- Email: [email protected]
Circadian susceptibility to soman poisoning
F U N D A M E N T A L A N D APPLIED T O X I C O L O G Y 1:238-241 (1981)
Circadian Susceptibility to Soman Poisoning T I M O T H Y F. E L S M O R E W a l t e r Reed A r m y I n s t i t u t e of R e s e a r c h , W a s h i n g t o n , D.C. 20012
INTRODUCTION This work was performed primarily by the members of the USAMRDC Neuroscience Working Group for Chemical Defense, consisting of representatives from both the Walter Reed Army Institute of Research (WRAIR) and the U.S. Army Biomedical Research Laboratory (BML). I particularly would like to acknowledge the help of Dr. John McDonough from the BML. Other representatives from the BML included Drs. David Lenz and Dan Rickett. WRAIRrepresentatives included Drs. Fred Tyner, Boyd Campbell, and myself. Other participants included Drs. To~ny Shih, Jim Romano, and David Penetar as well as Mr. Andy Kaminskis, and Donald Maxwell from the BML, and Drs. Frank Sodetz, Jim Meyerhoff, Steve Hursh, Lynn K a u f m a n , a n d Paul Newhouse from the WRAIR. MG William Aug~rson, Deputy Assistant Secretary of the Army for Health Research and Programs, also participated. This set of experiments arose out of observations that the toxicity of many drugs changes with time of day. This has been demonstrated w i t h a variety of drugs (Reinberg and Halberg, .197.1) including the organophosphate (OP) paraoxon, the active metabolite of parathion, and a potent inhibitor of cholinesterase (Mayersbach, 1974). There have been no reports, however of such variations in the toxic actions of OP chemical warfare agents. The finding of a circadian rhythm in susceptibility to "'nerve agent" poisoning would be interesting for several reasons. 9First, it would provide another direction in w h i c h to search for clues to the mechanism or mechanisms of action of these compounds. Secondly, it would have serious methodological implications for continued research into the mechanisms of action of OP agents, and antidotes. Finally, and perhaps most importantly, capitalizing upon and amplifying " n o r m a l " protective mechanisms which alter the toxicity of these drugs may provide alternatives to current approaches to prophylaxsis and therapy. The study dealt with circadian variability in the toxicity o f " S o m a n " or "'GD'" (3,3-dimethyl-2butyl methylphosphonofluoridate), a potent and rapid-acting OP "'nerve" agent.
The experimental plan was relatively simple, replication of a standard lethality experiment at six different times of the day. Table 1 shows the design and some of the major results of this experiment. 250 rats were the subjects, an~d, based on a pilot experiment, five intramuscular doses of Soman were chosen to bracket the observed LDhO of 95.5 pg/kg. Prior to and during the experiment, the animals were maintained under a 12-12 light-dark cycle with lights on from 0 6 0 0 h - 1 8 0 0 h and with dim red illumination presen! in the room during the dark phase for two days prior to injections. The number of animals for each cell in the table was ten. This table shows several things. First, the group that was injected at 2200h showed no signs of poisoning and none of the animals died. While it is possible that this represented a " t r u e " effect, since fresh dilutions of Soman were made for each time point, it is also possible that some problem occurred with this dilution, and therefore these data are excluded from remaining analyses. This time point was replicated in an experiment I will report on later. Even disregarding the 2200h group, overall lethality remained low with only 16% of the animals dying w i t h i n the 24 hours following injections, and only 36% of the animals dying in the high dose groups. The reasons for our failure to replicate the LDhO of the pilot experiment remain unclear. If you focus upon the right-hand column of this table, you can see that fewer animals died at 1800h, and that the greatest number died at lO00h.
SOMAN LETHALITY 150-
0200 0600 1000 1400 1800 2200
2 0 0 0 1 0
87.1
>I <
DARK
9
- - 140-
TABLE 1 L e t h a l i t i e s A f t e r 28 Hours 79.4
LIGHT
I
=E
T i m e of Day
<
130-
9
',
28 HOURS
'
10 DAYS
120
Dose (pglkg, i.m.) 95.5 104.7 114.8
Total
-" 1100 1 1 1 0 O.
2 0 1 1 0 0
4 1 6 2 .. 0 0
3 4 4 4 3 0
11 6 12 8 4 0
100
i
10
1'4
18
:2
2'2
TIME (HOURS} FIG. 1. L D 5 0 s at 28 h o u r s and 10 days.
Copyrighl 238
1951, Society of
Toxicology
Fundam. AppL ToxieoL (1)
3larch~April. 1981
SYMPOSIUM O N PROPtIYLAXIS A N D T R E A T M E N T OF O R G A N O P I t O S P t t A T E P O I S O N I N G
less of time of day, and time of day produced significant changes in body weight independent of dose.
TABLE 2 L e t h a l i t i e s A f t e r 10 D a y s T i m e of Day
79.4
87.1
0200 0600 1000 1400 1800 2200
3 1 0 2 l 0
0 2 l 2 0 0
Dose (/aglkg, i.m.) 95.5 104.7 114.8 4 2 4 3 1 0
4 3 6 3 4 0
4 6 6 5 4 0
Tolal 15 14 17 15 10 0
Table 2 shows cumulative lethalities after 10 days during w h i c h no treatment was given. An additional 30 animals died during this time, making a total of 28% overall, and 50% in the high dose groups. Again, the right-hand column shows g~:eatest lethality at 1000h and least lethality at 1800h. The lethality data are further illustrated in figure 1 which shows LD50s computed by a probit technique (Finney, 1971). This table shows that the lowest toxicity (Le. the highest LD50) occurred at 1800h or 0200h, depending on when lethalitywas assessed. The relatively low percentage of deaths in this experiment resulted in wide confidence limits for these LD50 values, but the data are at least suggestive of a change in toxicity as a function of time of day. The greatest lethality occurred during the light phase of the L/D cycle, which for the rat, a nocturnal animal, is the time of day during w h i c h the animal is normally inactive. Body weight changes in this experiment provided another index of toxicity. The animals were weighed at the time of injectien, and 24 hours later. Figure 2 shows body weight changes in the first 24 hours after injections. For purposes of the figure, all doses were pooled. This figure clearly illustrates the anamolous nature of the 2200h group. A two-way analysis of variance across dose and time (excluding the 2200h group) showed the effects of both dose (P<.O01) and time (p<.02) to be highly significant, with no significant interactions between the two factors (SAS Users Guide, 1979a). That is, dose of soman produced significant changes in body weight regard-
Since anamolous findings were obtained at 2200h, a replication experiment was performed in which animals were tested at 1000h and 2200h only. In this experiment, the same range of doses was used, with the addition of one higher dose. The trend obtained in the original experiment continued in this replication experiment. Figure 3 shows that the lethality doseeffect curve is shifted to the left at 1000h, relative to 2200h. The LD50 values at lO00h and 2200h were respectively, 104.8 and 116.0/~g/kg. A multivariate analysis performed by Dr. Romano of the BML showed that both dose and, more importantly, time, produced significant differences in lethality in this experiment. To summarize, the two experiments demonstrated significant changes as a function of time of day in the toxicity of Soman, as measured by both lethality and the non-lethal endpoint, body weight loss. Several mechanisms of circadian toxicity changes are possible. Changes in absorption and distribution (e.g. blood-brain barrier permeability), changes in the site of action (e.g. activity of cholinesterase), changes in detoxification mechanisms (e.g. liver enzyme systems), or changes in other aspects of the toxic response (e.g. receptor sensitivity, or cyclic nucleotide levels) may all occur. With regard to organophosphates, yon Meyersbach (1974) suggested that changes in brain cholinesterase levels, as well as in liver detoxification.mechanisms were responsible for the changes he observed in paraoxon toxicity as a function of time of day. . We explored one of these possibilit!es in a third experiment paralleling the original .around-the-clock experiment. In addition to the animals that received Soman, an additional 10 animals were sacrificed at each time point for analysis of blood and brain cholinesterase activity levels, and a seventh group of 10 animals was sacrificed at 0600h on the second day, repiicating the first 0600h group. These animals were anesthetized with pentobarbital, perfused with normal Saline, the brains removed immediately, placed on ice, dissected into eight regions, and frozen for assay. The brain tissues were unfrozen
24
HOURS
POST-DRUG
BODY WEIGHT LOSS rt " r
5"
DARK
LIGHT
9l I !
..,r
i
0
I-H
22a~
I--
-5W U O.
-10ul
;~ - 1 5
I~0
1~4 TIME
18
"T
2'2
OF INJECTION
FIG. 2. % w e i g h t c h a n g e in first 24 h o u r s . Fundamental and Applied Toxicology
(1) 3-4/81
LOG GO DOSE FIG. 3. Lethality p e r c e n t a g e s at 1000h and 2200h.
239
activity in the hypothalamus is plotted as a function of the time of sacrifice. In figure 4, the points represent mean activity, the vertical bars, the standard error, and the smooth curve, the best least-squares fit sinusoid. For the hypothalamus, shown in this figure, even though the fitted points fall close to the fitted curve, there were no significant differences as a function of time of day. Figure 5 shows similar data for brainstem. The points marked w i t h asterisks are significantly different from the lowest (1000h) point. Figure 6 shows similar data for the midbrain region, w h i c h als0 showed significant differences in cholinesterase activity as a function of time of day. Figure 7 presents the fitted curves for hypothalamus, brainstem, and midbrain to show the phasing of these rhythms. In this figure, the curves are plotted as percent of mean activity. For all three of these areas, w h i c h were the only ones to have rhythms approximating 24 hours, the lowest cholinesterase activity occurred during the light phase at the time when soman was observed to be most toxic, and highest cholinesterase activity occurred during the dark, when Soman was least toxic.
HYPOTHALAMUS
~
=': 210
~,
[
205
eL. 200]
10o ~.
--LIGHT
185~,
> <
,
6
10
14
18
"!
DARK
.
.
I
22
2
6
MIDBRAIN
! TIME (HOURS) FIG. 4. Cholinesterase activity in hypothalamus.
275" <
*
LIGHT
>
,c
DARK
>z I
:i
a.
. BRAINSTEM
z
;
270"
,
M=I
!
280
i-o
s
265z
275 260" IJJ
o... W
c~ 5 c~
255
250
LIGHT
J(
/ ~~ 260] .~ 2
6
x
DARK
>
6
1~0
1'4
22
89
6
TIME (HOURS) FIG. 6. Cholinesterase activity in midbrain.
5
1'0
5
1'4
~
18
2'2
2
CHOLINESTERASE ACTIVITY
6
<
105
TIME (HOURS)
UGHT
FIG. 5. Cholinesterase activity in brainstern. w i t h i n 2 4 hours, h o m o g e n i z e d w i t h a T r i t o n - s a l i n e solution, c e n t r i f u g e d , and t h e s u p e r n a t a n t d e c a n t e d and frozen for later a s s a y (Groff et al., 1976). T h e r e w e r e no significant d i f f e r e n c e s in blood c h o l i n e s t e r a s e activity a s a f u n c t i o n of t i m e of day. T h e data from brain r e g i o n s w a s a n a l y z e d in s e v e r a l w a y s . First, for e a c h r e g i o n a one-way analysis of variance was performed across time to determine if there were any differences between groups. In those regions where differences existed, a non-linear regression procedure (SAS Users Guide, 1979b) was used to fit a sinusoidal function to the data by the method of least squares. For three of the regions, brainstem, midbrain, and hypothalamus, the period of the best-fit curve approximated 24 hours. These data are shown in figure 4, in w h i c h cholinesterase 2-t0
18
103
9 9
DARK
~,
!
I
I
I
~r~
z
BRAINSTEM
.~ t0t-
MIDBRAIN HYPOTHALMUS
9997"
95
1'o
h
le
~2
TIME (HOURS)
~
e
FIG. 7. Fitted ChE activity curves for 3 brain regions. Fundam. AppL Toxicol. (!)
3larch~April, 1981
SYMPOSIUM ON PROPHYLAXIS AND TREATMENT OF ORGANOPHOSPHATE POISONING The differences demonstrated in Soman toxicity and cholinesterase activity as a function of time of day are not particularly large, but taken together, they make a point. It is suggested that the relationship between the toxicity and cholinesterase data may be as follows: The dose-effect curve for OP lethality is quite steep. Low doses of OPs deplete cholinesterase, w h i c h exists in excess. If a rhythm in brain cholinesterase exists, as we have shown for the midbrain and brainstem, this fluctuation would be reflected in the dose of an OP necessary to cross the threshold for toxic effects, and would appear as a rhythm in toxicity. This oversimplified picture is but one of many possible mechanisms, and should be regarded as merely a suggestion. Investigations into other possible mechanisms are underway. At the very least, the data presented have significant methodological implications for research in organophosphate toxicity in that they suggest that the time of day at which experiments are performed must be taken into consideration.
Fundamental and Applied Toxicology
(1) 3-4/81
REFERENCES
Fin n ey, D.J. (1971 ). Statist ical methods hz biological science, G ri ffi n Press, London. Cited in S A S Users Guide, 1979 Edition. S A S Institute, Raleigh, N.C., 1979, pp. 357-360. Groff, W.A., Kaminskis, A. and Ellin, R.I. (1976). lnterconversion of chofinesterase activity units by the manual delta pH method, and a recommended automated method. Clin. ToxicoL 9:353-358. Mayersbach, H.V. (1974). Circadian liver detoxication and acetylcholinesterase rhythmicity: two limiting factors in circadian E600 toxicity. In L.E. Scheving, F. Halberg and I.E. Pauly (Eds.), Chronobiolog.r, Igaku Shoin Ltd., Tokyo, pp. 191-196. Reinberg, A. and Halberg, F. (1971). Circadian chronopharmacol= ogy. Ann. Rev. PharmacoL 11:455-492. General linear models procedure (1979a). In S A S Users Gttide, 1979 Edition. SAS Institute, Raleigh, N.C., pp. 237-263. Non-linear regression (1979b). In S A S Users Guide, 1979 Edition. SAS Institute, Raleigh, N.C., pp. 317-329.
241
Circadian Susceptibility to Soman Poisoning T I M O T H Y F. E L S M O R E W a l t e r Reed A r m y I n s t i t u t e of R e s e a r c h , W a s h i n g t o n , D.C. 20012
INTRODUCTION This work was performed primarily by the members of the USAMRDC Neuroscience Working Group for Chemical Defense, consisting of representatives from both the Walter Reed Army Institute of Research (WRAIR) and the U.S. Army Biomedical Research Laboratory (BML). I particularly would like to acknowledge the help of Dr. John McDonough from the BML. Other representatives from the BML included Drs. David Lenz and Dan Rickett. WRAIRrepresentatives included Drs. Fred Tyner, Boyd Campbell, and myself. Other participants included Drs. To~ny Shih, Jim Romano, and David Penetar as well as Mr. Andy Kaminskis, and Donald Maxwell from the BML, and Drs. Frank Sodetz, Jim Meyerhoff, Steve Hursh, Lynn K a u f m a n , a n d Paul Newhouse from the WRAIR. MG William Aug~rson, Deputy Assistant Secretary of the Army for Health Research and Programs, also participated. This set of experiments arose out of observations that the toxicity of many drugs changes with time of day. This has been demonstrated w i t h a variety of drugs (Reinberg and Halberg, .197.1) including the organophosphate (OP) paraoxon, the active metabolite of parathion, and a potent inhibitor of cholinesterase (Mayersbach, 1974). There have been no reports, however of such variations in the toxic actions of OP chemical warfare agents. The finding of a circadian rhythm in susceptibility to "'nerve agent" poisoning would be interesting for several reasons. 9First, it would provide another direction in w h i c h to search for clues to the mechanism or mechanisms of action of these compounds. Secondly, it would have serious methodological implications for continued research into the mechanisms of action of OP agents, and antidotes. Finally, and perhaps most importantly, capitalizing upon and amplifying " n o r m a l " protective mechanisms which alter the toxicity of these drugs may provide alternatives to current approaches to prophylaxsis and therapy. The study dealt with circadian variability in the toxicity o f " S o m a n " or "'GD'" (3,3-dimethyl-2butyl methylphosphonofluoridate), a potent and rapid-acting OP "'nerve" agent.
The experimental plan was relatively simple, replication of a standard lethality experiment at six different times of the day. Table 1 shows the design and some of the major results of this experiment. 250 rats were the subjects, an~d, based on a pilot experiment, five intramuscular doses of Soman were chosen to bracket the observed LDhO of 95.5 pg/kg. Prior to and during the experiment, the animals were maintained under a 12-12 light-dark cycle with lights on from 0 6 0 0 h - 1 8 0 0 h and with dim red illumination presen! in the room during the dark phase for two days prior to injections. The number of animals for each cell in the table was ten. This table shows several things. First, the group that was injected at 2200h showed no signs of poisoning and none of the animals died. While it is possible that this represented a " t r u e " effect, since fresh dilutions of Soman were made for each time point, it is also possible that some problem occurred with this dilution, and therefore these data are excluded from remaining analyses. This time point was replicated in an experiment I will report on later. Even disregarding the 2200h group, overall lethality remained low with only 16% of the animals dying w i t h i n the 24 hours following injections, and only 36% of the animals dying in the high dose groups. The reasons for our failure to replicate the LDhO of the pilot experiment remain unclear. If you focus upon the right-hand column of this table, you can see that fewer animals died at 1800h, and that the greatest number died at lO00h.
SOMAN LETHALITY 150-
0200 0600 1000 1400 1800 2200
2 0 0 0 1 0
87.1
>I <
DARK
9
- - 140-
TABLE 1 L e t h a l i t i e s A f t e r 28 Hours 79.4
LIGHT
I
=E
T i m e of Day
<
130-
9
',
28 HOURS
'
10 DAYS
120
Dose (pglkg, i.m.) 95.5 104.7 114.8
Total
-" 1100 1 1 1 0 O.
2 0 1 1 0 0
4 1 6 2 .. 0 0
3 4 4 4 3 0
11 6 12 8 4 0
100
i
10
1'4
18
:2
2'2
TIME (HOURS} FIG. 1. L D 5 0 s at 28 h o u r s and 10 days.
Copyrighl 238
1951, Society of
Toxicology
Fundam. AppL ToxieoL (1)
3larch~April. 1981
SYMPOSIUM O N PROPtIYLAXIS A N D T R E A T M E N T OF O R G A N O P I t O S P t t A T E P O I S O N I N G
less of time of day, and time of day produced significant changes in body weight independent of dose.
TABLE 2 L e t h a l i t i e s A f t e r 10 D a y s T i m e of Day
79.4
87.1
0200 0600 1000 1400 1800 2200
3 1 0 2 l 0
0 2 l 2 0 0
Dose (/aglkg, i.m.) 95.5 104.7 114.8 4 2 4 3 1 0
4 3 6 3 4 0
4 6 6 5 4 0
Tolal 15 14 17 15 10 0
Table 2 shows cumulative lethalities after 10 days during w h i c h no treatment was given. An additional 30 animals died during this time, making a total of 28% overall, and 50% in the high dose groups. Again, the right-hand column shows g~:eatest lethality at 1000h and least lethality at 1800h. The lethality data are further illustrated in figure 1 which shows LD50s computed by a probit technique (Finney, 1971). This table shows that the lowest toxicity (Le. the highest LD50) occurred at 1800h or 0200h, depending on when lethalitywas assessed. The relatively low percentage of deaths in this experiment resulted in wide confidence limits for these LD50 values, but the data are at least suggestive of a change in toxicity as a function of time of day. The greatest lethality occurred during the light phase of the L/D cycle, which for the rat, a nocturnal animal, is the time of day during w h i c h the animal is normally inactive. Body weight changes in this experiment provided another index of toxicity. The animals were weighed at the time of injectien, and 24 hours later. Figure 2 shows body weight changes in the first 24 hours after injections. For purposes of the figure, all doses were pooled. This figure clearly illustrates the anamolous nature of the 2200h group. A two-way analysis of variance across dose and time (excluding the 2200h group) showed the effects of both dose (P<.O01) and time (p<.02) to be highly significant, with no significant interactions between the two factors (SAS Users Guide, 1979a). That is, dose of soman produced significant changes in body weight regard-
Since anamolous findings were obtained at 2200h, a replication experiment was performed in which animals were tested at 1000h and 2200h only. In this experiment, the same range of doses was used, with the addition of one higher dose. The trend obtained in the original experiment continued in this replication experiment. Figure 3 shows that the lethality doseeffect curve is shifted to the left at 1000h, relative to 2200h. The LD50 values at lO00h and 2200h were respectively, 104.8 and 116.0/~g/kg. A multivariate analysis performed by Dr. Romano of the BML showed that both dose and, more importantly, time, produced significant differences in lethality in this experiment. To summarize, the two experiments demonstrated significant changes as a function of time of day in the toxicity of Soman, as measured by both lethality and the non-lethal endpoint, body weight loss. Several mechanisms of circadian toxicity changes are possible. Changes in absorption and distribution (e.g. blood-brain barrier permeability), changes in the site of action (e.g. activity of cholinesterase), changes in detoxification mechanisms (e.g. liver enzyme systems), or changes in other aspects of the toxic response (e.g. receptor sensitivity, or cyclic nucleotide levels) may all occur. With regard to organophosphates, yon Meyersbach (1974) suggested that changes in brain cholinesterase levels, as well as in liver detoxification.mechanisms were responsible for the changes he observed in paraoxon toxicity as a function of time of day. . We explored one of these possibilit!es in a third experiment paralleling the original .around-the-clock experiment. In addition to the animals that received Soman, an additional 10 animals were sacrificed at each time point for analysis of blood and brain cholinesterase activity levels, and a seventh group of 10 animals was sacrificed at 0600h on the second day, repiicating the first 0600h group. These animals were anesthetized with pentobarbital, perfused with normal Saline, the brains removed immediately, placed on ice, dissected into eight regions, and frozen for assay. The brain tissues were unfrozen
24
HOURS
POST-DRUG
BODY WEIGHT LOSS rt " r
5"
DARK
LIGHT
9l I !
..,r
i
0
I-H
22a~
I--
-5W U O.
-10ul
;~ - 1 5
I~0
1~4 TIME
18
"T
2'2
OF INJECTION
FIG. 2. % w e i g h t c h a n g e in first 24 h o u r s . Fundamental and Applied Toxicology
(1) 3-4/81
LOG GO DOSE FIG. 3. Lethality p e r c e n t a g e s at 1000h and 2200h.
239
activity in the hypothalamus is plotted as a function of the time of sacrifice. In figure 4, the points represent mean activity, the vertical bars, the standard error, and the smooth curve, the best least-squares fit sinusoid. For the hypothalamus, shown in this figure, even though the fitted points fall close to the fitted curve, there were no significant differences as a function of time of day. Figure 5 shows similar data for brainstem. The points marked w i t h asterisks are significantly different from the lowest (1000h) point. Figure 6 shows similar data for the midbrain region, w h i c h als0 showed significant differences in cholinesterase activity as a function of time of day. Figure 7 presents the fitted curves for hypothalamus, brainstem, and midbrain to show the phasing of these rhythms. In this figure, the curves are plotted as percent of mean activity. For all three of these areas, w h i c h were the only ones to have rhythms approximating 24 hours, the lowest cholinesterase activity occurred during the light phase at the time when soman was observed to be most toxic, and highest cholinesterase activity occurred during the dark, when Soman was least toxic.
HYPOTHALAMUS
~
=': 210
~,
[
205
eL. 200]
10o ~.
--LIGHT
185~,
> <
,
6
10
14
18
"!
DARK
.
.
I
22
2
6
MIDBRAIN
! TIME (HOURS) FIG. 4. Cholinesterase activity in hypothalamus.
275" <
*
LIGHT
>
,c
DARK
>z I
:i
a.
. BRAINSTEM
z
;
270"
,
M=I
!
280
i-o
s
265z
275 260" IJJ
o... W
c~ 5 c~
255
250
LIGHT
J(
/ ~~ 260] .~ 2
6
x
DARK
>
6
1~0
1'4
22
89
6
TIME (HOURS) FIG. 6. Cholinesterase activity in midbrain.
5
1'0
5
1'4
~
18
2'2
2
CHOLINESTERASE ACTIVITY
6
<
105
TIME (HOURS)
UGHT
FIG. 5. Cholinesterase activity in brainstern. w i t h i n 2 4 hours, h o m o g e n i z e d w i t h a T r i t o n - s a l i n e solution, c e n t r i f u g e d , and t h e s u p e r n a t a n t d e c a n t e d and frozen for later a s s a y (Groff et al., 1976). T h e r e w e r e no significant d i f f e r e n c e s in blood c h o l i n e s t e r a s e activity a s a f u n c t i o n of t i m e of day. T h e data from brain r e g i o n s w a s a n a l y z e d in s e v e r a l w a y s . First, for e a c h r e g i o n a one-way analysis of variance was performed across time to determine if there were any differences between groups. In those regions where differences existed, a non-linear regression procedure (SAS Users Guide, 1979b) was used to fit a sinusoidal function to the data by the method of least squares. For three of the regions, brainstem, midbrain, and hypothalamus, the period of the best-fit curve approximated 24 hours. These data are shown in figure 4, in w h i c h cholinesterase 2-t0
18
103
9 9
DARK
~,
!
I
I
I
~r~
z
BRAINSTEM
.~ t0t-
MIDBRAIN HYPOTHALMUS
9997"
95
1'o
h
le
~2
TIME (HOURS)
~
e
FIG. 7. Fitted ChE activity curves for 3 brain regions. Fundam. AppL Toxicol. (!)
3larch~April, 1981
SYMPOSIUM ON PROPHYLAXIS AND TREATMENT OF ORGANOPHOSPHATE POISONING The differences demonstrated in Soman toxicity and cholinesterase activity as a function of time of day are not particularly large, but taken together, they make a point. It is suggested that the relationship between the toxicity and cholinesterase data may be as follows: The dose-effect curve for OP lethality is quite steep. Low doses of OPs deplete cholinesterase, w h i c h exists in excess. If a rhythm in brain cholinesterase exists, as we have shown for the midbrain and brainstem, this fluctuation would be reflected in the dose of an OP necessary to cross the threshold for toxic effects, and would appear as a rhythm in toxicity. This oversimplified picture is but one of many possible mechanisms, and should be regarded as merely a suggestion. Investigations into other possible mechanisms are underway. At the very least, the data presented have significant methodological implications for research in organophosphate toxicity in that they suggest that the time of day at which experiments are performed must be taken into consideration.
Fundamental and Applied Toxicology
(1) 3-4/81
REFERENCES
Fin n ey, D.J. (1971 ). Statist ical methods hz biological science, G ri ffi n Press, London. Cited in S A S Users Guide, 1979 Edition. S A S Institute, Raleigh, N.C., 1979, pp. 357-360. Groff, W.A., Kaminskis, A. and Ellin, R.I. (1976). lnterconversion of chofinesterase activity units by the manual delta pH method, and a recommended automated method. Clin. ToxicoL 9:353-358. Mayersbach, H.V. (1974). Circadian liver detoxication and acetylcholinesterase rhythmicity: two limiting factors in circadian E600 toxicity. In L.E. Scheving, F. Halberg and I.E. Pauly (Eds.), Chronobiolog.r, Igaku Shoin Ltd., Tokyo, pp. 191-196. Reinberg, A. and Halberg, F. (1971). Circadian chronopharmacol= ogy. Ann. Rev. PharmacoL 11:455-492. General linear models procedure (1979a). In S A S Users Gttide, 1979 Edition. SAS Institute, Raleigh, N.C., pp. 237-263. Non-linear regression (1979b). In S A S Users Guide, 1979 Edition. SAS Institute, Raleigh, N.C., pp. 317-329.
241