Neurotoxicologyand Teratology, Vol. 13, pp. 223-228. e PergamonPress plc, 1991. Printed in the U.S.A.
0892-0362/91 $3.00 + .00
Effects of Soman on Neuroendocrine and Immune Function G. JEAN KANT,* TSUNG-MING SHIH,t EDWARD W. BERNTON,* H E N R Y G. F E I N , ~ R O B E R T C. S M A L L R I D G E : ~ A N D E D W A R D H. M O U G E Y *
*Department of Medical Neurosciences and ~:Department of Clinical Physiology Walter Reed Army Institute of Research, Washington, DC 20307-5100 and y-Biochemical Pharmacology Branch, U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, MD 21010-5425 R e c e i v e d 2 M a y 1990
KANT, G. J., T.-M. SHIH, E. W. BERNTON, H. G. FEIN, R. C. SMALLRIDGE AND E. H. MOUGEY. Effects of soman on neuroendocrine and immunefunction. NEUROTOXICOL TERATOL 13(2) 223-228, 1991.--We have previously reported that plasma growth hormone (GH) and prolactin levels were markedly decreased in rats two weeks following a single dose (100 p.g/kg, SC) of soman. We have now conducted additional experiments to attempt to determine whether neuroendocrine responses to physiological or pharmacological challenge are altered in rat survivors of soman exposure, and whether immune function, which can be affected by circulating hormones, is altered in the soman-exposed rats. In the present study, basal prolactin levels were not significantly lower in the soman-treated rats although prolactin increases in response to physiological or pharmacological challenge were attenuated. Also, basal growth hormone levels in soman survivors were similar to control levels in 2 of 3 experiments in the present report. In the third experiment, growth hormone levels were lower in soman-treated animals. Endocrine abnormalities appeared to be related to the severity of soman insult as assessed by changes in body weight following exposure. Both ACTH and prolactin responses to stress were impaired in a severely affected subpopulation of soman survivors. The thymus, an important immune organ, was decreased in weight in severely affected soman survivors, but other tests of immune function did not show differences between control and soman-exposed rats. Soman Neuroendocrine Immune Metoclopramide Clonidine
Rat
Prolactin
SOMAN (pinacolyl methylphosphonofluoridate) is a potential chemical warfare nerve agent. Our laboratories are interested in assessing the long-term physiological effects of nerve agents to suggest possible pretreatments and antidotes that would protect soldiers against the adverse effects of these agents and to develop appropriate medical treatment of casualties. The primary mechanism of action of soman is believed to be the inhibition of the enzyme acetylcholinesterase, which is critical for normal neurotransmission at peripheral and central synapses utilizing the neurotransmitter acetylcholine (27,28). In addition to immediate pharmacological effects caused by excess synaptic acetylcholine, exposure to soman has been reported to cause long-lasting changes in physiology, biochemistry and behavior, and neuroanatomical lesions have been observed in rats which have survived soman exposure (10, 17, 20-22, 24-30). Cholinergic neurotransmission appears to be involved in the regulation of some hormones (3, 6, 9, 11, 18), and exposure to soman does cause immediate neuroendocrine responses in animals (4, 7, 8). However, it is difficult to separate specific cholinergic effects on the neuroendocrine system from the effects of the stress of the acute exposure (stress associated with the acute phys-
Growth hormone
ACTH
Corticosterone
iological responses to soman exposure including muscle fasciculations, ataxia, tremors, salivation, limb weakness, respiratory distress and seizures). Stress itself is a powerful stimulus for neuroendocrine responses (14, 16, 19). We have recently reported that neuroendocrine function in rats is abnormal two weeks following a single dose of soman (100 ixg/kg SC; approximately a 24 h 0.9 LDso), a time at which any stressful effects of the acute exposure on plasma hormones should no longer be present (15). Hormone levels were determined at 2h intervals around-the-clock to assess potential changes in circadian rhythms as well as changes in absolute levels. Substantial differences in plasma levels of some hormones were seen in comparing soman-treated vs. saline-treated rats. Levels of plasma prolactin were suppressed at all time points measured. In addition, normal episodic bursts of GH secretion were markedly suppressed. Both soman- and saline-treated rats displayed circadian rhythms in levels of plasma corticosterone, but the usual late afternoon rise in plasma corticosterone levels was shifted to earlier time points in the soman-treated rats. Levels of 13-endorphin and 13-1ipotrophic hormone were slightly but significantly suppressed in soman-treated rats at almost all time points. Adrenocorticotro-
~The views of the author(s) do not purport to reflect the position of the Department of the Army or the Department of Defense (para 4-3, AR 360-5). 2Research was conducted in compliance with the Animal Welfare Act, and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guidefor the Care and Use of Laboratory Animals, NIH publication 86-23, 1985 edition.
223
224
KANT ET AL.
pic hormone (ACTH) levels were increased in soman survivors,
but this difference was not statistically significant due to the high variability of the ACTH measurements. The present experiments were conducted to further investigate the neuroendocrine abnormalities found in the previous study. In particular, we were interested in assessing the dynamic response of prolactin and GH to challenges that should cause changes in the release of these hormones in normal animals, and in assessing immune function in the soman survivors since prolactin has been shown to be an immunopermissive hormone that might regulate normal immune function (1,2). METHOD
Animals Healthy male Crl:CDBR VAF/plus Sprague-Dawley rats
(Rattus norvegicus) weighing 250-300 g were used in this study. The rats were individually housed in polycarbonate shoebox cages. Food (Rodent Chow, Ziegler Brothers, Gardners, PA) and tap water were freely available. Animal rooms were maintained at 20-22°C. All animals were on a 12-hour light/dark full spectrum lighting cycle (lights on from 0600 to 1800 h). Animals were housed for two weeks prior to the injection of either saline or soman (95-100 ~g/kg SC, detailed for each experiment below). These experiments were designed to build upon and extend the findings of our previous study in which soman (100 Ixg/kg SC) was injected and 66% of the rats died within the 2-week postexposure period. Therefore, attempts were made to replicate these conditions by making slight adjustments in the soman dose between experiments to try to achieve a 66% lethality. The lethality of the soman exposure varied from experiment to experiment, due to the very steep lethality curve for soman (12). The dose injected and the lethality observed during the 2-week period following exposure for each experiment are detailed in the Results section below. Following soman injection, animals were observed and deaths were recorded hourly for 7 hours and daily thereafter. Body weights were recorded dally. All other experimental procedures were conducted two weeks following exposure on saline-treated rats and soman survivors. Experiments were conducted and animals were sacrificed at the U.S. Army Medical Research Institute of Chemical Defense (USAMRICD) at Aberdeen Proving Ground, MD. Animals were sacrificed between 0830 and 1130 h to minimize circadian effects on hormonal levels (15,16).
Experiment 1: Effects of Soman Exposure on Basal Hormone Levels Rats were injected with saline (n = 28) or 100 Ixg/kg soman (n = 84). All 28 of the saline-treated animals and 10 of the animals injected with soman survived for two weeks following exposure. Deaths occurred during the entire 2-week period, although 54 of the 74 deaths occurred within the first 24 h. Two weeks after exposure, 10 rats from the saline-injected group and 10 rats from the soman-injected group were sacrificed by decapitation immediately upon removal from their home cage, and trunk blood was collected and processed as detailed below for assay of plasma hormones (one soman animal was excluded from the data analysis because of extreme weight loss; this animal lost 191 grams during the 2-week period).
Experiment 2: Effects of Soman Exposure on Plasma Hormone Stress Response, and Immune Function Due to the high mortality observed in the first experiment, the dose of soman used in this experiment was reduced to 95 txg/kg.
One hundred and ten animals were injected with soman and 60 survived for the 2-week period. All saline-injected rats survived for 2 weeks. For this experiment, 10 soman survivors with the largest acute weight loss following exposure were selected as representing severely affected soman survivors and 10 additional soman survivors were picked randomly from the remaining 50 survivors as a less severely affected group. Each of the two soman groups (n = 10/group) and the saline group (n = 20) were then divided into 2 groups. One group was sacrificed immediately upon removal from the home cage (unstressed) and the other group was stressed for 15 min by placing each animal in a restraint device. The device (Centrap Cage, Fisher Scientific, cat #01-282-10) consisted of plastic "fingers" which could be adjusted around each animal's torso so that the animal could not move. Animals were not squeezed but were held snugly in a normal upright position. After 15 min of restraint stress, the rats were sacrificed by decapitation. Trunk blood was collected from all animals, processed as detailed below, frozen and transported to the Walter Reed Army Institute of Research (WRAIR) for hormone assays. Pituitaries, spleens and thymi were dissected from the unstressed animals only and weighed. Spleens were processed as detailed below and transported to WRAIR for mitogen-stimulated splenocyte proliferation assays.
In Vitro Immune Function Tests The spleens were removed, weighed and cells disassociated over steel mesh. Spleen cells from each half spleen were suspended in 15 ml RPMI 1640 containing 5% fetal bovine serum for transport. After arrival at WRAIR, the splenocyte suspensions were washed twice in transport buffer and adjusted to 4,000,000 cells/m1 for plating into 96 well culture plates (0.1 ml/well). Cell proliferation in response to the T cell mitogens, concanavalin A (CON; 1 p~g/ml) and phytohemagglutinin (PHA; 5 ~g/ml) and the B cell mitogen lipopolysaccharide (LPS; 5 Ixg/ml) were each used to assess cellular incorporation of 3H-methyl-thymidine (5 Ci/ mmol, Dupont) at 60 h (1).
Experiment 3: Effects of Soman Exposure on Plasma Hormone Responses to Pharmacological Challenge Rats were injected with soman (98 I~g/kg SC) or saline in this experiment. Thirty-four of 105 injected soman animals survived the 2 weeks following injection. All 36 saline-injected rats survived for the 2-week period following injection. Two weeks following soman exposure, both soman and saline animals were subdivided into 3 groups. One subgroup was injected with saline, one with metoclopramide HC1 (0.5 mg/kg) to stimulate prolactin release and the third group with clonidine HC1 (0.5 mg/kg) to stimulate growth hormone release. There were between 9 and 14 animals in each subgroup (the n for each group is listed in Table 6). All injections were given IP. Thirty min following injection, rats were sacrificed by decapitation and trunk blood was processed as detailed below. Spleens, thymi and pituitary glands were weighed but no further experiments were performed using these tissues.
Blood Collection and Processing Rats were decapitated using a guillotine within 30 s of removal from the home cage or the restraint device in Experiment 2. Trunk blood was collected in heparinized beakers; Aprotinin (a peptidase inhibitor) was added and the blood was centrifuged. Plasma was then frozen on dry ice, transported to WRAIR, and stored at - 4 0 ° C until assayed for plasma hormones.
Hormone Assays Plasma prolactin was measured by radioimmunoassay (RIA)
EFFECT OF SOMAN ON NEUROENDOCRINE FUNCTION
225
TABLE 2 EXPERIMENT 1: PLASMAHORMONES TWO WEEKSFOLLOWINGSOMAN
TABLE 1 LETHALITYAND BODY WEIGHTGAIN FOLLOWINGSOMANEXPOSURE Expt
(l~g/kg) of Soman
Expt No, 1 Saline Soman
(100)
Expt No. 2 Saline Soman Expt No. 3
(95)
% Lethality in 24 h
% Lethality in 2 Weeks
Increase in Body Weight (g)
0 62
0 88
64 _ 4 14 - 24
0 26
0 46
77 --+ 4 45 ± 13
0 50
0 68
75 ± 2 16 ±- 14
(98)
Saline Soman
Male rats were injected with saline or soman as described in the text. Values represent the mean ± SEM. Change in body weight covers the 2 weeks between saline or soman injection and sacrifice.
as described previously (19). Materials for the prolactin assay were provided by the National Institutes of Health (Bethesda, MD) through the National Hormone and Pituitary Program (NHPP). Intraassay variation was < 8 % and interassay variation
0892-0362/91 $3.00 + .00
Effects of Soman on Neuroendocrine and Immune Function G. JEAN KANT,* TSUNG-MING SHIH,t EDWARD W. BERNTON,* H E N R Y G. F E I N , ~ R O B E R T C. S M A L L R I D G E : ~ A N D E D W A R D H. M O U G E Y *
*Department of Medical Neurosciences and ~:Department of Clinical Physiology Walter Reed Army Institute of Research, Washington, DC 20307-5100 and y-Biochemical Pharmacology Branch, U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, MD 21010-5425 R e c e i v e d 2 M a y 1990
KANT, G. J., T.-M. SHIH, E. W. BERNTON, H. G. FEIN, R. C. SMALLRIDGE AND E. H. MOUGEY. Effects of soman on neuroendocrine and immunefunction. NEUROTOXICOL TERATOL 13(2) 223-228, 1991.--We have previously reported that plasma growth hormone (GH) and prolactin levels were markedly decreased in rats two weeks following a single dose (100 p.g/kg, SC) of soman. We have now conducted additional experiments to attempt to determine whether neuroendocrine responses to physiological or pharmacological challenge are altered in rat survivors of soman exposure, and whether immune function, which can be affected by circulating hormones, is altered in the soman-exposed rats. In the present study, basal prolactin levels were not significantly lower in the soman-treated rats although prolactin increases in response to physiological or pharmacological challenge were attenuated. Also, basal growth hormone levels in soman survivors were similar to control levels in 2 of 3 experiments in the present report. In the third experiment, growth hormone levels were lower in soman-treated animals. Endocrine abnormalities appeared to be related to the severity of soman insult as assessed by changes in body weight following exposure. Both ACTH and prolactin responses to stress were impaired in a severely affected subpopulation of soman survivors. The thymus, an important immune organ, was decreased in weight in severely affected soman survivors, but other tests of immune function did not show differences between control and soman-exposed rats. Soman Neuroendocrine Immune Metoclopramide Clonidine
Rat
Prolactin
SOMAN (pinacolyl methylphosphonofluoridate) is a potential chemical warfare nerve agent. Our laboratories are interested in assessing the long-term physiological effects of nerve agents to suggest possible pretreatments and antidotes that would protect soldiers against the adverse effects of these agents and to develop appropriate medical treatment of casualties. The primary mechanism of action of soman is believed to be the inhibition of the enzyme acetylcholinesterase, which is critical for normal neurotransmission at peripheral and central synapses utilizing the neurotransmitter acetylcholine (27,28). In addition to immediate pharmacological effects caused by excess synaptic acetylcholine, exposure to soman has been reported to cause long-lasting changes in physiology, biochemistry and behavior, and neuroanatomical lesions have been observed in rats which have survived soman exposure (10, 17, 20-22, 24-30). Cholinergic neurotransmission appears to be involved in the regulation of some hormones (3, 6, 9, 11, 18), and exposure to soman does cause immediate neuroendocrine responses in animals (4, 7, 8). However, it is difficult to separate specific cholinergic effects on the neuroendocrine system from the effects of the stress of the acute exposure (stress associated with the acute phys-
Growth hormone
ACTH
Corticosterone
iological responses to soman exposure including muscle fasciculations, ataxia, tremors, salivation, limb weakness, respiratory distress and seizures). Stress itself is a powerful stimulus for neuroendocrine responses (14, 16, 19). We have recently reported that neuroendocrine function in rats is abnormal two weeks following a single dose of soman (100 ixg/kg SC; approximately a 24 h 0.9 LDso), a time at which any stressful effects of the acute exposure on plasma hormones should no longer be present (15). Hormone levels were determined at 2h intervals around-the-clock to assess potential changes in circadian rhythms as well as changes in absolute levels. Substantial differences in plasma levels of some hormones were seen in comparing soman-treated vs. saline-treated rats. Levels of plasma prolactin were suppressed at all time points measured. In addition, normal episodic bursts of GH secretion were markedly suppressed. Both soman- and saline-treated rats displayed circadian rhythms in levels of plasma corticosterone, but the usual late afternoon rise in plasma corticosterone levels was shifted to earlier time points in the soman-treated rats. Levels of 13-endorphin and 13-1ipotrophic hormone were slightly but significantly suppressed in soman-treated rats at almost all time points. Adrenocorticotro-
~The views of the author(s) do not purport to reflect the position of the Department of the Army or the Department of Defense (para 4-3, AR 360-5). 2Research was conducted in compliance with the Animal Welfare Act, and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guidefor the Care and Use of Laboratory Animals, NIH publication 86-23, 1985 edition.
223
224
KANT ET AL.
pic hormone (ACTH) levels were increased in soman survivors,
but this difference was not statistically significant due to the high variability of the ACTH measurements. The present experiments were conducted to further investigate the neuroendocrine abnormalities found in the previous study. In particular, we were interested in assessing the dynamic response of prolactin and GH to challenges that should cause changes in the release of these hormones in normal animals, and in assessing immune function in the soman survivors since prolactin has been shown to be an immunopermissive hormone that might regulate normal immune function (1,2). METHOD
Animals Healthy male Crl:CDBR VAF/plus Sprague-Dawley rats
(Rattus norvegicus) weighing 250-300 g were used in this study. The rats were individually housed in polycarbonate shoebox cages. Food (Rodent Chow, Ziegler Brothers, Gardners, PA) and tap water were freely available. Animal rooms were maintained at 20-22°C. All animals were on a 12-hour light/dark full spectrum lighting cycle (lights on from 0600 to 1800 h). Animals were housed for two weeks prior to the injection of either saline or soman (95-100 ~g/kg SC, detailed for each experiment below). These experiments were designed to build upon and extend the findings of our previous study in which soman (100 Ixg/kg SC) was injected and 66% of the rats died within the 2-week postexposure period. Therefore, attempts were made to replicate these conditions by making slight adjustments in the soman dose between experiments to try to achieve a 66% lethality. The lethality of the soman exposure varied from experiment to experiment, due to the very steep lethality curve for soman (12). The dose injected and the lethality observed during the 2-week period following exposure for each experiment are detailed in the Results section below. Following soman injection, animals were observed and deaths were recorded hourly for 7 hours and daily thereafter. Body weights were recorded dally. All other experimental procedures were conducted two weeks following exposure on saline-treated rats and soman survivors. Experiments were conducted and animals were sacrificed at the U.S. Army Medical Research Institute of Chemical Defense (USAMRICD) at Aberdeen Proving Ground, MD. Animals were sacrificed between 0830 and 1130 h to minimize circadian effects on hormonal levels (15,16).
Experiment 1: Effects of Soman Exposure on Basal Hormone Levels Rats were injected with saline (n = 28) or 100 Ixg/kg soman (n = 84). All 28 of the saline-treated animals and 10 of the animals injected with soman survived for two weeks following exposure. Deaths occurred during the entire 2-week period, although 54 of the 74 deaths occurred within the first 24 h. Two weeks after exposure, 10 rats from the saline-injected group and 10 rats from the soman-injected group were sacrificed by decapitation immediately upon removal from their home cage, and trunk blood was collected and processed as detailed below for assay of plasma hormones (one soman animal was excluded from the data analysis because of extreme weight loss; this animal lost 191 grams during the 2-week period).
Experiment 2: Effects of Soman Exposure on Plasma Hormone Stress Response, and Immune Function Due to the high mortality observed in the first experiment, the dose of soman used in this experiment was reduced to 95 txg/kg.
One hundred and ten animals were injected with soman and 60 survived for the 2-week period. All saline-injected rats survived for 2 weeks. For this experiment, 10 soman survivors with the largest acute weight loss following exposure were selected as representing severely affected soman survivors and 10 additional soman survivors were picked randomly from the remaining 50 survivors as a less severely affected group. Each of the two soman groups (n = 10/group) and the saline group (n = 20) were then divided into 2 groups. One group was sacrificed immediately upon removal from the home cage (unstressed) and the other group was stressed for 15 min by placing each animal in a restraint device. The device (Centrap Cage, Fisher Scientific, cat #01-282-10) consisted of plastic "fingers" which could be adjusted around each animal's torso so that the animal could not move. Animals were not squeezed but were held snugly in a normal upright position. After 15 min of restraint stress, the rats were sacrificed by decapitation. Trunk blood was collected from all animals, processed as detailed below, frozen and transported to the Walter Reed Army Institute of Research (WRAIR) for hormone assays. Pituitaries, spleens and thymi were dissected from the unstressed animals only and weighed. Spleens were processed as detailed below and transported to WRAIR for mitogen-stimulated splenocyte proliferation assays.
In Vitro Immune Function Tests The spleens were removed, weighed and cells disassociated over steel mesh. Spleen cells from each half spleen were suspended in 15 ml RPMI 1640 containing 5% fetal bovine serum for transport. After arrival at WRAIR, the splenocyte suspensions were washed twice in transport buffer and adjusted to 4,000,000 cells/m1 for plating into 96 well culture plates (0.1 ml/well). Cell proliferation in response to the T cell mitogens, concanavalin A (CON; 1 p~g/ml) and phytohemagglutinin (PHA; 5 ~g/ml) and the B cell mitogen lipopolysaccharide (LPS; 5 Ixg/ml) were each used to assess cellular incorporation of 3H-methyl-thymidine (5 Ci/ mmol, Dupont) at 60 h (1).
Experiment 3: Effects of Soman Exposure on Plasma Hormone Responses to Pharmacological Challenge Rats were injected with soman (98 I~g/kg SC) or saline in this experiment. Thirty-four of 105 injected soman animals survived the 2 weeks following injection. All 36 saline-injected rats survived for the 2-week period following injection. Two weeks following soman exposure, both soman and saline animals were subdivided into 3 groups. One subgroup was injected with saline, one with metoclopramide HC1 (0.5 mg/kg) to stimulate prolactin release and the third group with clonidine HC1 (0.5 mg/kg) to stimulate growth hormone release. There were between 9 and 14 animals in each subgroup (the n for each group is listed in Table 6). All injections were given IP. Thirty min following injection, rats were sacrificed by decapitation and trunk blood was processed as detailed below. Spleens, thymi and pituitary glands were weighed but no further experiments were performed using these tissues.
Blood Collection and Processing Rats were decapitated using a guillotine within 30 s of removal from the home cage or the restraint device in Experiment 2. Trunk blood was collected in heparinized beakers; Aprotinin (a peptidase inhibitor) was added and the blood was centrifuged. Plasma was then frozen on dry ice, transported to WRAIR, and stored at - 4 0 ° C until assayed for plasma hormones.
Hormone Assays Plasma prolactin was measured by radioimmunoassay (RIA)
EFFECT OF SOMAN ON NEUROENDOCRINE FUNCTION
225
TABLE 2 EXPERIMENT 1: PLASMAHORMONES TWO WEEKSFOLLOWINGSOMAN
TABLE 1 LETHALITYAND BODY WEIGHTGAIN FOLLOWINGSOMANEXPOSURE Expt
(l~g/kg) of Soman
Expt No, 1 Saline Soman
(100)
Expt No. 2 Saline Soman Expt No. 3
(95)
% Lethality in 24 h
% Lethality in 2 Weeks
Increase in Body Weight (g)
0 62
0 88
64 _ 4 14 - 24
0 26
0 46
77 --+ 4 45 ± 13
0 50
0 68
75 ± 2 16 ±- 14
(98)
Saline Soman
Male rats were injected with saline or soman as described in the text. Values represent the mean ± SEM. Change in body weight covers the 2 weeks between saline or soman injection and sacrifice.
as described previously (19). Materials for the prolactin assay were provided by the National Institutes of Health (Bethesda, MD) through the National Hormone and Pituitary Program (NHPP). Intraassay variation was < 8 % and interassay variation
