Bushnell Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, United States Received 10 July 2006; received in revised form 12 September 2006; accepted 16 October 2006 Available online 24 October 2006
Abstract Toluene is a hazardous air pollutant that can be toxic to the nervous and cardiovascular systems. The cardiotoxicity data for toluene come from acute studies in anesthetized animals and from clinical observations made on toluene abusers and there is little known on the response of the cardiovascular and other autonomic processes to graded doses of toluene. This study assessed the effects of toluene (0.4, 0.8, and 1.2 g/kg; gavage) on heart rate (HR), blood pressure, core temperature (Tc), and motor activity (MA) in unrestrained, male Long-Evans rats monitored by telemetry. Toluene doses of 0.8 and 1.2 g/kg elicited significant elevations in HR, characterized by a transient 100 beats/min increase in HR lasting 1 h followed with a steady state tachycardia lasting N 6 h. Overall, HR increased by 25 and 50 beats/min in the 0.8 and 1.2 g/kg groups, respectively. MA increased markedly in the 0.8 and 1.2 g/kg groups but the tachycardia persisted in spite of recovery of MA in the 0.8 g/kg group. There was a small (b 0.5 °C) increase in Tc above controls in rats dosed with 0.8 g/kg toluene, whereas 1.2 g/kg toluene elicited a transient reduction in Tc followed by a small elevation lasting several hours. In a second study, rats were implanted with transmitters to monitor blood pressure (BP), and were administered with toluene as in the first study. HR, Tc, and MA were also monitored. The tachycardic effects of toluene at 0.8 and 1.2 g/kg were associated with a rise in blood pressure. Doses of 0.8 and 1.2 g/kg elicited a mean BP elevation of 6 and 16 mm Hg, respectively, for 7-hour post-dosing. The biphasic tachycardia to toluene suggests multiple sites for eliciting the cardiotoxic effects of this toxicant. © 2006 Elsevier Inc. All rights reserved. Keywords: Radiotelemetry; Heart rate; Tachycardia; Volatile organic compounds
1. Introduction Toluene is a volatile organic compound (VOC) widely used in industrial settings nd has been shown to have a variety of adverse health effects. VOC's are prevalent in urban air and are generated by several types of regulated sources. In addition, many commercial products contain VOC's in the form of organic solvents, including paints, waxes, inks, cleaning solutions, and pesticides. The nervous and cardiovascular systems have been shown to be ☆
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. B105-04, U.S. EPA, 109 S. T.W. Alexander Drive, Research Triangle Park, NC 27711, United States. Tel.: +1 919 541 1509; fax: +1 919 541 4416. E-mail address: [email protected] (C.J. Gordon). 0892-0362/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2006.10.004
markedly susceptible to acute exposure to toluene vapors and other VOC's [2]. Furthermore, recent research suggests that relatively low doses of inhaled toluene can have adverse effects on brain neurochemistry [16]. There are renewed efforts to understand the toxicity of relatively low concentrations of toluene. The clinical reports on the treatment of abusers of toluenebased products (i.e., “glue sniffers”) provides a considerable data base on the hazardous effects of acute toluene on the cardiovascular system. The heart appears to be a sensitive target to toluene and other volatile organic compounds; there are several clinical case reports of ventricular sensitization and arrhythmic effects along with tachycardia or bradycardia in cases of acute toluene abuse [1,5,14]. There are surprisingly few studies on the cardiotoxic effects of toluene in experimental animals. The few studies that have been performed utilized anesthetized preparations subjected to
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acute doses of inhaled or parenterally administered toluene [12,19]. For example, a tachycardic response was seen in anesthetized rats exposed to a nearly saturated atmosphere of toluene or benzene [19]. Morvai et al. [12] showed an appreciable fall in blood pressure of anesthetized rats administered toluene intravenously. On the other hand, anesthetized mice exposed to high doses of inhaled toluene develop bradycardia and atrialventricular block [18]. The depressant action of anesthetics on the CNS and cardiovascular system deems the use of anesthetized rodents as unacceptable models to study the toxicity of VOC's in most cases. Radiotelemetry technology that utilizes surgically implanted radiotransmitters is an ideal means of monitoring cardiovascular and other autonomic processes in awake, undisturbed rodents. The unrestrained, awake rodent is probably the most sensitive model to study the autonomic effects of a toxicant [8,9]. The purpose of this study is to assess the effects of toluene exposure on blood pressure, heart rate, core temperature, and motor activity in the unrestrained, awake rat. It is important to comment on our selection of an oral route of exposure. Inhalation exposure would be most relevant to study the cardiotoxic effects of an air pollutant. Oral route of exposure has often been used in acute toluene studies because it is less costly and time-consuming than inhalation systems. Inhalation “nose-only” systems involve prolonged restraint of the test subject. The stress of restraint is clearly not conducive for collecting physiological data that are free of stress artifacts. A single oral dose of toluene elicits a prolonged elevation in blood toluene that can be used to model the acute effects of inhalation exposure [10,17]. Blood toluene levels can be manipulated in rats dosed orally to match that when they are exposed to given air concentrations [10,17] (see Discussion). In the current study, telemetry is used to monitor the physiological response to an acute, oral dose of toluene that can be used to predict physiological responses to equivalent inhaled doses. 2. Materials and methods Animals used in these studies were male rats of the LongEvans strain acquired from Charles River Laboratory (Raleigh, NC) at approximately 2 months of age. The rats were housed individually in plastic cages with pine wood shavings at an ambient temperature of 22 °C and 50% relative humidity with a 12:12 light:dark photoperiod with lights on at 0600 h. 2.1. Surgery In the first study, heart rate, core temperature, and motor activity were monitored in seven unrestrained rats using radiotelemetry (model TA11CTA-F40; Data Sciences Int., St. Paul, MN). Details of the surgical procedure have been published [8]. Briefly, rats were anesthetized with Nembutal (50 mg/kg; IP). A midline incision was made in the abdominal musculature and a transmitter with electrocardiogram leads was secured inside the abdominal cavity and sutured to the midline. The ECG leads were tunneled under the skin and positioned on the left and right sides of the thorax allowing for the best detection of the ECG
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signal. The skin was closed with wound clips and the rat was administered an analgesic (buprenorphine; 0.03 mg/kg; SC) and an antibiotic (penicillin; ∼ 1000 units; IM) and allowed to recover for at least 10 days prior to study. In a second study, five rats were implanted with radiotransmitters (model TL11M2-C50-PXT; Data Sciences) to monitor blood pressure, ECG, heart rate, core temperature, and motor activity. Details on the surgical procedure have been published [15]. The transmitters were implanted by the staff of Charles River Laboratories in Raleigh, NC. Once the rats had recovered from the surgery for at least 7 days, they were shipped to our facility for study. These transmitters have a catheter that is placed into the lumen of the descending aorta to measure aortic blood pressure. A gel in the tip of the catheter inhibits clot formation in most cases. The transmitter provides a continuous measure of systolic, diastolic, and mean blood pressure. Heart rate is derived from the pressure pulse. Core temperature and motor activity are also monitored. The QA interval, a measure of cardiac contractility, was also monitored at 5 min intervals. The QA interval is defined as the interval between the depolarization of the ECG and the upswing of aortic pressure and is calculated automatically by the telemetry software [15]. 2.2. Protocol Rats were dosed with toluene diluted in corn oil (dosing volume = 4 mL/kg). All rats were given corn oil, 0.4, 0.8, and 1.2 g/kg toluene (99.5% spectrophotometric grade, SigmaAldrich Chemical Co., St. Louis, MO). Each rat received the control and three toluene treatments in a different, randomlyselected sequence. The telemetry parameters were monitored at 5 min intervals in rats left undisturbed in the animal facility. The day before dosing, the rats were weighed and placed into a clean cage of wood shaving bedding. Animals were allowed food and water ad libitum. On the day of dosing, all rats were administered with toluene within 10 min of 1100 h and were then left undisturbed for 72 h. Toluene administered by oral gavage is completely cleared to nearly undetectable levels in the circulation within 24 h after dosing [10,17]. Moreover, the telemetric parameters appeared to be near fully recovered within 48 h after dosing. Hence, it was decided that a recovery interval of at least 3 days was adequate. Rotating the animals through each control and toluene treatment allowed for the experiment to be performed with a minimum number of animals. Mean body weight of rats from the start to the end of each study was 433 to 490 g in Study 1 and 341 to 402 g in Study 2. 2.3. Statistical analysis The average response for each telemetry parameter, beginning 30 min after dosing (1130 h) until the onset of the dark phase (6 pm), was averaged for each animal. These mean responses over the 6.5-hour interval were analyzed for statistical significance using repeated measures, one-way analysis of variance (RMANOVA) (Sigma Stat® 3.1). If there was a significant treatment effect for each telemetry parameter, then a
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comparison of each toluene treatment group to the control was performed using the Hold–Sidak method (Sigma Stat) with an overall significance level set a p = 0.05.
Table 1 Analysis of the overall effects of toluene on each telemetry parameter when compared to the change from control values Treatment
Time
Heart rate, beats/min
Core temperature, °C
Motor activity, counts/min
Control 0.4 g/kg 0.8 g/kg 1.2 g/kg Control 0.4 g/kg 0.8 g/kg 1.2 g/kg
Day Day Day Day Night Night Night Night
338.18 ± 17.62 346.44 ± 6.18 363.05 ± 20.51⁎ 378.81 ± 13.49⁎ 390.65 ± 18.08 375.46 ± 22.45 368.91 ± 13.50⁎ 361.46 ± 21.68⁎
37.34 ± 0.098 37.47 ± 0.154 37.47 ± 0.154 37.44 ± 0.290 38.09 ± 0.076 37.94 ± 0.10 37.95 ± 0.10⁎ 37.89 ± 0.18⁎
1.91 ± 0.67 2.29 ± 0.75 3.42 ± 1.12 6.01 ± 2.57⁎ 5.45 ± 2.48 4.90 ± 1.73 4.97 ± 1.89 4.49 ± 2.07
3. Results 3.1. Study 1 (heart rate, core temperature, and activity) Long-Evans rats displayed distinct circadian rhythms of heart rate, blood pressure, core temperature, and motor activity when left undisturbed in the animal facility (Fig. 1). Prior to dosing, heart rate was at minimal daytime levels of 300– 325 beats/min; core temperature was 37.0 to 37.2 °C and motor activity was minimal (Fig. 1B, C). Administering the corn oil vehicle and toluene led to abrupt increases in all telemetry variables, a response attributed to the stress of handling and dosing. In the first 30 min after dosing, the heart rate of control rats increased to over 400 beats/min then recovered rapidly to a level of ∼ 325 beats/min for the remainder of the light phase. The heart rate of rats dosed with 0.4, 0.8, and 1.2 g/kg peaked at 400 to 425 beats/min during the first 30 min after dosing. These acute tachycardic responses were transient, but heart rate remained elevated above control levels throughout the light phase in rats dosed with 0.8 and 1.2 g/kg. Overall, heart rate during the light phase was significantly elevated by toluene, increasing by 25 and 50 beats/min over the 6.5-hour period at doses of 0.8 and 1.2 g/kg, respectively (Table 1). During the dark phase, heart rate showed a notable reduction in the toluene groups with overall significant reductions at doses of 0.8 and 1.2 g/kg. Heart rate was not significantly affected by 0.4 g/kg. However, this reduction in nocturnal heart rate was not
Data expressed as mean ± S.D. Asterisks indicate significant difference from control ( p b 0.05).
replicated in Study 2 (see below). The electrodes implanted in one rat did not provide a clear ECG signal; hence, the heart rate data for this animal were excluded from the analyses, leaving an N of 6 for the heart rate and N of 7 for temperature and activity. Core temperature increased transiently following dosing with corn oil, 0.4, and 0.8 g/kg toluene (Fig. 1B). Temperature of the 0.4 g/kg group was similar to that of controls whereas the 0.8 g/ kg exhibited an elevated temperature that persisted through much of the light phase. Rats dosed with 1.2 g/kg toluene underwent a transient decrease in temperature and did not exhibit the stress-induced rise in core temperature that was seen in the controls and the 0.4 and 0.8 g/kg treatment groups. At approximately 2.5 h after dosing, core temperature of the 1.2 g/ kg group increased above control levels, reaching 38 °C by 5 h after dosing. Overall, there were no significant effects of toluene on core temperature when it was assessed over the 6.5-hour
Fig. 1. Time-course of heart rate (A), core temperature (B), and motor activity (C) in rats dosed by oral gavage with the corn oil vehicle, 0.4, 0.8, and 1.2 g/kg toluene. Each plot represents the response of a given dose of toluene compared to that of the controls. N = 7 for core temperature and activity; N = 6 for heart rate.
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period after dosing. Nevertheless, exploratory analysis of the core temperature during the periods of maximum hypothermia and hyperthermia after dosing suggested an attenuation of the normal circadian temperature rhythm. There was a significant reduction in core temperature in the highest dose groups during the night after toluene exposure. Motor activity increased markedly during the first hour after dosing with the corn oil vehicle and toluene treatments (Fig. 1C). The peak rise in activity was notably higher in rats dosed with 0.8
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and 1.2 g/kg. Motor activity in the 0.8 g/kg group remained elevated for about an hour after dosing and then returned to control levels. On the other hand, motor activity of rats dosed with 1.2 g/kg was elevated for 2 h after dosing, decreased to near control levels, then increased again for another 3 h. Overall, motor activity in the 1.2 g/kg dose group increased by 4.1 counts/min over the 6.5-hour period after dosing (Table 1). At night, there was a slight reduction in motor activity in rats dosed with toluene but the reduction was not significant (Table 1).
Fig. 2. Time-course of blood pressure heart rate (A), systolic (B), mean (C), and diastolic blood pressure (D), QA interval (E), core temperature (F), and motor activity (G) of rats administered the corn oil vehicle or toluene at doses of 0.4, 0.8, or 1.2 g/kg. N = 5 for all parameters except blood pressure and QA interval where N = 4 for the 1.2 g/kg treatment.
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after dosing. In the 0.8 g/kg group, the elevated blood pressure was maintained for about 3 h and then equaled control levels at the onset of the dark phase. Following administration of the 1.2 g/kg dose, the recording of blood pressure gave spurious readings in one rat and those data were excluded from analysis. In the other four rats administered 1.2 g/kg, the transient increase in blood pressure observed immediately after dosing was higher than the control response. Blood pressure was maintained above control values after dosing and the hypertension persisted for several hours into the dark phase (Fig. 2B, C, D). QA interval was unaffected by toluene (Fig. 2E). Although there were no significant treatment effects on QA interval, there was a trend in the high dose group for a reduction in QA interval that was associated with time of maximal increase in blood pressure. Core temperature of rats dosed with 1.2 g/kg decreased transiently then rebounded abruptly beginning 2.5 h after dosing and increased by nearly 1.0 °C over the next 90 min (Fig. 2F). The time course of core temperature of the 0.4 and 0.8 g/kg groups was similar to that of the control group. 3.3. Heart rate vs. motor activity Fig. 2 (continued ).
3.2. Study 2 (blood pressure, heart rate, core temperature, QA, and activity) The acute effects of toluene on heart rate and motor activity observed in Study 1 were replicated using the blood pressure transmitter system. One exception was a higher mean heart rate in the control group which was ∼20 b/min higher over the 6.5-hour period as compared to the controls in the first study (Fig. 2A). Nonetheless, the significant effects of toluene doses of 0.8 and 1.2 g/kg were quite similar as in Study 1 with heart rate over the 6.5-hour period increasing an average of 38 and 54 b/min above controls, respectively (Table 2). Motor activity was significantly elevated by the 0.8 and 1.2 g/kg toluene treatments. The 0.4 g/kg dose had no significant effects on heart rate or motor activity. The tachycardic effects of toluene were associated with significant elevations in blood pressure (Fig. 2B, C, D; Table 2). Systolic, mean, and diastolic pressure changed in parallel fashion. Toluene doses of 0.8 and 1.2 g/kg elicited elevations in blood pressure that were maintained throughout the light phase
An interesting aside in both studies is the comparison of heart rate and motor activity in the rats dosed with 0.8 g/kg. The time scales have been expanded around the time of dosing to view the dynamics of heart and motor activity (Fig. 3A, B). The time course of heart rate following 0.8 g/kg was remarkably similar in both studies. Heart rate increased rapidly after toluene and remained at a steady level of 425 beats/min (Study 1) and 450 beats/min (Study 2) for 1 h. Heart rate then dropped precipitously, reaching a new steady state level of 40–50 beats/min above controls that persisted for most of the light phase. The abrupt decrease in heart rate in the first hour coincided with a marked drop in motor activity (denoted by dashed arrows in Fig. 3). 4. Discussion Exposure to toluene via oral gavage in the awake, unrestrained rat elicited a marked tachycardia and hyperactivity with relatively little change in body temperature. We suspected that the tachycardic effects of toluene would be more easily detectable when the rat was studied in a resting state. The rat is nocturnal and exhibits a circadian rhythm of heart rate, core temperature, and motor activity. During the daytime, heart rate approaches basal
Table 2 Analysis of effects of toluene on blood pressure, heart rate, QA interval, core temperature, and motor activity Parameter
Control
0.4 g/kg
0.8 g/kg
1.2 g/kg
Mean pressure, mm Hg Diastolic pressure, mm Hg Systolic pressure, mm Hg Heart rate, beats/min QA interval, ms Core temperature, °C Motor activity, counts/min
103.87 ± 4.23 90.66 ± 3.676 119.18 ± 6.35 361.08 ± 23.61 40.27 ± 2.73 37.61 ± 0.121 0.78 ± 0.53
108.08 ± 6.35 95.67 ± 6.94 122.34 ± 7.76 370.64 ± 34.35 40.37 ± 2.61 37.58 ± 0.199 1.06 ± 0.67
110.30 ± 6.86 96.99 ± 5.12 125.06 ± 8.92 399.92 ± 21.74⁎ 39.90 ± 2.91 37.63 ± 0.217 3.52 ± 2.50⁎
119.05 ± 9.82⁎ 104.41 ± 8.77⁎ 134.95 ± 11.21⁎ 415.76 ± 22.19⁎ 39.47 ± 3.78 37.60 ± 0.264 6.58 ± 2.13⁎
Data averaged over 7-hour period from time of dosing until onset of dark phase. N = 5. Asterisks indicate significant difference from control ( p b 0.05).
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Fig. 3. Expanded view of the time-course of heart rate and motor activity in rats dosed with 0.8 g/kg toluene in Study 1 (A) and Study 2 (B). Note the abrupt decrease in heart rate coinciding with secondary peak in motor activity (denoted by dashed arrows) that occurs approximately 1 h after dosing.
levels of approximately 325 beats/min when the rat is housed at an ambient temperature of 22 °C on a wire-screen floor [22]. At night, heart rate increases to over 400 beats/min. Hence, by exposing resting rats to toluene during the daytime when housed on pine shaving bedding that affords insulation and more favorable thermal environment, we contend that the cardiovascular system is close to basal, resting conditions and dosedependent tachycardia and hypertension should be detected at lower doses of toluene. The tachycardic effects of toluene at doses of 0.8 and 1.2 g/ kg were apparent within 15 min after dosing as evidenced by the recovery of the telemetric parameters in rats given corn oil (i.e., stress response) but a persistent elevation in heart rate of the toluene-treated animals. One novel finding in this study is the biphasic heart rate response characterized by an initial tachycardia followed by the establishment of a lower, steady state heart rate that was significantly elevated above controls for at least 6 h after dosing. Another interesting observation was the inability to observe recovery of heart rate. That is, the recovery from tachycardia could not be seen because, at the onset of the dark phase, heart rate of controls and toluene-treated animals increased together, thus eliminating the ability to view a recovery from toluene. Finally, when plotted side-by-side, blood pressure and heart rate essentially rise and fall simultaneously following dosing with toluene (Fig. 4). This would suggest that the hypertensive response to toluene is driven primarily by the tachycardia but this will require further
investigation. Core temperature was mildly affected by toluene with a transient hypothermia that was seen only after exposure to the highest dose, and was followed by a delayed hyperthermia that may be associated with the period of hypertension. There have been a few studies on the effects of toluene and other VOC's on heart rate and blood pressure but the
Fig. 4. Expanded view of the time-course of mean heart rate and blood pressure of rats dosed with 1.2 g/kg toluene. Note how blood pressure tracks closely with the changes in heart rate. N = 5 for heart rate; N = 4 for pressure.
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measurements were made in anesthetized rats [12,19]. These studies reported on tachycardic, bradycardic, and arrhythmogenic effects following acute exposure to VOC's but the results should be viewed with caution in view of the use of anesthesia. Lethal effects of acute toluene in anesthetized dogs can be a result of fatal arrhythmia as a result of direct effects of the VOC on the myocardium [6]. Exposure to near lethal quantities of toluene and benzene in chloralose-anesthetized rats leads to tachycardia [19]. No evidence of hypertension was observed in anesthetized rats administered acute doses of toluene intravenously [12]. To the best of our knowledge, this is the first report of using radiotelemetry to assess the effects of graded doses of toluene on cardiovascular and thermoregulatory parameters in resting, undisturbed rats. It is clear that the cardiovascular responses observed under the conditions of the present study are not parallel with those seen in anesthetized animals. In our opinion, telemetry is the best approach to collect data on the physiological effects of VOC's in experimental animals that can be extrapolated to humans. There has been one study of toluene-induced tachycardia in awake rats monitored by radiotelemetry. A preliminary report by Oshiro et al. [13] used radiotelemetry to determine the effects of inhaled toluene on heart rate and body temperature of LongEvans rats. Heart rate and core temperature were monitored by radiotelemetry in rats trained to perform an operant signal detection task. Rats inhaled toluene vapor for 1 h either while sedentary or while performing the behavioral task. In sedentary rats, 2000 ppm toluene increased heart rate from about 250 to ∼ 350 b/min by 1 h of exposure. Toluene concentrations in the brain under these conditions increased to ∼70 μg/mL over the course of the hour of exposure (unpublished observations). In rats actively performing the task, heart rate in rats breathing clean air was ∼370 beats/min, and toluene increased it by only 20 to about 390 beats/min. Brain toluene concentrations in these active rats reached about 100 μg/mL. Thus, the magnitude of the toluene-induced tachycardia depended more on the control baseline heart rate than upon the concentration of toluene in the brain. In contrast to the present evidence for toluene-induced hyperthermia, inhaled toluene induced a hypothermic response in these trained rats, observed against a ∼ 1 °C increase in temperature associated with performing the task. This difference could reflect the route of exposure or the temperature of the animals in the absence of toluene. How would the no observed adverse effects level (NOAEL) and lowest observed adverse effects level (LOAEL) doses for oral toluene exposure in the current study (i.e., 0.4 and 0.8 g/kg, respectively) compare to the estimated concentrations of inhaled toluene? Two studies provide estimates that used toluene dissolved in corn oil. Sullivan and Conolly [17] compared blood toluene levels in Sprague-Dawley rats exposed to toluene via oral gavage to that of a 6-hour inhalation. Toluene doses of 0.433 g/kg and 0.867 g/kg elicited elevations in blood toluene that reached an asymptote at approximately 2 h after dosing. Blood toluene levels in rats given the lower dose of toluene fell slowly after reaching the asymptote over the next 4 h, whereas rats given the higher dose of toluene exhibited near steady state blood toluene levels for several hours after reaching the
asymptote. A correlation between oral and inhalation routes shows that a concentration of 607 ppm is approximately equal to an oral dose of 0.4 g/kg; 1274 ppm is equivalent to 0.8 g/kg [17]. Gospe and Al-Bayati [10] found that the asymptote for blood toluene in the rat dosed orally with 0.741 and 0.911 g/kg was 4.33 and 6.3 h, respectively. These investigators used a 3-hour exposure as compared to the 6-hour inhalation exposure described above [17]. Blood toluene levels reached their asymptote within 2 h of exposure to ambient concentrations of 99 to 1145 ppm [10]. The analysis of the relationship between oral and inhaled toluene differed substantially from Sullivan and Conolly [17]; 2500 ppm was equivalent to an oral dose of 0.4 g/ kg and 5200 ppm was equivalent to 0.8 g/kg. It is interesting to note that the pattern of tachycardia does not correlate with the predicted elevations in blood and brain toluene concentrations reported by others [10,17]. For example, we found in both studies that an oral dose of 0.8 g/kg toluene resulted in a 100 beats/min increase in heart rate that was maintained for the first hour after dosing. This was followed with a drop in heart rate and maintenance at a steady level above controls for several hours. Hence, the tachycardic response subsides to a steady state level prior to blood and brain toluene reaching their peak concentrations. This would suggest that the cardiac responses to ingested toluene involves multiple points of sensory and effector response to the toxicant. Clinical case reports on accidental and occupational exposure to toluene point to tachycardic and ventricular arrhythmias. Japanese painters exposed to toluene and other VOC's exhibit a variety of CNS maladies [11]. Two symptoms suggestive of cardiovascular dysfunction are cold hands and feet as well as dizziness. Chronic toluene abuse (i.e., “glue-sniffing”) in humans is associated with cardiac sensitization, which generally results in tachyarrhythmias, but some bradycardic events have also been noted [7]. Rats exposed chronically to relatively low concentrations of toluene (40 to 80 ppm) underwent changes in CNS neurochemistry (dopaminergic and serotonergic pathways) as well as behavioral alterations [3,16,20]. Brain regions showing marked changes in catecholamine biosynthesis are involved in neurovegetative and cardiorespiratory regulations. Overall, the use of radiotelemetry allows for assessment of cardiovascular and other autonomic effects of toluene in the undisturbed rat. There is a dose-related tachycardia and hypertensive response that persists for at least 7 h after dosing. It is not clear how long the tachycardia effects persist because the night time elevation in heart rate of control rats obviates any effects of the toxicant on baseline heart rate The literature on cardiotoxic effects of toluene and other VOC's implies that the tachycardic and arrythmogenic effects of these compounds arise from their direct effects on the cardiac tissue. On the other hand, the well known effects of acute toluene on catecholamine pathways in the CNS would suggest that the cardiovascular effects arise from a central origin. Several studies suggest central activation of serotonergic pathways in rats exposed acutely to toluene [4,21]. Such activation of serotonergic pathways could be responsible for the tachcardic effects of this VOC. The unusual pattern of tachycardia that was most
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pronounced in rats dosed with 0.8 g/kg toluene illustrates the peculiar sensory-effector response to toluene. Future studies will discern the mechanisms of action and site of origin (i.e., central and/or peripheral nervous system) for mediation of toluene-induced tachycardia and hypertension. Acknowledgement We thank Drs. L. Leon and V. Benignus for their review of the manuscript. We also thank P. Becker for providing technical assistance. References [1] M. Bass, Sudden sniffing death, J. Am. Med. Assoc. 212 (1970) 2075–2079. [2] V.A. Benignus, Health effects of toluene: a review, Neurotoxicology 2 (1981) 567–588. [3] P. Berenguer, C. Soulage, D. Perrin, J.-M. Pequignot, J.H. Abraini, Behavioral and neurochemical effects induced by subchronic exposure to 40 ppm toluene in rats, Pharmacol. Biochem. Behav. 74 (2003) 997–1003. [4] L. Castilla-Serna, M.G. Barragan-Mejia, R.A. Rodriguez-Perez, A. Carcia Rillo, C. Reyes-Vazquez, Effects of acute and chronic toluene inhalation on behavior, monoamine metabolisms and specific binding (3H-serotonin and 3H-norepinephrine) of rat brain, Arch. Med. Res. 24 (1993) 169–176. [5] S. Einav, Y. Amitai, J. Reichman, D. Geber, Bradycardia in toluene poisoning, J. Toxicol. Clin. Toxicol. 35 (1997) 295–299. [6] N. Ideda, H. Takahashi, K. Umetsu, T. Suzuki, The course of respiration and circulation in “toluene-sniffing”, Forensic Sci. Int. 44 (1990) 151–158. [7] R.D. Gerkin Jr., F. LoVecchio, Rapid reversal of life-threatening tolueneinduced hypokalemia with hemodialysis, J. Emerg. Med. 16 (1998) 723–725. [8] C.J. Gordon, Acute and delayed effects of diisopropyl fluorophosphate (DFP) on body temperature, heart rate, and motor activity in awake, unrestrained rats, J. Toxicol. Environ. Health 39 (1993) 247–260. [9] C.J. Gordon, Temperature and Toxicology: An Integrative, Comparative, and Environmental Approach, CRC Press, Boca Raton, FL, 2005.
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