Effects of methanol on autonomic thermoregulation of rats at different ambient temperatures

Toxicology Letters, 52 (1990) 153-162

153

Elsevier

TOXLET

02349

Effects of methanol on autonomic thermoregulation of rats at different ambient temperatures*

Forrest S. Mohler and Christopher J. Gordon U.S. Environmental

Protection Agency, Health Effects Research Laboratory,

Neurotoxicology

Division,

Research Triangle Park, NC (U.S.A.) (Received

18 October

(Accepted

18 February

1989) 1990)

Key words: Methanol; rate; Evaporative

Toxicity;

Autonomic

water loss; Dry thermal

thermoregulation; conductance;

Body temperature;

Activity;

Metabolic

Rats

SUMMARY To measure

the effect of methanol

traperitoneally chamber,

set at an ambient

rate (MR),

evaporative

the rat was removed a significant

temperature

water

thermoregulation,

from the chamber

hypothermia

following

35°C. At a T, of 25°C MR was elevated,

and dry thermal

and colonic

conductance

temperature

methanol

Total activity

caused

decreased

a significant

rats, and methanol

reduction reduced

tance)

and MR were related.

was not significant

following

to a significant

hypothermia

in rats by an inhibitory

*This research

has been reviewed

by the Health

3 g/kg methanol

0378-4274/90/S

conductance

hypothermia

15 to were

and its depressive

on the heat loss effecters Thus, methanol

action on heat production

Laboratory,

exposure

U.S. Environmental

U.S. EPA, HERL,

B.V. (Biomedical

(EWL, conducleads

pathways.

of trade names or commercial

3.50 @ 1990 Elsevier Science Publishers

at T.‘s from

had no effect, but at the other T,‘s, where

of heat conservation.

Address for correspondence: Dr. Christopher J. Gordon, Triangle Park, NC 27711, U.S.A. Tel. (919) 541-1509.

The rats developed

in MR. EWL and thermal

Effects Research

tion Agency and approved for publication. Mention stitute endorsement or recommendation for use.

metabolic

After 60 min.

EWL at this T, while it had no effect on conduc-

The effect of methanol

or was in the direction

were measured.

inin a

at T,‘s from 5 to 25”C, while the change

tance at this or any other T,. These data suggest that the methanol-induced effect on activity

rats were injected

The rats were then placed

(T,) was measured.

the 3 g/kg dosage of methanol

MR was at basal levels and methanol

at 35°C in control

male Fischer

(20% w/v in saline).

(T,) of 5, 15,25, or 35°C. for 60 min while total activity,

loss (EWL),

in T, at 35°C was not significant.

elevated

on autonomic

with saline or 1 or 3 g/kg methanol

NTD

Division)

products

Protec-

does not con-

(MD-74B),

Research

154 INTRODUCTION

Methanol exposure causes a number of toxic symptoms when ingested by humans or experimental mammals, and symptoms such as blindness [ 1,2] indicate that methanol is neurotoxic. Because distinctions are made between the metabolism of methanol in primates and rodents [3,4], it is important to study and understand the characteristics of methanol intoxication in a number of species. Body temperature and whole body metabolism can be very sensitive indicators of acute toxicity [5,6]. For example, heavy metals caused a decrease in the metabolic rate and colonic temperature (T,) of mice at dosages N 10% or less of the LDsa values [6]. When studying the thermoregulatory response to a toxicant, it is important to understand whether the body temperature (Tb) changes are forced or regulated [S]. A forced change in Tb is one in which the animal, if given the means, will prevent the change; this would indicate that the toxicant caused a direct change in one or more of the autonomic thermoregulatory effecters without an effect on the central processing. On the other hand, a regulated change in Tb is one in which the animal promotes the change through a coordinated autonomic or behavioral thermoregulatory response, and this would indicate that the toxicant had a direct effect on the CNS mechanisms of thermoregulation. A previous study from this laboratory showed that methanol caused a decrease in the T, of rats allowed to thermoregulate behaviorally, but the results did not show a clear decrease in the preferred ambient temperature (T,). Therefore, the present study was designed to measure the autonomic thermoregulatory responses of rats given methanol at a range of T,‘s from 5°C where metabolic heat production is used to maintain normothermia, to 35°C where heat dissipation such as increased evaporation and conductance is necessary to maintain normothermia. MATERIALS AND METHODS

Animals The subjects used in this experiment were male Fischer-344 rats (Charles River Labs) aged 85-100 days. The rats were housed in groups of 2-3 at a T, of 22°C with a relative humidity of 50 96under a 12112 light/dark photoperiod. Water and rat chow were available ad libitum.

Injections Absolute methanol (Sigma) was diluted in physiological saline to a concentration of 20% (w/v). The methanol solution was injected intraperitoneally (i.p.) at appropriate volumes to deliver 1 or 3 g/kg methanol. Saline was injected i.p. in a volume equivalent to a 3 g/kg dose of methanol as a control.

155

Autonomic measurements

Measurements were made in a ldliter stainless-steel chamber immersed in a water bath maintained at a T, of 5, 15, 25 or 35°C. Dry air with a dew-point temperature (Tdp) of N -20°C was pulled through the chamber at a rate of 4.1 l/min. A fraction of the effluent chamber air was drawn through a dew-point hygrometer (General Eastern, model 1200 APS), dried, and pulled through an oxygen analyzer (Applied Electrochemistry, model S-3A). A layer of mineral oil at the bottom of the environmental chamber prevented moisture from urine and feces from entering into the measurement of T+. Whole body metabolic rate (MR) in dimensions of W/kg was calculated from the percent oxygen, and evaporative water loss (EWL) in dimensions of W/kg was calculated from the T+ as described previously [7]. Activity was monitored using a Doppler-type movement detector. This detector measures disturbances in a low-power microwave field, and activity was measured in relative units of volts of output from the movement detector. Dry thermal conductance (C) in the dimensions of W/kg/C was calculated using the equation: ,__,MR-EWL T, - T, where MR is the metabolic rate in W/kg, EWL is the evaporative water loss in W/kg, T, is colonic temperature in “C, and T, is ambient temperature in “C. Protocol

The environmental chamber was set to the desired temperature, and measurements of ambient SO, and T+ of the air entering the chamber were collected. A naive rat then received an injection of saline or methanol. The rat was immediately placed in the chamber, and SO2 and Td,, of the effluent air, and activity were measured continuously. At the end of 60 min, the chamber was opened, and the rat’s T, was measured with a thermocouple (Physitemp, RET-3) inserted 6 cm past the anal sphincter. Statistical

analysis

A two-way ANOVA was calculated for each of the thermoregulatory measures (T,, activity, MR, EWL, conductance) to determine the effects of T, and methanol dose. Where a significant dose-effect was indicated, individual t-tests were performed to calculate the effective dosages. The effect of T, under control conditions was calculated for each of the autonomic measures by use of a one-way ANOVA. RESULTS

Colonic temperature

The T, of control rats was not significantly affected by T, in the range of 535°C (Fig. 1): i.e., the saline-treated rats were able to regulate a similar T, across the range

156 39.4 , 39.2

1

39.0 -

TT

T

Cl

q

Saline 1 g/kg

MeOH

3 g/kg MeOH

38.8 38.6 38.4 -

TT

38.2 38.0 37.8 37.6 37.4 37.2 37.0 t

Fig. 1. The effect of an i.p. injection of saline or I or 3 g/kg methanol on the colonic temperature(TJ of Fischer rats at T,‘s from 5 to 35°C. Data plotted as mean + standard error of 5 animals. A significant effect of methanol was measured at T,‘s of 5, 15and 25°C.

of T,‘s tested. At T,‘s from 5 to 25X, the rats injected with saline were able to maintain a T, around 38.438.8”C. However, methanol had a significant dose-effect on T,. While injection of 1 g/kg of methanol had no significant effect on T,, rats treated with 3 g/kg methanol developed a significant hypothermia within 60 min. The magnitude of the hypothermia was greater at the cooler T,‘s with T, dropping 1.7, 1.3, and 0.8”C at 5, 15, and 25°C T,, respectively. At the warm T, of 35°C T, of the rats treated with 3 g/kg methanol dropped 0.3”C, although this change was not significant. The magnitude of the hypothermia following 3 g/kg methanol was described by a linear relationship with T, using the equation LIT, = - 1.94 + 0.046 x T,, having an r2 of 0.995. Activity

T, had no effect on the activity of control rats in the present experiment (Fig. 2).

157

300 Cl

Saline

fl

lgkg MeOH 3 g/kg MeOH

260 240 2

220 -

: >r, .% .s 2

200 1801603 140 120100-r 5

25

15

35

$,“C ** significant at p < .OOS Fig. 2. The effect of an i.p. injection of saline or 1 or 3 g/kg methanol on the activity of Fischer rats at T,‘s from 5 to 35°C. Data plotted as mean + standard error of 5 animals. A significant effect of methanol was measured at T.‘s of 15,25 and 35°C.

However, methanol intoxication in general caused a reduction in the total activity of rats. At the cold T, of 5°C methanol caused a slight reduction in total activity, but the changes were not significant. While 1 g/kg of methanol did not affect activity at any T,, 3 g/kg caused a significant decline in activity in the T, range of 15-35°C. The effect of methanol on activity was apparently greater near the rat’s preferred T, than at Ta’s presenting a greater thermal stress. Metabolic

rate

T, had a significant effect on the MR of control rats (Fig. 3) with metabolic heat production increasing as T, decreased from 25 to 5°C. At a T, of 25°C where the MR was lowest, there was no significant effect of methanol. However, at T,‘s where MR was elevated, 3 g/kg of methanol caused a significant hypometabolism. Evaporative

water loss

The EWL of control rats was significantly affected by T, (Fig. 4) with the rats in-

r

Cl

q

Saline 1 g/kg MeOt i 3 g/kg MeOt i

6

Fig. 3. The effect of an i.p. injection rats at T,‘s from

25

15

5

5 to 35°C. Data

of saline or 1 or 3 g/kg methanol plotted

of methanol

as mean

was measured

+ standard

error

35

on the metabolic of 5 animals.

rate of Fischer

A significant

effect

at T,‘s of 5, 15 and 35°C.

creasing evaporation to maintain normothermia in the heat challenge of 35°C T,. At T,‘s of 5_25”C, average EWL ranged from 0.66 - 1.08 W/kg. At a T, of 35”C, the rats significantly increased their rate of evaporation to an EWL of 3.2 W/kg. There was no effect of methanol on the EWL of rats at T,‘s of 5 or 25°C. However, at the warm T, (35°C) where EWL was elevated, methanol in dosages of 1 and 3 g/kg caused a significant depression in EWL of 0.9 and 1.5 W/kg, respectively. At 15°C methanol caused a statistically significant decrease in EWL, but the physiological significance of a 0.4 W/kg change in EWL is questionable. Conductance

Dry thermal conductance is a measure of an animal’s ability to lose heat to the environment and is, thus, the opposite of insulation. The thermal conductance of control rats was significantly affected by T, (Fig. 5) with rats increasing their conduc-

159

3.0 -

0

q

a $

2s

z s t

2.0-

E s! '3 c

Saline 1 g/kg MeOH 3 g/kg MeOH

1.5 -

0

4

l.O-

3 0.5 -

0.0 T 5

15

T

0P

25

Fig. 4. The effect of an i.p. injection of saline or 1 or 3 g/kg methanol on the evaporative water loss of Fischer rats at T,‘s from 5 to 35°C. Data plotted as mean + standard error of 5 animals. A significant effect of methanol was measured at T,‘s of 15 and 35°C.

tance under the thermal challenge of thermal conductance of rats at T,‘s of to a T, of 35°C increased significantly affect the thermal conductance of rats

35°C T,. While there was no difference in the 5, 15 or 25°C the conductance of rats exposed by N 0.3 W/kg/C. Methanol exposure did not at any temperature or any dosage.

DISCUSSION

Methanol exposure caused a significant hypothermia in rats at T,‘s from 525°C. The autonomic thermoregulation of the rat at these Ta’s facilitated the generation of this hypothermia (see Table I). Under a cold stress of 5°C T,, MR was elevated and EWL and thermal conductance were at a minimum as the rat maintained normothermia. Methanol treatment tended to reduce activity and cause a significant decrease in MR, while EWL and thermal conductance were unaffected at this T,. At

160

0.8

0.7 -

q

0.6-

Cl

Saline

H

1 g/kg 3 g/kg

MsOH MeOH

% 5

0.5-

8 s 25

OA-

8 2 P

0.3 -

0.2 -

0.1 -+

-I 5

15

25

Ta, ‘c Fig. 5. The effect of an i.p. injection of saline or 1 or 3 g/kg methanol on the dry thermal conductance of Fischer rats at T,‘s from 5 to 35°C. Data plotted as mean + standard error 5 animals. No significant effect of methanol was measured at any T,.

a T, of 15°C EWL and thermal conductance were still at a minimum, and the rats increased MR (to a lesser magnitude than at 5°C) to maintain normothermia. Similar to the changes at 5”C, methanol caused a significant reduction in activity and MR while having no effect on dry conductance. Methanol caused a significant decrease in the EWL at this T,, but the contribution of this effect to the overall thermal balance was questionable. A T, of 25°C is near the preferred T, of Fischer rats [7] and EWL, thermal conductance, and MR were all near minimum. At a T, of 25”C, methanol caused a significant decrease in T, and activity but did not affect MR, possibly indicating an inability of the rat to reduce MR further. Interestingly, EWL and dry conductance were not elevated to promote heat loss and facilitate the development of hypothermia in methanol-treated rats. Different thermoregulatory mechanisms were utilized at the warm T, of 35°C to maintain normothermia. In saline-treated rats, MR was near minimum, and both dry conductance and EWL were elevated to increase heat loss. Methanol exposure had no significant effect on T,. Methanol caused a significant reduction in total activity

161 TABLE I COMPARISON OF THE EFFECTS OF METHANOL AND ETHANOL ON THE AUTONOMIC AND BEHAVIORAL THERMOREGULATION OF THE RAT A. Autonomic measures Ethanol 25-26 T,, (“C) 17-18 T,

lkg

ldS

vo2

1s

19-d

--bs

_d8

T,,

32-36 - fg L”

18

TC MR Cond.

Methanol 5

15

25

35

1 1 -

1 1 -

1 -

1 -

B. Behavioral measures Preferred T, Activity

Ethanol

Methanol

lSk _b

;’

aGordon et ai. [15]; bLomax and Bajorek [9]; “Lomax et al. [I I]; dLomax et al. [IO]; =MohIer and Gordon [12]; ?vfyers [13]; SSpiers et al. f14]. Qualitative changes are indicated as a decrease (1) or no change (-) in the parameter. T, = colonic temperature; V02 = oxygen consumption; TsI = skin temperature.

and MR. However, methanol also caused a si~ificant reduction in EWL. Rats lack the ability to pant or sweat and, thus, they increase EWL predominantly by actively spreading saliva on their fur [8]. The methanol-induced decrease in total activity may have interfered with saliva spreading, therefore decreasing the rat’s EWL. Because changes in body temperature result from an imbalance between heat production and heat loss, the lack of changes in T, of methanol-treated rats suggested that the changes in heat production (MR) were counterbalanced by opposite changes in heat loss. This is illustrated by the decrease in EWL of 1.4 W/kg counteracting the decrease in metabolic heat production of 1.5 W/kg following injection of 3 g/kg methanol. Thus, the ability of the rat to decrease T, was compromised. In rats, the thermoregulatory effect of methanol was similar to that of ethanol (Table I). At sufhcient dosages, both alcohols caused significant hypothe~ia except at elevated T,‘s 19-147. At a T, where MR was near basal levels f -25”C), ethanol either decreased MR [14] or had no effect [lo]; methanol had no effect on MR. However, at T,‘s where MR was elevated, both ethanol [l4] and methanol caused a significant reduction in MR. Because no studies measured the effect of ethanol on thermal conductance, a comparison to skin temperature (Tsk) was made. Conductance is a broader measure than Tsk, including many factors such as skin blood flow and piloerection, but both measures evaluate the animal’s ability to dissipate internal heat to the environment. Ethanol caused a reductin in Tsk at a T, of 35°C [14], but at lower T,‘s it had no effect [9,10,14] while methanol had no effect on dry conductance at any T*. Behaviorally, rats chose T,‘s which promoted the hypothe~ia following ethanol treatment

162

[lO,l 1,151. After methanol injection, rats in a temperature gradient did not prefer lower T,‘s, but they also did not prevent the development of hypothermia by selecting warmer T,‘s [ 121. In conclusion, methanol caused hypothermia and a reduction in activity and MR. At higher T,‘s, methanol-treated rats did not develop hypothermia because of the counteracting effect of methanol on heat loss effecters. Thus, methanol apparently causes hypothermia by an inhibitory action on the heat production pathways in the rat. On the other hand, a previous study [12] showed that when methanol-treated rats were allowed to thermoregulate behaviorally, they chose T,‘s at which they developed hypothermia. These results indicated that methanol caused a regulated hypothermia. More work is needed to understand better the mechanisms of the methanolinduced changes in thermoregulation. REFERENCES 1 Bennett, Jr., I.L., Cary, F.H., Mitchell, Jr., G.L., and Cooper, M.N. (1953) Acute methyl alcohol poisoning: a review based on experiences in an outbreak of 323 cases. Medicine 32,431463. 2 Dethlefs, R. and Naraqi, S. (1978) Ocular manifestations and complications of acute methyl alcohol intoxication. Med. J. Aust. 2,483485. 3 Gilger, A.P. and Potts, A.M. (1955) Studies on the visual toxicity of methanol. V. The role of acidosis in experimental methanol poisoning. Am. J. Ophthalmol. 39,63-86. 4 Tephly, T.R. and McMartin, K.E. (1984) Methanol metabolism and toxicity. In: L.D. Stegink and L.J. Filer, Jr. (Eds.), Aspartame: Physiology and Biochemistry. Marcel Dekker, New York, pp. 11l140. 5 Gordon, C.J., Mohler, F.S., Watkinson, W.P. and Rezvani, A.H. (1988) Temperature regulation following acute toxic insult. Toxicology 53, 161-178. 6 Gordon, C.J., Fogelson, L. and Highfill, J.W. (1990) Hypothermia and hypometabolism: sensitive indices of whole-body toxicity’following exposure to metallic salts in the mouse. J. Toxicol. Environ. Health 29, 1855200. 7 Gordon, C.J. (1987) Relationship between preferred ambient temperature and autonomic thermoregulatory function in the rat. Am. J. Physiol. 252, Rll3cRll37. 8 Hainsworth, F.R. (1967) Saliva spreading, activity, and body temperature regulation in the rat. Am. J. Physiol. 212, 1288-1292. 9 Lomax, P. and Bajorek, J.G. (1980) Comparative thermoregulatory effects of ethanol in rats, mice, and gerbils. Proc. West. Pharmacol. Sot. 23,219-223. 10 Lomax, P., Bajorek, J.G., Bajorek, T.-A. and Chaffee, R.R.J. (1981) Thermoregulatory mechanisms and ethanol hypothermia. Eur. J. Pharmacol. 71,483487. 11 Lomax, P., Bajorek, J.G., Chesarek, W.A. and Chaffee, R.R.J. (1980) Ethanol-induced hypothermia in the rat. Pharmacology 21,288-294. 12 Mohler, F.S. and Gordon, C.J. (1990) Thermoregulatory effects of methanol in Fischer and Long Evans rats. Neurotoxicol. Teratol. 12,41-45. 13 Myers, R.D. (1981) Alcohol’s effect on body temperature: hypothermia, hyperthermia, or poikilothermia? Brain Res. Bull. 7,209-220. 14 Spiers, D.E., Threatte, R.M. and Fregly, M.J. (1984) Response to thermal stress in the rat following acute administration of ethanol. Pharmacology 28, 155-170. 15 Gordon, C.J., Fogelson, L., Mohler, F., Stead, A.G. and Rezvani, A.H. (1988) Behavioral thermoregulation in the rat following the oral administration of ethanol. Alcohol Alcohol. 23, 383-390.