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Effect of body temperature on acute ethanol toxicity
Life Sciences, Vol . 25, pp . 547-552 Printed in the II .S .A .
Pergasson Press
EFFC{T OF BODY TEMPERATURE ON ACLRE ETHANOL TOXICITY T .K .H . Dirih md L. Gailis Quebec Heart Institute and Department of Medicine Laval University, Quebec Quebec, Canada GN 4G5 (ßeceived in final fora July 5,
1979)
wMMARY We studied the effect of elevated rectal temperature an eflwrd taucieify and the effect of erharrd av the mean lethal twrrperatvre (LT50) . The rectal temperature was maintained at a preseleefed level fc 4 h. EFhand (23% w/v) w~ injeehd i .p . In ebarrtrol mice amstMtized with pentobarbital, the 4 h LT50 wrs 41 . 8 + 0.1 C (mean± riarrdard error) . In mice which had received a non-lethal ethand dose ~6 sib), the LT50 was deceased to 39 . 0 + 0. 2o C ( pc 0. 001) . In carMrd ~mice, whose temperature dropped, tire 4 h ethanol LDSO was 8.5 + 0. 8 s/Ica. ff tM recto) tengerafure of the mice was maintained irssteäd of a maxinrurn of 38 .5 - 40 C, the LD50 was deceased to 5 . 7 + 0. 8 ~ ( p < 0. 0~ . TM resrilt: show that eMwrd increases tl+e arswptlbili~y of mice ro hyperthermic damage, and oawersely, that hypertherrria increases the toxicity of eH,arrd. Our previous work has shown that ethavd con poterrtiah the heat inactivation of protein mrd RNA synthesis in tissw dices (1, 2) . To determine whethr this intaadian operates in tM intact mirrrol, and is red just a tissue artifact, wf studied tM effeà of body temperature m acute ethand toxicity in mice . MATBtWLS AND METHODS Female Swig albino mice (Quebec Breeding Farms, AAontryal) hwirp free access ro food and water were used . Their wwight was apprarimoNly 25 gi in arry individual exprimevt, mice within o weight tangs of + p.5 g were randomly aaigned to groups â 10 . Ethand (24% w/v in solive) wau injected irrtraperitaneally . The letlwl temperature was established by placing groups of mice an a dtde warmer and mainroinirq their rectal tavperatures at prealected level fc 4 h. Temperatures were measured of 15 min intervals tlroughout the eo~peerivrevts . The ocwrence of death was recorded (death being defined as respiratory arrest Imting mce than 1 min) . The experirrrental group received a single van-lethal etFrarrd dose (6 sib) just pric ro warning; the eartrols received an equal vdume of cline and pentobarbital anestheda (60 ma/kg, 2-3 times) . In anesthetized mice (either by ethavd 0024-3205/79/060547-05$02 .00/0 Copyright (e) 1979 Pergamon Press Ltd
548
Temperature sect Ethanol Toxicity
Vol . 25, No . 6, 1979
ETt1ANOL DOSE ( y /k9)
FIG .
1
Effect of worming aker ethanol injection . Control mice roeeived no treatment after ethanol injection, while the rectal temperature of the "warmed" group : was maintained at 38 . 5 - 40° C maximum (the maximum temperaturo reached by any single mouse of a group at any time during the experiment) . Number of mia per curve: Control, 100; warmed, 90 . ar pentobarbital), the range of temperatures within wry group at any one time was reasonably uniform, nevi exceeding 1 ° C . Ethanol LD50 was established in paired groups of mice which either roceived no treatment after the ethanol injection, allowing tMir temperaturo to drop (control :), or were placed an a slide warmer to keep their rectal ternperaturo at a mean of 38 .5 - 39°C ( 9~P)~ (3) .
The fitting of curves and the calculartian of standard errors was done by probit analysis RESULTS
Effect of rectal temperaturo on ethanol taocicity . The mean temperatures of individual groups of mice prior to the experiment : ranged The higher from 37 . 7 - 38 . 0 C, with individual values ranging from 36 . 7 - 38 . 5 . temperatures were seen in physically active mice . Following ethanol injection, the recta) temperature of mice left in their cages dropped lJnder these canditiau, the ethanol LD50 after to 34 - 29° C in a dose related fashion . 4 h was 8 .5 + 0 . 8 g/k9 (Fig i) . If instead, the temperature of the mice was increased by placing them an a slide
Vol . 25, No . 6, 1979
Temperature and Ethaaol Tozicity
549
MAXIRAUM RECTAL TERAPERATURE 1°Cl
FIG .
2
Effect of ethand (6 q/icp)'an lethal rs#al temperature (4 h) . Filled cirelss<: Pentobarbital anathettzed control (tarot rwrrlber : 34) Clpen circles: non-aneatFhetized controls (17 mia) . Craw : ethand treated (40 miw) . warmer, the ethand LD50 was deceased to 5 .7 + 0. 8 s/kp . The difference is statistically significant ( p < 0.0~ (3). The rectal terrlperature â a^Y of the mice in a group never exceeded 38 .5 - 40. 0° C (dependirp on the experiment) . The mean rectal temperature fa the 4 h in any :angle experiment carped from 38 .1 - 38 .6 ° C. fffect of ethand on I~thal terrrp~eraturat . In a few exprinent: with urMreated, conscious mia, eansiderable difficulties were encountered in moinroining a high rectal terrlperature. Mia in a warmed cape wen obviously stressed and attempted to escape . TMir fur was moist throughout the expriment and shy lost 20% of body weight in 4 h . To circumvent these problems, ww anesthetized mice with pentobarbital . The 50% letlwl temperature (LT50) of anesthetized miu was 41 . 8 + 0.1 ° C (mean + standard error) (Fig . 2) . The temperature used fa plotting was thï mmdmum r~ectaT terriperoture reached aF any time during the expriment (mean tertrperotun was 0.8 - 1 .2o C lower) . Reaps from a small number of car:ciaus mia (Fig . 2) suggest that the pentobarbital does not greatly d+ange their sensitivity to elevated ternperaturr . Mia which had received a 6 a/kg ethand dose, which was not lethal in controls, showed a 2. 8° C deaeme in the LT50, to 39 . 0 + 0. 2° C . Thi: decreae wa statistical ly ügnfficant ( p < 0.001) . Neithr pentobarbital nor ethanol treated mice last weight during the exprimert.
550
Temperature and Ethanol Toxicity
Vol . 25, No . 6, 1979
D ISCU551 ON Tha letFwl tenrperatura that we obtained (41 . 8° C) is lower than that reported for the mouse (~3 .3° C (4) ). Part of the discrepancy may be dw to the method of mesthesia a aonfinernant . Howwer, the choice of 4 h, rather than a shortxoperiod may be tM main contributing factor, sine wan at the highest tunparatura (43 C), we abased no deatFa beforo 90 min . The mitt are unlikely to have suffered looolized thermal damage duo to contact with the :lide warn~er, since the temperature of the wrfacs wm close to the rectal temperotun of the mia . For example, in the experiments establishing the LDSO~af ethanol, the plate temperature did not exceed ~1° C. Ctur LD50 (8.5 g/kg) is significantly higher than the 7.0 a/kg obtained by Weatherby and Ciemenis, who used a similar ethanol cancentratim (5) . The difference may refleà difference in the method of injection and in the origin of the miu . Another unknown differenu is the ambierM condition: after injection ; since, es drawn by our result:, the toxicity of ethanon is highly tamperatun dependent, diffennca in the rote of heat lea could produce differences in mortality . Since blood ethanol value ware not meawnd, the paaibility that the temperature may have influenced the rate of ethmol absorption cannot be excluded . However, the worming only took place agar sthand injection, and although the miu wan placed an the slide wamer within five min, they ware brought up to tire final temperature slowly, over a ~periad of 15~30 min . Cmja and Kalarrt (6) reported that aRx i . p. injection in rots, the blood alcohol Iwel peaked in 2 min . Although they used a Iwwr ethorrol eancentrotian and daa than we did, it is likely that the ethanol absorption would oleo be rapid under our conditions . Changes in ethand metabolism may conceivably be imdved in the increased toxicity . Warming could aeaelerate the formation of awtaldehyde or, by destroying ethanol metabolizing enzymes, block ethanol ascidafian. However, it has been demoratr+ated, at least in vitro, that ethanol metabolism is not saential for the interoctian with hyperthermia: the effect is observed in heart tissw (2), which does naf significantly metabolize ethanol (~, and in tumor calls (8) . The effect is also seen with solvent: other than ethanol (1, 2, 8). Ths interaction is most likely to bs duo to the ohanges in membrane fluidity (9) and volume (10) produced by ethanol, dwnges which may decrease membrone and protein stability and render them mare susceptible to heat denaturatian . It should be pointed auf that the temperatures at which this interaction with ethanol occurs (e g 39. 0° C), while slightly above nomwl, w by no means unusual and can be produced by a slight fwer ar by moderate physical activity . Tha rosults point out the importance of the eloa control of tamperoture in ethanol studies, and the paaible danger of mixing alcohol and hyperthermia. They also wggest that the hypothermia produced bY high doss of ethanol may exert a protective effect . Although the doses used were high and eorrapond to quantities only rarely ingested by human:, it sans plausible that some tissue damage could also occur at sublathal doss, aspeciolly since 40 mM ethanol has been shown to have an effect in vitro (2) .
Vol . 25, No . 6, 1979
Temperature and Ethaaol Toxicity
551
REFERENCES 1.
L. GAILIS, Raunt Advance: in Studies on Cardia~ Structure and Metabolian, 7 443-449(1976 . 2. L. GAll15, Heat inactivation of Iwcine- 14 C incorporation into heart :liar: Potentialion by ethanol . Gen . Phamiacol . 10 57-61 (1979) . D. J. FINNEY, Probit Auf, 3rd~h Cambridge University fres:, 3. Cambridge (1971) . 4. H. PRECHT, J. CHRISTOPHERSEN, H. HENSEL and W. LARCHER, Te~nture . and Life, p . 658. Springer-Verlag, New Yak (1973) . -VV ATHERBY and E. L. CLEMENTS, Quart . J. Stud . Alc . 2139499 (19160). 5. J. C. CZAJA and H . KALANT, Can. J. Bi ysi . _39 327-334 (19161). 6. 7. L. GAll15 and M. VERDY, Can. J. Bioehern . 49 227-233 (1971) . 8. G . C. LI, G. M. HAHN, and E. C SHIU, J . C~11 . . iol . 93 331-334 (1977) . 1_435-441 (1977) ' 9. J. H. CHIN and D . B. GOLDSTEIN, Molee~amo 10 . P. SEEMAN, Experimentia 30 759-760 1974 .
3,
F I.
Pergasson Press
EFFC{T OF BODY TEMPERATURE ON ACLRE ETHANOL TOXICITY T .K .H . Dirih md L. Gailis Quebec Heart Institute and Department of Medicine Laval University, Quebec Quebec, Canada GN 4G5 (ßeceived in final fora July 5,
1979)
wMMARY We studied the effect of elevated rectal temperature an eflwrd taucieify and the effect of erharrd av the mean lethal twrrperatvre (LT50) . The rectal temperature was maintained at a preseleefed level fc 4 h. EFhand (23% w/v) w~ injeehd i .p . In ebarrtrol mice amstMtized with pentobarbital, the 4 h LT50 wrs 41 . 8 + 0.1 C (mean± riarrdard error) . In mice which had received a non-lethal ethand dose ~6 sib), the LT50 was deceased to 39 . 0 + 0. 2o C ( pc 0. 001) . In carMrd ~mice, whose temperature dropped, tire 4 h ethanol LDSO was 8.5 + 0. 8 s/Ica. ff tM recto) tengerafure of the mice was maintained irssteäd of a maxinrurn of 38 .5 - 40 C, the LD50 was deceased to 5 . 7 + 0. 8 ~ ( p < 0. 0~ . TM resrilt: show that eMwrd increases tl+e arswptlbili~y of mice ro hyperthermic damage, and oawersely, that hypertherrria increases the toxicity of eH,arrd. Our previous work has shown that ethavd con poterrtiah the heat inactivation of protein mrd RNA synthesis in tissw dices (1, 2) . To determine whethr this intaadian operates in tM intact mirrrol, and is red just a tissue artifact, wf studied tM effeà of body temperature m acute ethand toxicity in mice . MATBtWLS AND METHODS Female Swig albino mice (Quebec Breeding Farms, AAontryal) hwirp free access ro food and water were used . Their wwight was apprarimoNly 25 gi in arry individual exprimevt, mice within o weight tangs of + p.5 g were randomly aaigned to groups â 10 . Ethand (24% w/v in solive) wau injected irrtraperitaneally . The letlwl temperature was established by placing groups of mice an a dtde warmer and mainroinirq their rectal tavperatures at prealected level fc 4 h. Temperatures were measured of 15 min intervals tlroughout the eo~peerivrevts . The ocwrence of death was recorded (death being defined as respiratory arrest Imting mce than 1 min) . The experirrrental group received a single van-lethal etFrarrd dose (6 sib) just pric ro warning; the eartrols received an equal vdume of cline and pentobarbital anestheda (60 ma/kg, 2-3 times) . In anesthetized mice (either by ethavd 0024-3205/79/060547-05$02 .00/0 Copyright (e) 1979 Pergamon Press Ltd
548
Temperature sect Ethanol Toxicity
Vol . 25, No . 6, 1979
ETt1ANOL DOSE ( y /k9)
FIG .
1
Effect of worming aker ethanol injection . Control mice roeeived no treatment after ethanol injection, while the rectal temperature of the "warmed" group : was maintained at 38 . 5 - 40° C maximum (the maximum temperaturo reached by any single mouse of a group at any time during the experiment) . Number of mia per curve: Control, 100; warmed, 90 . ar pentobarbital), the range of temperatures within wry group at any one time was reasonably uniform, nevi exceeding 1 ° C . Ethanol LD50 was established in paired groups of mice which either roceived no treatment after the ethanol injection, allowing tMir temperaturo to drop (control :), or were placed an a slide warmer to keep their rectal ternperaturo at a mean of 38 .5 - 39°C ( 9~P)~ (3) .
The fitting of curves and the calculartian of standard errors was done by probit analysis RESULTS
Effect of rectal temperaturo on ethanol taocicity . The mean temperatures of individual groups of mice prior to the experiment : ranged The higher from 37 . 7 - 38 . 0 C, with individual values ranging from 36 . 7 - 38 . 5 . temperatures were seen in physically active mice . Following ethanol injection, the recta) temperature of mice left in their cages dropped lJnder these canditiau, the ethanol LD50 after to 34 - 29° C in a dose related fashion . 4 h was 8 .5 + 0 . 8 g/k9 (Fig i) . If instead, the temperature of the mice was increased by placing them an a slide
Vol . 25, No . 6, 1979
Temperature and Ethaaol Tozicity
549
MAXIRAUM RECTAL TERAPERATURE 1°Cl
FIG .
2
Effect of ethand (6 q/icp)'an lethal rs#al temperature (4 h) . Filled cirelss<: Pentobarbital anathettzed control (tarot rwrrlber : 34) Clpen circles: non-aneatFhetized controls (17 mia) . Craw : ethand treated (40 miw) . warmer, the ethand LD50 was deceased to 5 .7 + 0. 8 s/kp . The difference is statistically significant ( p < 0.0~ (3). The rectal terrlperature â a^Y of the mice in a group never exceeded 38 .5 - 40. 0° C (dependirp on the experiment) . The mean rectal temperature fa the 4 h in any :angle experiment carped from 38 .1 - 38 .6 ° C. fffect of ethand on I~thal terrrp~eraturat . In a few exprinent: with urMreated, conscious mia, eansiderable difficulties were encountered in moinroining a high rectal terrlperature. Mia in a warmed cape wen obviously stressed and attempted to escape . TMir fur was moist throughout the expriment and shy lost 20% of body weight in 4 h . To circumvent these problems, ww anesthetized mice with pentobarbital . The 50% letlwl temperature (LT50) of anesthetized miu was 41 . 8 + 0.1 ° C (mean + standard error) (Fig . 2) . The temperature used fa plotting was thï mmdmum r~ectaT terriperoture reached aF any time during the expriment (mean tertrperotun was 0.8 - 1 .2o C lower) . Reaps from a small number of car:ciaus mia (Fig . 2) suggest that the pentobarbital does not greatly d+ange their sensitivity to elevated ternperaturr . Mia which had received a 6 a/kg ethand dose, which was not lethal in controls, showed a 2. 8° C deaeme in the LT50, to 39 . 0 + 0. 2° C . Thi: decreae wa statistical ly ügnfficant ( p < 0.001) . Neithr pentobarbital nor ethanol treated mice last weight during the exprimert.
550
Temperature and Ethanol Toxicity
Vol . 25, No . 6, 1979
D ISCU551 ON Tha letFwl tenrperatura that we obtained (41 . 8° C) is lower than that reported for the mouse (~3 .3° C (4) ). Part of the discrepancy may be dw to the method of mesthesia a aonfinernant . Howwer, the choice of 4 h, rather than a shortxoperiod may be tM main contributing factor, sine wan at the highest tunparatura (43 C), we abased no deatFa beforo 90 min . The mitt are unlikely to have suffered looolized thermal damage duo to contact with the :lide warn~er, since the temperature of the wrfacs wm close to the rectal temperotun of the mia . For example, in the experiments establishing the LDSO~af ethanol, the plate temperature did not exceed ~1° C. Ctur LD50 (8.5 g/kg) is significantly higher than the 7.0 a/kg obtained by Weatherby and Ciemenis, who used a similar ethanol cancentratim (5) . The difference may refleà difference in the method of injection and in the origin of the miu . Another unknown differenu is the ambierM condition: after injection ; since, es drawn by our result:, the toxicity of ethanon is highly tamperatun dependent, diffennca in the rote of heat lea could produce differences in mortality . Since blood ethanol value ware not meawnd, the paaibility that the temperature may have influenced the rate of ethmol absorption cannot be excluded . However, the worming only took place agar sthand injection, and although the miu wan placed an the slide wamer within five min, they ware brought up to tire final temperature slowly, over a ~periad of 15~30 min . Cmja and Kalarrt (6) reported that aRx i . p. injection in rots, the blood alcohol Iwel peaked in 2 min . Although they used a Iwwr ethorrol eancentrotian and daa than we did, it is likely that the ethanol absorption would oleo be rapid under our conditions . Changes in ethand metabolism may conceivably be imdved in the increased toxicity . Warming could aeaelerate the formation of awtaldehyde or, by destroying ethanol metabolizing enzymes, block ethanol ascidafian. However, it has been demoratr+ated, at least in vitro, that ethanol metabolism is not saential for the interoctian with hyperthermia: the effect is observed in heart tissw (2), which does naf significantly metabolize ethanol (~, and in tumor calls (8) . The effect is also seen with solvent: other than ethanol (1, 2, 8). Ths interaction is most likely to bs duo to the ohanges in membrane fluidity (9) and volume (10) produced by ethanol, dwnges which may decrease membrone and protein stability and render them mare susceptible to heat denaturatian . It should be pointed auf that the temperatures at which this interaction with ethanol occurs (e g 39. 0° C), while slightly above nomwl, w by no means unusual and can be produced by a slight fwer ar by moderate physical activity . Tha rosults point out the importance of the eloa control of tamperoture in ethanol studies, and the paaible danger of mixing alcohol and hyperthermia. They also wggest that the hypothermia produced bY high doss of ethanol may exert a protective effect . Although the doses used were high and eorrapond to quantities only rarely ingested by human:, it sans plausible that some tissue damage could also occur at sublathal doss, aspeciolly since 40 mM ethanol has been shown to have an effect in vitro (2) .
Vol . 25, No . 6, 1979
Temperature and Ethaaol Toxicity
551
REFERENCES 1.
L. GAILIS, Raunt Advance: in Studies on Cardia~ Structure and Metabolian, 7 443-449(1976 . 2. L. GAll15, Heat inactivation of Iwcine- 14 C incorporation into heart :liar: Potentialion by ethanol . Gen . Phamiacol . 10 57-61 (1979) . D. J. FINNEY, Probit Auf, 3rd~h Cambridge University fres:, 3. Cambridge (1971) . 4. H. PRECHT, J. CHRISTOPHERSEN, H. HENSEL and W. LARCHER, Te~nture . and Life, p . 658. Springer-Verlag, New Yak (1973) . -VV ATHERBY and E. L. CLEMENTS, Quart . J. Stud . Alc . 2139499 (19160). 5. J. C. CZAJA and H . KALANT, Can. J. Bi ysi . _39 327-334 (19161). 6. 7. L. GAll15 and M. VERDY, Can. J. Bioehern . 49 227-233 (1971) . 8. G . C. LI, G. M. HAHN, and E. C SHIU, J . C~11 . . iol . 93 331-334 (1977) . 1_435-441 (1977) ' 9. J. H. CHIN and D . B. GOLDSTEIN, Molee~amo 10 . P. SEEMAN, Experimentia 30 759-760 1974 .
3,
F I.