221
Toxicology Letters, 51 (1990) 221-225 Elsevier
TOXLET
02310
Sequence of cardiorespiratory effects of soman altered by route of administration*
David R. Franz and Roger Hilaski Physiology Branch, Pathophysiology Division, United States Army Medical Research, Institute of Chemical Defense, Aberdeen Proving Ground. MD (U.S.A.) (Received
18 October
(Accepted
2 November
Ke_vwords: Soman;
1989) 1989)
Guinea-pig;
Route of challenge;
Cardiorespiratory;
Vagus
SUMMARY Dilute travenous
soman
was administered
(IV), subcutaneous
to anesthetized
routes were used. Times to ventilatory (IP), 34.8 + 5.1 min (UAW), block occurred of absorption pared
(30 pg/kg/lO
min); in-
(IT), and upper-airway
(UAW)
failure were 8.8 & 1.3 min (IV), 10.5 k 1.2 min (LT.), 17.3 k 2.8 min
failure with all routes of challenge
began after one-half
and noncritical-site
and suggest
by slow infusion
(IP), intratracheal
and 36.0 +4.4 min (SC) for the 5 routes
before ventilatory
AV block typically
guinea-pigs
(SC), intraperitoneal
reduction
binding
that the pathogenesis
in minute volume.
of the organophosphorus of intoxication
soman is infused into regions heavily enervated
of exposure.
Atrioventricular
(AV)
except IT and IP; when it did occur, The results reflect the effects of rate compound
in the anesthetized
when all routes are com-
guinea-pig
is different
when
by vagal afferents.
INTRODUCTION
The effect of route of challenge on the pathophysiology of organophosphorus intoxication is not clearly understood. Freedman and Himwich [I] showed in rabbits that injection of diisopropylphos-
*The opinions
and assertions
contained
strued as official or as reflecting In conducting
the research
described
mal Facilities and Care as promulgated NAS/NRC. Address for correspondence: Frederick,
MD 21701-5011,
037&4274/90/S
herein are the private views of the authors
the views of the Department
David
in this report,
the authors
by the Committee
R. Franz,
adhered
on the Guide for Laboratory
Pathophysiology
Division,
B.V. (Biomedical
of Defense.
to the Guide for Laboratory Animal
USAMRIID,
U.S.A. Tel. 301-663-7181.
3.50 @ 1990 Elsevier Science Publishers
and are not to be con-
of the Army or the Department
Division)
Ani-
Resources,
Fort
Detrick,
phonofluoridate
(DFP) in both carotids
(0.1 mglkg). one carotid
(0.4 mg,‘kg). a femo-
ral artery (0.86 mgl’kg). or the portal vein (2.3 mg/kg) results in death of 50% of the animals tested. Fredriksson et al. [2] reported little difference in the progression of signs and mean time-to-death when anesthetized and awake dogs were challenged with sarin
by inhalation,
percutaneous,
and intravenous
exposure.
Ainsworth
and
Shephard [3] showed that 98% of sarin (isopropylmethylphosphonofluoridatc) vapor inhaled by rabbits and 9376 of that inhaled by dogs is absorbed cephalad of the bronchi, suggesting that inhalation of such materials results primarily in upper-airways exposure. Reasoning that the site of absorption, primarily dependent on the route of exposure, might affect the onset or sequence of the pathophysiologic manifestations of intoxication. we compared the effects of dilute soman (methylphosphonofluoridic acid 1.2,2-trimethylpropyl ester), delivered via intravenous (IV), subcutaneous (SC), intraperitoneal (IP). intratracheal (IT), and upper-airways (UAW) administration. The aim was to describe the effects of soman, by these 5 routes of challenge. on the development of the selected cardiorespiratory signs. MATERIALS
AND METHODS
Male Hartley albino guinea-pigs (Charles River Laboratories, Wilmington, MA) weighing 40&500 g were anesthetized by IP injection of pentobarbital sodium (35 mgjkg) and chloral hydrate (160 mg/kg) [4]. One ventral tracheostomy was made just caudad of the larynx and a second, 0.5 cm cephalad of the anterior aperture of the thorax. Lengths of polyethylene tubing (PE200), used as tracheal cannulae, were heat-flared slightly on one end to provide a snug fit when placed through the l/3 diameter incisions between two tracheal rings. The proximal tracheal cannula was attached to a 000 Fliesch pneumotachograph and Validyne differential transducer via a ‘T’ tube bias flow system; the artificial anatomical dead space was adjusted to within normal limits. A 20 ga indwelling catheter was fixed in the abdominal vena cava without ligation of the vessel and a PE 10 cannula was sutured into the peritoneal space. Stainless steel electrocardiograph (ECG) leads were attached to the limbs. Tidal volume was derived from electronic integration of the flow signal. Heart rate was obtained from the ECG tracing. Following 20 min stabilization, a IO-min infusion (0.022 ml/min) of 5% normal saline was begun in one of the 5 selected sites of exposure (40 min infusion of saline had no effect on parameters measured). Soman was then infused at the same rate, resulting in the delivery of 30 ,ug/kg body wt./l0 min. For administration by the UAW or IT routes, PElO tubing was prethreaded through the appropriate tracheal cannula and extended beyond the end of the cannula by 2 mm. IP and IV administration were through the preplaced cannulae. The tip of a stainless steel hypodermic needle was placed beneath the skin over the scapulae for SC administration. All animals, therefore, received soman at the same rate; only the route by which the soman was administered varied. Treatment groups were compared by ANOVA and the Scheffe procedure. P-C 0.05 was considered significant.
223
4038 36 34 32 30 22 r-
RCUTE CF CIiALLENGE
Fig. 1. Time to reduction failure
0 by route
of minute volume by one-half
of soman
challenge
in anesthetized
0, atrioventricular guinea
block 0, and total ventilatory
pigs. Values are represented
as mean
k
SEM. n=6.
RESULTS
Figure
1 illustrates
the effect of soman
intoxication
by the 5 routes
of exposure.
Reduction of minute volume by one-half occurred earlier by IV than by IP, UAW or SC challenge, and earlier by IT challenge than by SC or UAW exposure (P < 0.05) (Table I). Atrioventricular (AV) block occurred later by SC and UAW exposure than by the IV route (PcO.05). Total ventilatory failure occurred first in animals challenged by the IV, IT and IP routes and later in those receiving test agent via the UAW and SC routes (PcO.05). Atrioventricular block occurred before total ventilatory failure in all animals challenged by IV, UAW or SC routes; a normal sinus rhythm was present at the time of total respiratory failure in all animals challenged by either IT or IP routes. AV block began after one-half reduction in minute volume in all 6 animals challenged by the IV route and in 5 and 4 of 6 animals challenged by the UAW and SC routes, respectively.
224
TABLE
I
TIME (MIN) BLOCK,
TO REDUCTION
AND
TOTAL
ANESTHETIZED
OF MINUTE
VENTILATORY
VOLUME
FAILURE
BY ONE-HALF,
BY ROUTE
ATRIOVENTRICULAR
OF SOMAN
CHALLENGE
Minute volume12
Route IV
5.0*0.6’
IT
7.2rf: 1.6’
AV block 6.7kO.91
Ventdatory 8.8 *
1.Y
IP
16.2+2.6’ 23.7k3.5
28.7k3.7
34x+
SC
26.7k2.5
28.2k2.6
36.Ok4.4
17.3 * 2.84
to IP, UAW, and SC; ‘P < 0.05 when compared
pared to UAW and SC; 4P< Values are represented
0.05 when compared
failure
10.5&1.24
UAW
‘PC 0.05 compared
IN
GUINEA-PIGS.
5. I
to UAW and SC; ‘P < 0.05
when com-
to UAW and SC.
as mean + SEM. n = 6.
DISCUSSION
The point at which minute volume was reduced to one-half of pre-exposure volume is assumed to show severe respiratory system intoxication. The onset of AV block is assumed to indicate significant cardiac intoxication. There was no visible evidence of respiratory effort at the time airflow ceased. Failure of central drive rather than neuromuscular block is assumed to have occured [5]. In this slow-infusion model, differences in response to route of challenge did exist, both in chronology and sequence of events. The relative time at which pathophysiologic signs developed. when comparing routes of challenge, was generally consistent with rates of absorption from each of the sites. IV and IT routes provide direct access to the bloodstream and large, vascular surface areas for absorption respectively. It is likely that some vaporization occurred in the trachea, enhancing distribution of soman within the lung. UAW and SC administration leads to slower absorption because they provide a more limited access to vascular beds; the prolonged cardiorespiratory response is most like that described following accidental human exposure. The rate of absorption from the IP space should have been at least as rapid as with IT administration. Because IP absorption is primarily through the portal circulation [6], the test agent must pass through the liver to reach vital cardiorespiratory system tissues. Although little metabolism of the organophosphorus compounds occurs in the liver, this organ is a source of noncritical binding sites: alesterases, carboxyesterases, and mixed-function oxidases [7]. The number of noncritical sites that must be filled before the agent can bind to critical sites is important and may explain the slowed onset of signs when the IP route was used. We have not determined why challenge by IT and IP routes resulted in total respiratory failure even before the onset of AV block. Time-to-death in IT exposure was similar to that with IV challenge. Death occurred later in IP exposure, yet in neither
22s
IT nor IP did AV block occur. The IT and IP routes are alike in that both the lung and the abdominal to the respiratory
organs
are richly enervated
by the vagus nerve; vagal afferents
center may play a role in the relatively
rapid total ventilatory
fail-
ure that occurs with IT and IP challenge. REFERENCES Freedman,
A.M. and Himwich,
H.E. (1949) Site of injection
and variation
in response.
Am. J. Physiol.
156, 1255128. Fredriksson,
T., Hansson,
aesthetized
dog following
Pharmacodyn. Ainsworth,
C. and Holmstedt, inhalation,
B. (1960) Effects of sarin in the anaesthetized
percutaneous
absorption
and intravenous
infusion.
and unanArch.
Int.
126,2888302. M. and Shephard,
R.J. (1961) The intrabronchial
rates of gas flow. In: C.N. Davies
(Ed.), Inhaled
Particles
distribution
of soluble vapours
and Vapors.
Pergamon
at selected
Press, New York,
pp. 233-247. Valenstein,
E.S. (1961) A note on anesthetizing
DeCandole,
C.A., Douglas,
W.W., Evans, CL.,
son, K.M. (1953) The failure of respiration
rats and guinea pigs. J. Exp. Anal. Behav. 4. 6. Holmes,
R., Spencer,
K.E.V., Torrance,
in death by anticholinesterase
poisoning.
R.W. and Wil-
Br. J. Pharmacol.
8,466415. Lukas.
G.. Brindle, S.D. and Greengard.
istered compounds,
J. Pharmacol.
P. (1971) The route of absorption
of intraperitoneally
admin-
Exp. Ther. 187, 5622566.
Clement,
J.G. (1983) Effect of pretreatment
Biochem.
Pharmacol32,
1411~1415.
with sodium phenobarbital
on the toxicity of soman in mice.
Toxicology Letters, 51 (1990) 221-225 Elsevier
TOXLET
02310
Sequence of cardiorespiratory effects of soman altered by route of administration*
David R. Franz and Roger Hilaski Physiology Branch, Pathophysiology Division, United States Army Medical Research, Institute of Chemical Defense, Aberdeen Proving Ground. MD (U.S.A.) (Received
18 October
(Accepted
2 November
Ke_vwords: Soman;
1989) 1989)
Guinea-pig;
Route of challenge;
Cardiorespiratory;
Vagus
SUMMARY Dilute travenous
soman
was administered
(IV), subcutaneous
to anesthetized
routes were used. Times to ventilatory (IP), 34.8 + 5.1 min (UAW), block occurred of absorption pared
(30 pg/kg/lO
min); in-
(IT), and upper-airway
(UAW)
failure were 8.8 & 1.3 min (IV), 10.5 k 1.2 min (LT.), 17.3 k 2.8 min
failure with all routes of challenge
began after one-half
and noncritical-site
and suggest
by slow infusion
(IP), intratracheal
and 36.0 +4.4 min (SC) for the 5 routes
before ventilatory
AV block typically
guinea-pigs
(SC), intraperitoneal
reduction
binding
that the pathogenesis
in minute volume.
of the organophosphorus of intoxication
soman is infused into regions heavily enervated
of exposure.
Atrioventricular
(AV)
except IT and IP; when it did occur, The results reflect the effects of rate compound
in the anesthetized
when all routes are com-
guinea-pig
is different
when
by vagal afferents.
INTRODUCTION
The effect of route of challenge on the pathophysiology of organophosphorus intoxication is not clearly understood. Freedman and Himwich [I] showed in rabbits that injection of diisopropylphos-
*The opinions
and assertions
contained
strued as official or as reflecting In conducting
the research
described
mal Facilities and Care as promulgated NAS/NRC. Address for correspondence: Frederick,
MD 21701-5011,
037&4274/90/S
herein are the private views of the authors
the views of the Department
David
in this report,
the authors
by the Committee
R. Franz,
adhered
on the Guide for Laboratory
Pathophysiology
Division,
B.V. (Biomedical
of Defense.
to the Guide for Laboratory Animal
USAMRIID,
U.S.A. Tel. 301-663-7181.
3.50 @ 1990 Elsevier Science Publishers
and are not to be con-
of the Army or the Department
Division)
Ani-
Resources,
Fort
Detrick,
phonofluoridate
(DFP) in both carotids
(0.1 mglkg). one carotid
(0.4 mg,‘kg). a femo-
ral artery (0.86 mgl’kg). or the portal vein (2.3 mg/kg) results in death of 50% of the animals tested. Fredriksson et al. [2] reported little difference in the progression of signs and mean time-to-death when anesthetized and awake dogs were challenged with sarin
by inhalation,
percutaneous,
and intravenous
exposure.
Ainsworth
and
Shephard [3] showed that 98% of sarin (isopropylmethylphosphonofluoridatc) vapor inhaled by rabbits and 9376 of that inhaled by dogs is absorbed cephalad of the bronchi, suggesting that inhalation of such materials results primarily in upper-airways exposure. Reasoning that the site of absorption, primarily dependent on the route of exposure, might affect the onset or sequence of the pathophysiologic manifestations of intoxication. we compared the effects of dilute soman (methylphosphonofluoridic acid 1.2,2-trimethylpropyl ester), delivered via intravenous (IV), subcutaneous (SC), intraperitoneal (IP). intratracheal (IT), and upper-airways (UAW) administration. The aim was to describe the effects of soman, by these 5 routes of challenge. on the development of the selected cardiorespiratory signs. MATERIALS
AND METHODS
Male Hartley albino guinea-pigs (Charles River Laboratories, Wilmington, MA) weighing 40&500 g were anesthetized by IP injection of pentobarbital sodium (35 mgjkg) and chloral hydrate (160 mg/kg) [4]. One ventral tracheostomy was made just caudad of the larynx and a second, 0.5 cm cephalad of the anterior aperture of the thorax. Lengths of polyethylene tubing (PE200), used as tracheal cannulae, were heat-flared slightly on one end to provide a snug fit when placed through the l/3 diameter incisions between two tracheal rings. The proximal tracheal cannula was attached to a 000 Fliesch pneumotachograph and Validyne differential transducer via a ‘T’ tube bias flow system; the artificial anatomical dead space was adjusted to within normal limits. A 20 ga indwelling catheter was fixed in the abdominal vena cava without ligation of the vessel and a PE 10 cannula was sutured into the peritoneal space. Stainless steel electrocardiograph (ECG) leads were attached to the limbs. Tidal volume was derived from electronic integration of the flow signal. Heart rate was obtained from the ECG tracing. Following 20 min stabilization, a IO-min infusion (0.022 ml/min) of 5% normal saline was begun in one of the 5 selected sites of exposure (40 min infusion of saline had no effect on parameters measured). Soman was then infused at the same rate, resulting in the delivery of 30 ,ug/kg body wt./l0 min. For administration by the UAW or IT routes, PElO tubing was prethreaded through the appropriate tracheal cannula and extended beyond the end of the cannula by 2 mm. IP and IV administration were through the preplaced cannulae. The tip of a stainless steel hypodermic needle was placed beneath the skin over the scapulae for SC administration. All animals, therefore, received soman at the same rate; only the route by which the soman was administered varied. Treatment groups were compared by ANOVA and the Scheffe procedure. P-C 0.05 was considered significant.
223
4038 36 34 32 30 22 r-
RCUTE CF CIiALLENGE
Fig. 1. Time to reduction failure
0 by route
of minute volume by one-half
of soman
challenge
in anesthetized
0, atrioventricular guinea
block 0, and total ventilatory
pigs. Values are represented
as mean
k
SEM. n=6.
RESULTS
Figure
1 illustrates
the effect of soman
intoxication
by the 5 routes
of exposure.
Reduction of minute volume by one-half occurred earlier by IV than by IP, UAW or SC challenge, and earlier by IT challenge than by SC or UAW exposure (P < 0.05) (Table I). Atrioventricular (AV) block occurred later by SC and UAW exposure than by the IV route (PcO.05). Total ventilatory failure occurred first in animals challenged by the IV, IT and IP routes and later in those receiving test agent via the UAW and SC routes (PcO.05). Atrioventricular block occurred before total ventilatory failure in all animals challenged by IV, UAW or SC routes; a normal sinus rhythm was present at the time of total respiratory failure in all animals challenged by either IT or IP routes. AV block began after one-half reduction in minute volume in all 6 animals challenged by the IV route and in 5 and 4 of 6 animals challenged by the UAW and SC routes, respectively.
224
TABLE
I
TIME (MIN) BLOCK,
TO REDUCTION
AND
TOTAL
ANESTHETIZED
OF MINUTE
VENTILATORY
VOLUME
FAILURE
BY ONE-HALF,
BY ROUTE
ATRIOVENTRICULAR
OF SOMAN
CHALLENGE
Minute volume12
Route IV
5.0*0.6’
IT
7.2rf: 1.6’
AV block 6.7kO.91
Ventdatory 8.8 *
1.Y
IP
16.2+2.6’ 23.7k3.5
28.7k3.7
34x+
SC
26.7k2.5
28.2k2.6
36.Ok4.4
17.3 * 2.84
to IP, UAW, and SC; ‘P < 0.05 when compared
pared to UAW and SC; 4P< Values are represented
0.05 when compared
failure
10.5&1.24
UAW
‘PC 0.05 compared
IN
GUINEA-PIGS.
5. I
to UAW and SC; ‘P < 0.05
when com-
to UAW and SC.
as mean + SEM. n = 6.
DISCUSSION
The point at which minute volume was reduced to one-half of pre-exposure volume is assumed to show severe respiratory system intoxication. The onset of AV block is assumed to indicate significant cardiac intoxication. There was no visible evidence of respiratory effort at the time airflow ceased. Failure of central drive rather than neuromuscular block is assumed to have occured [5]. In this slow-infusion model, differences in response to route of challenge did exist, both in chronology and sequence of events. The relative time at which pathophysiologic signs developed. when comparing routes of challenge, was generally consistent with rates of absorption from each of the sites. IV and IT routes provide direct access to the bloodstream and large, vascular surface areas for absorption respectively. It is likely that some vaporization occurred in the trachea, enhancing distribution of soman within the lung. UAW and SC administration leads to slower absorption because they provide a more limited access to vascular beds; the prolonged cardiorespiratory response is most like that described following accidental human exposure. The rate of absorption from the IP space should have been at least as rapid as with IT administration. Because IP absorption is primarily through the portal circulation [6], the test agent must pass through the liver to reach vital cardiorespiratory system tissues. Although little metabolism of the organophosphorus compounds occurs in the liver, this organ is a source of noncritical binding sites: alesterases, carboxyesterases, and mixed-function oxidases [7]. The number of noncritical sites that must be filled before the agent can bind to critical sites is important and may explain the slowed onset of signs when the IP route was used. We have not determined why challenge by IT and IP routes resulted in total respiratory failure even before the onset of AV block. Time-to-death in IT exposure was similar to that with IV challenge. Death occurred later in IP exposure, yet in neither
22s
IT nor IP did AV block occur. The IT and IP routes are alike in that both the lung and the abdominal to the respiratory
organs
are richly enervated
by the vagus nerve; vagal afferents
center may play a role in the relatively
rapid total ventilatory
fail-
ure that occurs with IT and IP challenge. REFERENCES Freedman,
A.M. and Himwich,
H.E. (1949) Site of injection
and variation
in response.
Am. J. Physiol.
156, 1255128. Fredriksson,
T., Hansson,
aesthetized
dog following
Pharmacodyn. Ainsworth,
C. and Holmstedt, inhalation,
B. (1960) Effects of sarin in the anaesthetized
percutaneous
absorption
and intravenous
infusion.
and unanArch.
Int.
126,2888302. M. and Shephard,
R.J. (1961) The intrabronchial
rates of gas flow. In: C.N. Davies
(Ed.), Inhaled
Particles
distribution
of soluble vapours
and Vapors.
Pergamon
at selected
Press, New York,
pp. 233-247. Valenstein,
E.S. (1961) A note on anesthetizing
DeCandole,
C.A., Douglas,
W.W., Evans, CL.,
son, K.M. (1953) The failure of respiration
rats and guinea pigs. J. Exp. Anal. Behav. 4. 6. Holmes,
R., Spencer,
K.E.V., Torrance,
in death by anticholinesterase
poisoning.
R.W. and Wil-
Br. J. Pharmacol.
8,466415. Lukas.
G.. Brindle, S.D. and Greengard.
istered compounds,
J. Pharmacol.
P. (1971) The route of absorption
of intraperitoneally
admin-
Exp. Ther. 187, 5622566.
Clement,
J.G. (1983) Effect of pretreatment
Biochem.
Pharmacol32,
1411~1415.
with sodium phenobarbital
on the toxicity of soman in mice.
