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Toxicologic and physiologic effects of bromotrifluoromethane in hyperbaric atmospheres
TOXICOLOGY
AND APPLIED
PHARMACOLoGY
Toxicologic Bromotrifluoromethane
21, 1-l 1 (1972)
and
Physiologic Effects of in Hyperbaric Atmospheres1
LEON J. GREENBAUM,JR.,* LARRY G. DICKSON, DAVID L. JACKSON, AND DELBERTE. EVANS~ Department of Environmental Naval Medical Research Bethesda, Maryland Received
July
Biosciences, Institute, 20014
28, 1970
Toxicologic and Physiologic Effects of Bromotrifluoromethane in Hyperbaric Atmospheres. GREENBAUM, LEON J., JR., DICKSON, LARRY G., JACKSON, DAVID L., and EVANS, DELBERT E. (1972). Toxicol. Appl. Pharmacol. 21, l-11. Electrocardiograms, evoked visual potentials and blood gases were recorded in cats breathing bromotrifluoromethane at 165 ft of seawater (73 psig). The cats breathed the bromotrifluoromethane (228 mm Hg) in air for 5 min. Heart rates were not significantly altered, but the QRS intervals were increased from a control value of 0.05 to 0.06 sec. The increase in QRS interval was associated with frequent nodal beats. Respiratory rate and the Pace, increased. Small reductions in the amplitude and increased latency of the lateral geniculate response were recorded. In cats sacrificed immediately after exposure, electron microscopic examination of the lungs demonstrated minimal engorgement of alveolar capillaries and small numbers of acute inflammatory cells. The alveolar basement membrane was thickened with vacuolation of alveolar capillary endothelium and alveolar epithelium. No morphologic changes were seenin cats sacrificed 5 days after exposure. Bromotrifluoromethane4 has been successfully employed as a fire extinguishant in hypobaric (Haun et al., 1967), normobaric(E. I. duPont, report), and hyperbaric atmospheres (U.S. Navy, 1969) when the oxygen tension exceeded 150 mm Hg (oxygen equivalent for air at atmospheric pressure). Recent physiologic and toxicologic studies with bromotrifluoromethane at sea level pressure have demonstrated some cardiac irregularities (VanStee and Back, personal communication) and decrement in performance (Carter et al., 1970). Since the use of bromotrifluoromethane may be considered as a practical and efficient means of avoiding catastrophic fires in hyperbaric chambers, the following study was ’ The opinions or assertionscontained herein are the private onesof the authors and are not to be construed as official or reflectingthe viewsof the Navy Department or the naval serviceat large. The experimentsreported herein were conducted according to the principles set forth in the “Guide for Laboratory Animal Facilitiesand Care” preparedby the Committee on the Guide for Laboratory Animal Resources,National Academy of Sciences-National ResearchCouncil. ’ National Institute of Neurological Diseasesand Stroke, NIH, Bethesda,Md. 3 Department of Pharmacology,Schoolof Medicine, Georgetown University, Washington, D.C. ’ E. I. duPont Inc., Wilmington, Delaware. Q 1972 by Academic Press, Inc. 1
2
C;REENBAUM, JR. ET AL.
undertaken to investigate the physiological and toxicologic effects of 5:; bromotrifluoromethane in air administered for 5 min to cats pressurized to 73 psig i 165 ft seawater). METHODS
Twelve cats weighing 2.5-3.5 kg were anesthetized with a halothane/nitrous oxide mixture containing 20 y0 oxygen. After tracheal cannulation, polyethylene catheters were placed in the right and left femoral arteries and the right saphenous vein. The cat was positioned in a stereotaxic apparatus,4 and inhalation anesthesia was discontinued. In the initial phase of the study, 6 cats received pentobarbital sodium (initial dose 25 mgjkg) given iv over 2 hr. The rate was then adjusted to maintain light anesthesia without marked respiratory or cardiovascular depression. The remaining 6 cats in the study were maintained on iv drip or urethane (750 mg/kg) and chloralose (75 mg/kg) to avoid the cardiovascular and respiratory depression seen with the barbiturates. The scalp was incised in the midline, and the skin flaps were deflected laterally. A 2-cm opening was trephined in the skull over the left posterior lateral gyrus, and a bipolar stainless steel electrode was lowered in the lateral geniculate, according to the stereotaxic coordinates of Marshall and Marsan. Evoked potentials from the lateral geniculate were induced by a strobe light5 flashing into thecat’s eyes at a rate of I /sec. The responses were amplified 1000 times anddisplayed on an oscilloscope.6 One hundred consecutive responses were averaged on a computer of average transients’ and plotted on an X- Y recorder.* The left femoral artery catheter was connected to a pressure transducer,p and a continuous recording of the animal blood pressure was displayed on a polygraph.‘O The right femoral artery and right saphenous vein catheter were connected to a blood gas analyzer” forming an arterial venous shunt, which, after the cat was heparinized, permitted continuous measurement of arterial P,,. The electrodes were calibrated at surface pressure before and after each dive. The fire/explosion hazard imposed by the 1 I O-V electrical supply to power the blood gas analyzer inside the chamber was obviated by pressurizing the chamber with an inert gas (helium). The Po, recorded inside the chamber was maintained below 80 mm Hg. The animal was maintained on a separate closed-circuit breathing system, in which the breathing gas mixture could be easily changed with a minimum of equipment respiratory dead space. A pediatric nonrebreathing valve with a bag reservoir was attached to the tracheal cannula. This system allowed a rapid change in the breathing media from air to 5% bromotrifluoromethane in air. A small thermister in the tracheal cannula was employed to record respiratory rate. Rectal temperature was monitored with a thermister which was lead to a digital thermometer. I2 Core temperature was controlled at 37 rt 0.5”C by a hot water coil under the animal. ’ David Kopf, Tujunga, California. 5 Slaughter Co., Ardmore, 6 Tektronix, Beaver-ton, Oregon. ’ Technical Measurement Corporation, North Haven, Connecticut. s Hewlett Packard Co., Palo Alto, California. 9 Statham Co., Hato Rey, Puerto Rico. lo Grass Co., Quincy, Massachusetts. I1 Instrumentation Laboratory, Boston, Massachusetts. I* United Systems Corp., Dayton, Ohio.
Oklahoma.
BROMOTRIFLUOROMETHANE
IN CATS
3
Skin electrodes were placed on the legs for standard ECG recording. The ECG, like the blood pressure and respiratory rate, was continuously recorded on a polygraph. Each cat was placed in the hyperbaric chamber, and the breathing gas reservoir was connected to an air supply line. The chamber was slowly pressurized at a rate of 25 ft/min to a depth of 165 ft of seawater (73 psig). After a control period of 10 min at depth, during which measurements of ECG, blood pressure, respiratory rate, Pa,,,, Pao,, and averaged evoked visual potentials were recorded, the breathing medium was changed to a mixture of 5% bromotrifluoromethane in air. The 100 responses evoked by the strobe light at 1/set, after amplification and visual display, were averaged by the computer of average transients and then plotted on the X- Y plotter. At this time partial pressures of the gases at 73 psig were 228 mm Hg of bromotrifluoromethane, 866 mm Hg of 02, and 3466 mm Hg of Nz. The physiological parameters noted above were then recorded at 1-min intervals for the 5 min of bromotrifluoromethane exposure, after which the breathing medium was changed back to air. Appropriate recordings were taken for 5 and 10 min after cessation of bromotrifluoromethane exposure. Each cat was then decompressed over the next 30 min, including 10 min stops at 20 and 10 ft. During the final50 ft of ascent to the surface, lOO’% oxygen was used as a breathing medium to facilitate safe decompression. After a period of 10 min at surface pressure, a final complete series of physiological measurements were made. Two of the 12 cats were sacrificed for pathological study, one immediately after exposure to bromotrifluoromethane and one 5 days after the exposure. In addition, a control cat was subjected to the complete experimental protocol with the deletion of the bromotrifluoromethane from the breathing mixture at 165 ft. Underpentobarbital anesthesia, 2.5 “/, glutaraldehyde in 250 milliosmoles s-collodine buffer was instilled into the lungs via a catheter inserted through the tracheostomy tube. Cubes of inflation-fixed lung for electron microscopy were immersed in the same fixative for 2 hr at 4°C postfixed with I .O% osmium tetroxide and stained with 2 % uranyl acetate solution. Specimens were processed through graded ethanol and xylene and embedded in PGE-22 epoxy resin. Sections were cut at a thickness of 60 mm on an ultramicrotome and stained with 0.2 ‘A lead citrate and examined in an electron microscopei at 80 kV. Portions of tissue for electron microscopy of alveolar acid mucopolysaccharide surface lining material were first stained with Muller’s colloidal iron solution, and then processed in a manner similar to the method described above. Tissue for light microscopy was placed in 10% neutral buffered formalin, processed in ethanol and xylene and embedded in paraffin. Sections were cut at 5-p intervals and stained with hematoxylin and eosin. Additional sections for light microscopy were prepared from tissue embedded in epoxy resin for electronmicroscopy. These were 0.5 to 1.5 p in thickness and stained with 0.2% toluidine blue. RESULTS Electrocardiograms were recorded continuously on all 12 cats. During the control period at depth (165 ft SW), the mean heart rate was 212 beats/min (Table 1). The I3 Ivan Sorvall Inc., Norwalk, Connecticut. I4 Siemens Corp., Iselin, New Jersey.
4
GREENBAUM,
JR. ET AL.
range of values recorded was 178-272 beats/min. The rate did not vary significantly during the 5 min exposure to bromotrifluoromethane. It should be noted that the mean heart rate in these experiments was 57 beats/min greater than that recorded in normal, unanesthetized cats (Hamlin et al., 1963). The mean PR interval in these experiments was 0.08 set with a range of 0.06-o. 12 sec. These values are similar to the values seen in normal unanesthetized cats, and the PR interval did not change during the exposure to bromotrifluoromethane. The mean QRS duration, a rough measure of the rapidity of ventricular activation, was 0.05 set during the control period (range of 0.04-0.06 set). This is greater than the value recorded in normal cats (Hamlin et al., 1963) of 0.037 with a range of 0.35-0.04 sec. After 5 min of exposure to bromotrifluoromethane, the mean QRS duration had slightly increased to 0.06 set, with a range of 0.04-0.08 sec. TABLE COMPARISON CATS AND
OF HEART RATE, PR INTERVAL, AND QRS DURATION IN NORMAL IN CATS BEFORE, DURING, AND AFTER BROMOTRIFLUOROMETHANE EXPOSURE AT 165 FT SEAWATER
Rate
Group Normal Control 2 min on Fe 1301 5 min on Fe 1301 1 min on air
1
(mean with range) 145 (105-194)
212 (178-272) 212 (160-272) 212 (160-270) 212 (155-288)
PR interval (mean with range) (se4
QRS duration (mean with range) (se4
0.08 0.08 0.08 0.08 0.09
0.037 0.050 0.056 0.060 0.059
(0.065-0.09) (0.06-o. 12) (0.07-0.12) (0.06-O. 12) (0.07-O. 12)
(0.035-0.040) (0.04-0.06) (0.04-0.07) (0.04-0.08) (0.04-0.08)
The small increase of QRS duration seen when all 12 cats are treated as a single group can be seen to be entirely accounted for by the aberrancy in ventricular conduction seen in 3 of the 12 animals (Fig. 1). This disturbance in ventricular conduction was associated with frequent nodal beats (+++ in Table 2). In 2 cats, no disturbance in cardiac rhythm was recorded (- in Table 2). In the remaining 7 animals, supraventricular conduction defects, ranging from infrequent premature atria1 contractions to frequent nodal beats, were recorded. In these animals, however, there was no aberrancy in ventricular conduction (-t- to ++ in Table 2). Blood pressure was recorded continuously in 10 of 12 animals. The values during the control period ranged from 140/60 to 180/130 (mean value was 160/l 15), During the exposure to bromotrifluoromethane, the blood pressure range was 90/40 to 166/130 with a mean of 148/96. There were no significant changes in respiratory rate (Table 2) during bromotrifluoromethane exposure, but the arterial carbon dioxide (Pace,) was elevated in 7 of the 10 cats from whom reliable blood gas values were obtained. The blood gas values recorded in Table 2 represent the maximum deviations from the control. The PaoZ decreased in nine of the 10 cats, and the drop in oxygen tension was a slow but steady decline during the bromotrifluoromethane inhalation. The rate of rise of Pa,,, was also slow. The arterial oxygen and carbon dioxide tensions returned to control levels within 2 to 3 min following the return to air breathing.
BROMOTRIFLUOROMETHANE
IN CATS
B.
CAT 119: A)
EKG
CONTROL
6)
EKG
AFTER 4 MIN
PERIOD
AT DEPTH (IGS FT. SW.)-NORMAL
EXPOSURE
INTERVAL WITH CHANGE ABERRENCY c)
EKG AFTER SOSEC SINUS RHYTHM.
FIG. 1. Electrocardiogram at depth.
SINUS RHYTHM
TO 5Y. FREON 1301- FREQUENT
IN PRS CONFIGURATION
BREATHING
recording
AIR (AFTER
NODAL EEATS,DECREASE
SUGGESTIVE
CESSATION
OF VENTRICULAR
OF FREON
IN
PR
COF(DUCTION
1301 EXPOSURE):
during control period and bromotrifluoromethane
NORMAL
exposure
The electrical response of the lateral geniculate neurons to the bromotrifluoromethane exposure was minimal reduction in amplitude and increased latency (Fig. 2). The reductions were variable and for most animals did not exceed 10%. Three animals were sacrificed for detailed examination of pulmonary histology, both by light and electron microscopy. The control animal was prepared in an identical manner to all experimental animals and underwent an identical hyperbaric exposure, with only the elimination of bromotrifluoromethane from the breathing medium. Examination of the pulmonary histology (Fig. 3) revealed normal alveolar capillaries and alveolar walls, with no evidence of acute inflammation. In the one cat that was sacrificed immediately following the bromotrifluoromethane exposure, the alveolar spaces appeared to be of normal size. The alveolar walls were thickened to 1.5 times normal and alveolar capillaries were engorged with erythrocytes
7 9 13 24 36 13 15 19 13 20 20 15
100 101 104 105 106 110 112 113 115 117 118 119
0 See text. b Off scale.
Control
Cat
Respiration Freon
90140 160/130 135180 160/110 166/110 180/100 130185 15OjlOO 150/100 160/100
-
140160 170/140 140/105 170/l 35 -
170/120 185/120 150/85 18Ojl20 180/130 180/130
15 8 16 24 42 18 20 18 9 23 19 17 47 33.5 60 35 62 25.5
43 39.5 46.5 20 -
__- &0, Control
2
705 515 09 660 760 645 700 OS 745 OS
38 45.4 18.5 49 36 68 38.5 69 31
670 695 625 760 735 710
320 790 640 -
625 -
Pao,I____(mm W Control Freon 210 250 272 211 200 200 212 230 186 230 178 182
-I ,. .-; j .: .L. I-I 1~. :. .!
214 197 240 214 255 160 240 198 200
T +-’ j
ECG” abnormalities 217 240 288
Freon
Heart rate
(73 PSIG)
Control
1301 AT 165 FT SEA WATER
48.5 -
Freon
(mm W
TO BROMOTRIFLUOROMETHANE
Control
Blood pressure (mm Hg)
RESPONSES
Freon
rate
PHYSIOLOGICAL
TABLE
BROMOTRIFLUOROMETHANE
7
IN CATS
-
AIR AT 73 PSIG
-----
5% FREON AT
73PSlG
FIG. 2. Electrical response of lateral geniculate neurons (average of 100 responses) during control period and bromotrifluoromethane exposure.
FIG. 3. Pathological specimens of lung tissue from control cat (no bromotrifluoromethane exposure). (A) Essentially normal alveolar capillary. (B) Normal alveolar capillary with normal intraluminal plate let. ~23,100.
8
GREENBAUM, JR. E‘T AL.
and smaller numbers of acute inflammatory cells (Fig. 4A, D). Electron microscopic examination showed the same features, with vacuolization of alveolar capillary endothelium and alveolar epithelium. Numerous neutrophils and platelets were adherent to endothelial surfaces with focal dissolution of opposed unit membranes. The alveolar
FIG. 4. Pathologic specimens of lung tissue from cat sacrificed immediately following bromotrifluoromethane exposure. (A) Alveolar septae are widened. Alveolar capillary engorgement is prominent. Toluidine blue stain. (B) Alveolar capillary showing intraluminal neutrophil with loss of unit membrane integrity of the alveolar capillary endothelial cells. (C) Alveolar wall showing swelling of endothelium and epithelium. A platelet is inside the capillary lumen and adherent to the endothelial cell at a small area of lost integrity of the endothelial unit membrane. (D) Colloidal iron stain of alveolar wall showing irregular acid mucopolysaccharide alveolar lining layer. A, x 1230; B, x5600; C, i: 14,600; D, x18.700.
basement membrane was thickened (Fig. 4B, C). The alveolar surface acid mucopolysaccharide was thinned and occasionally absent (Fig. 4D). One week after exposure, light microscopic examination revealed lung morphology within normal limits. The alveolar wall was much less edematous and there was less vacuolization of alveolar and capillary lining cells (Fig. 5A, B). Occasional margination of platelets still was present (Fig. 32). The alveolar surface acid mucopolysaccharide lining was normal (Fig. 5D).
BROMOTRIFLUOROMETHANE
IN CATS
9
FIG. 5. Pathological specimens of lung tissue from cat sacrificed 1 week after bromotrifluoromethane exposure. (A) Alveolar septum which is engorged. This is the most severely affected area seen; most areas showed no residual abnormality. Toluidine blue stain. (B) Essentially normal alveolar capillary. (C) Margination of platelet against damaged, thickened alveolar endothelial cell. (D) Colloidal iron stain of alveolar wall showing normal amount and distribution of alveolar surface acid mucopolysaccharide. B, ~16,000; C, x19,000; D, ~18,700.
DISCUSSION
The ability of halogenated hydrocarbons to sensitize the myocardium to the arrhythmogenic action of epinephrine is well documented (DiPalma, 1965). The cardiotoxic potential of bromotrifluoromethane in high concentrations (lo-80 %) was demonstrated by Van Stee and Back (1969). They recorded spontaneous cardiac arrhythmias when dogs and monkeys breathed bromotrifluoromethane in concentrations of 40 % or more at atmospheric pressure (14.7 psia). Generally, the higher concentrations evoked the more serious arrhythmias, with ventricular fibrillation noted after the injection of epinephrine (5-10 pug/kg). They also recorded a mild to moderate hypotension and tachycardia which preceded the onset of the arrhythmias. The results of our experiments, utilizing 5 % bromotrifluoromethane at 165 ft (6 atmospheres absolute pressure), show a broad spectrum of effects on cardiac function. In nearly all animals measured (10 of 12), there was a mild-to-moderate hypotension during the bromotrifluoromethane exposure (10-50 mm Hg drop in systolic and/or
10
GREENBAIJM,
JR. ET
AL.
diastolic pressure when compared with control period at depth). There was a mean drop of 12 mm Hg in systolic pressure and a mean drop of 19 mm Hg in diastolic pressure. In 2 of the 12 animals there were no alterations in cardiac rhythm, while various degrees of supraventricular arrhythmias were manifested by the remaining 10 cats. These abnormalities ranged from occasional premature atria1 contractions and/or nodal beats to frequent nodal beats associated with mild aberrancy in ventricular conduction (3 of 12). The control heart rate (mean = 212 beats/min) was higher than that found for normal unanesthetized cats (mean = 145 beats/min). This is suggestive of a relatively high level of sympathetic activity, especially in view of the fact that there was no appreciable increase in the heart rate during the bromotrifluoromethane exposure, despite a drop in blood pressure of 10 to 50 mm Hg (systolic and/or diastolic). This was not confirmed, as no quantitative measurements of circulating catecholamines or levels of sympathetic activity were made. The possible relationship between the observed arrythmias and circulating catecholamines in the presence of bromotrifluoromethane should be evaluated in future experiments. From a cardiovascular standpoint, exposure of cats for 5 min to an atmosphere containing 5% bromotrifluoromethane in air at 165 ft, in the face of the potentially fatal hazard of a chamber flash fire, would seem to be an acceptable risk. The question of use in manned chambers may require further study from a physiological and safety standpoint. There was a tendency for the respiratory rate in cats to increase during the bromotrifluoromethane exposure. Other investigators working with this gas have not reported any significant respiratory effects, even when bromotrifluoromethane concentrations as high as 80 % were employed. Usually, the increase in the respiratory rate paralleled the elevated Pace, (Table 2), but 4 of the animals had rates that did not differ significantly from the control values. The few studies on the central nervous system effects of bromotrifluoromethane which have been reported have been confined to changes in primate performance (Carter et al., 1970). None of these investigations has dealt with alterations in the electrical activity of the nervous system. Fluorinated hydrocarbons are known to produce central nervous system depression sufficient to produce anesthesia (DiPalma, 1965), but in general the concentrations exceed those used in this study. Small increases in response latency and minimal decrements in amplitude of response were recorded during the 5 min of bromotrifluoromethane inhalation. The changes usually did not exceed 10 %. The common denominator for these 2 phenomena, latency and amplitude, is that temporal summation is dependent on the average firing potentials of those elements recorded. During bromotrifluoromethane exposures, we have recorded an increase in latency and a decrease in amplitude. Both of these changes represent manifestations of the same basic phenomenon, a diminution in the responsiveness of the neuron pool. Carter et al. (1970) exposed monkeys to 20-25 % bromotrifluoromethane at the surface and recorded significant decrements in operant behavior. No visible signs of central nervous system depression or analgesia accompanied the loss of ability to perform conditioned performance tasks. They suggested that the mechanism by which bromotrifluoromethane causes impaired performance differs from the central nervous system depression and analgesia produced by the fluorinated anesthetics. The concentrations
BROMOTRIFLUOROMETHANE IN CATS
11
used in our study slightly exceeded those (6 atm x 5.0 % = 228 mm Hg or 30 % surface equivalent) used by Carter et al. (1970). ACKNOWLEDGMENTS We would like to expressour appreciation to Captain Robert D. Workman, MC, USN for scientific support and technical advice and also to the personnel of the Taylor Diving Company, New Orleans, for their discussion of the operational aspectsof fire prevention, It is also a pleasure to acknowledge the excellent technical assistanceof HMl N. L. Everhart, USN, HM3 D. W. Steckel, USN, and Mr. H. C. Langworthy. REFERENCES Report from E. I. duPont DeNemours and Co., Inc. Freon 1301. Wilmington, Delaware. CARTER,V. L., JR., FARRER,D. N., and BACK, K. C. (1970). The effect of bromotrifluoromethane on operant behavior in monkeys. Proc. 9th Ann. Meeting Sot. Toxicol. p. 71. DIPALMA, J. R. (Ed.) (1965). Drill’s Pharmacology in Medicine. McGraw-Hill, New York. ELLIOTT,H. W., and HINE, C. H. (1968). Clinical toxicologic studies of Freon 1301. Hine Laboratory Report, San Francisco, California. HAMLIN, R. L., SMETZER,D. L., and SMITH, C. R. (1963). The electrocardiogram, phonocardiogram and derived ventricular activation process in domestic cats. Amer. J. Vet. Res.
241,792-802. HAUN, C. C., VERNOT, E. H., MACEWEN, J. D., GEIGER,D. L., MCNERNEY,J. M., and GECKLER,R. P. (1967). Inhalation toxicity of pyrolysis products of monobromomonochloromethane (CB) and monobromomonotritluoromethane (CBrF,). Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, Ohio. Rept. TR-66-240. U.S. Navy. (1969). Navy Diving Program Review. Fire protection in hyperbaric chambers. Report 3-70, U.S. Navy Supervisor of Diving, Washington, D.C. VAN STEER,E. W., and BACK,K. C. (1969). Short-term inhalation exposure to bromotrifluoromethane. Toxicol. Appl. Pharmacol. 15, 164174.
AND APPLIED
PHARMACOLoGY
Toxicologic Bromotrifluoromethane
21, 1-l 1 (1972)
and
Physiologic Effects of in Hyperbaric Atmospheres1
LEON J. GREENBAUM,JR.,* LARRY G. DICKSON, DAVID L. JACKSON, AND DELBERTE. EVANS~ Department of Environmental Naval Medical Research Bethesda, Maryland Received
July
Biosciences, Institute, 20014
28, 1970
Toxicologic and Physiologic Effects of Bromotrifluoromethane in Hyperbaric Atmospheres. GREENBAUM, LEON J., JR., DICKSON, LARRY G., JACKSON, DAVID L., and EVANS, DELBERT E. (1972). Toxicol. Appl. Pharmacol. 21, l-11. Electrocardiograms, evoked visual potentials and blood gases were recorded in cats breathing bromotrifluoromethane at 165 ft of seawater (73 psig). The cats breathed the bromotrifluoromethane (228 mm Hg) in air for 5 min. Heart rates were not significantly altered, but the QRS intervals were increased from a control value of 0.05 to 0.06 sec. The increase in QRS interval was associated with frequent nodal beats. Respiratory rate and the Pace, increased. Small reductions in the amplitude and increased latency of the lateral geniculate response were recorded. In cats sacrificed immediately after exposure, electron microscopic examination of the lungs demonstrated minimal engorgement of alveolar capillaries and small numbers of acute inflammatory cells. The alveolar basement membrane was thickened with vacuolation of alveolar capillary endothelium and alveolar epithelium. No morphologic changes were seenin cats sacrificed 5 days after exposure. Bromotrifluoromethane4 has been successfully employed as a fire extinguishant in hypobaric (Haun et al., 1967), normobaric(E. I. duPont, report), and hyperbaric atmospheres (U.S. Navy, 1969) when the oxygen tension exceeded 150 mm Hg (oxygen equivalent for air at atmospheric pressure). Recent physiologic and toxicologic studies with bromotrifluoromethane at sea level pressure have demonstrated some cardiac irregularities (VanStee and Back, personal communication) and decrement in performance (Carter et al., 1970). Since the use of bromotrifluoromethane may be considered as a practical and efficient means of avoiding catastrophic fires in hyperbaric chambers, the following study was ’ The opinions or assertionscontained herein are the private onesof the authors and are not to be construed as official or reflectingthe viewsof the Navy Department or the naval serviceat large. The experimentsreported herein were conducted according to the principles set forth in the “Guide for Laboratory Animal Facilitiesand Care” preparedby the Committee on the Guide for Laboratory Animal Resources,National Academy of Sciences-National ResearchCouncil. ’ National Institute of Neurological Diseasesand Stroke, NIH, Bethesda,Md. 3 Department of Pharmacology,Schoolof Medicine, Georgetown University, Washington, D.C. ’ E. I. duPont Inc., Wilmington, Delaware. Q 1972 by Academic Press, Inc. 1
2
C;REENBAUM, JR. ET AL.
undertaken to investigate the physiological and toxicologic effects of 5:; bromotrifluoromethane in air administered for 5 min to cats pressurized to 73 psig i 165 ft seawater). METHODS
Twelve cats weighing 2.5-3.5 kg were anesthetized with a halothane/nitrous oxide mixture containing 20 y0 oxygen. After tracheal cannulation, polyethylene catheters were placed in the right and left femoral arteries and the right saphenous vein. The cat was positioned in a stereotaxic apparatus,4 and inhalation anesthesia was discontinued. In the initial phase of the study, 6 cats received pentobarbital sodium (initial dose 25 mgjkg) given iv over 2 hr. The rate was then adjusted to maintain light anesthesia without marked respiratory or cardiovascular depression. The remaining 6 cats in the study were maintained on iv drip or urethane (750 mg/kg) and chloralose (75 mg/kg) to avoid the cardiovascular and respiratory depression seen with the barbiturates. The scalp was incised in the midline, and the skin flaps were deflected laterally. A 2-cm opening was trephined in the skull over the left posterior lateral gyrus, and a bipolar stainless steel electrode was lowered in the lateral geniculate, according to the stereotaxic coordinates of Marshall and Marsan. Evoked potentials from the lateral geniculate were induced by a strobe light5 flashing into thecat’s eyes at a rate of I /sec. The responses were amplified 1000 times anddisplayed on an oscilloscope.6 One hundred consecutive responses were averaged on a computer of average transients’ and plotted on an X- Y recorder.* The left femoral artery catheter was connected to a pressure transducer,p and a continuous recording of the animal blood pressure was displayed on a polygraph.‘O The right femoral artery and right saphenous vein catheter were connected to a blood gas analyzer” forming an arterial venous shunt, which, after the cat was heparinized, permitted continuous measurement of arterial P,,. The electrodes were calibrated at surface pressure before and after each dive. The fire/explosion hazard imposed by the 1 I O-V electrical supply to power the blood gas analyzer inside the chamber was obviated by pressurizing the chamber with an inert gas (helium). The Po, recorded inside the chamber was maintained below 80 mm Hg. The animal was maintained on a separate closed-circuit breathing system, in which the breathing gas mixture could be easily changed with a minimum of equipment respiratory dead space. A pediatric nonrebreathing valve with a bag reservoir was attached to the tracheal cannula. This system allowed a rapid change in the breathing media from air to 5% bromotrifluoromethane in air. A small thermister in the tracheal cannula was employed to record respiratory rate. Rectal temperature was monitored with a thermister which was lead to a digital thermometer. I2 Core temperature was controlled at 37 rt 0.5”C by a hot water coil under the animal. ’ David Kopf, Tujunga, California. 5 Slaughter Co., Ardmore, 6 Tektronix, Beaver-ton, Oregon. ’ Technical Measurement Corporation, North Haven, Connecticut. s Hewlett Packard Co., Palo Alto, California. 9 Statham Co., Hato Rey, Puerto Rico. lo Grass Co., Quincy, Massachusetts. I1 Instrumentation Laboratory, Boston, Massachusetts. I* United Systems Corp., Dayton, Ohio.
Oklahoma.
BROMOTRIFLUOROMETHANE
IN CATS
3
Skin electrodes were placed on the legs for standard ECG recording. The ECG, like the blood pressure and respiratory rate, was continuously recorded on a polygraph. Each cat was placed in the hyperbaric chamber, and the breathing gas reservoir was connected to an air supply line. The chamber was slowly pressurized at a rate of 25 ft/min to a depth of 165 ft of seawater (73 psig). After a control period of 10 min at depth, during which measurements of ECG, blood pressure, respiratory rate, Pa,,,, Pao,, and averaged evoked visual potentials were recorded, the breathing medium was changed to a mixture of 5% bromotrifluoromethane in air. The 100 responses evoked by the strobe light at 1/set, after amplification and visual display, were averaged by the computer of average transients and then plotted on the X- Y plotter. At this time partial pressures of the gases at 73 psig were 228 mm Hg of bromotrifluoromethane, 866 mm Hg of 02, and 3466 mm Hg of Nz. The physiological parameters noted above were then recorded at 1-min intervals for the 5 min of bromotrifluoromethane exposure, after which the breathing medium was changed back to air. Appropriate recordings were taken for 5 and 10 min after cessation of bromotrifluoromethane exposure. Each cat was then decompressed over the next 30 min, including 10 min stops at 20 and 10 ft. During the final50 ft of ascent to the surface, lOO’% oxygen was used as a breathing medium to facilitate safe decompression. After a period of 10 min at surface pressure, a final complete series of physiological measurements were made. Two of the 12 cats were sacrificed for pathological study, one immediately after exposure to bromotrifluoromethane and one 5 days after the exposure. In addition, a control cat was subjected to the complete experimental protocol with the deletion of the bromotrifluoromethane from the breathing mixture at 165 ft. Underpentobarbital anesthesia, 2.5 “/, glutaraldehyde in 250 milliosmoles s-collodine buffer was instilled into the lungs via a catheter inserted through the tracheostomy tube. Cubes of inflation-fixed lung for electron microscopy were immersed in the same fixative for 2 hr at 4°C postfixed with I .O% osmium tetroxide and stained with 2 % uranyl acetate solution. Specimens were processed through graded ethanol and xylene and embedded in PGE-22 epoxy resin. Sections were cut at a thickness of 60 mm on an ultramicrotome and stained with 0.2 ‘A lead citrate and examined in an electron microscopei at 80 kV. Portions of tissue for electron microscopy of alveolar acid mucopolysaccharide surface lining material were first stained with Muller’s colloidal iron solution, and then processed in a manner similar to the method described above. Tissue for light microscopy was placed in 10% neutral buffered formalin, processed in ethanol and xylene and embedded in paraffin. Sections were cut at 5-p intervals and stained with hematoxylin and eosin. Additional sections for light microscopy were prepared from tissue embedded in epoxy resin for electronmicroscopy. These were 0.5 to 1.5 p in thickness and stained with 0.2% toluidine blue. RESULTS Electrocardiograms were recorded continuously on all 12 cats. During the control period at depth (165 ft SW), the mean heart rate was 212 beats/min (Table 1). The I3 Ivan Sorvall Inc., Norwalk, Connecticut. I4 Siemens Corp., Iselin, New Jersey.
4
GREENBAUM,
JR. ET AL.
range of values recorded was 178-272 beats/min. The rate did not vary significantly during the 5 min exposure to bromotrifluoromethane. It should be noted that the mean heart rate in these experiments was 57 beats/min greater than that recorded in normal, unanesthetized cats (Hamlin et al., 1963). The mean PR interval in these experiments was 0.08 set with a range of 0.06-o. 12 sec. These values are similar to the values seen in normal unanesthetized cats, and the PR interval did not change during the exposure to bromotrifluoromethane. The mean QRS duration, a rough measure of the rapidity of ventricular activation, was 0.05 set during the control period (range of 0.04-0.06 set). This is greater than the value recorded in normal cats (Hamlin et al., 1963) of 0.037 with a range of 0.35-0.04 sec. After 5 min of exposure to bromotrifluoromethane, the mean QRS duration had slightly increased to 0.06 set, with a range of 0.04-0.08 sec. TABLE COMPARISON CATS AND
OF HEART RATE, PR INTERVAL, AND QRS DURATION IN NORMAL IN CATS BEFORE, DURING, AND AFTER BROMOTRIFLUOROMETHANE EXPOSURE AT 165 FT SEAWATER
Rate
Group Normal Control 2 min on Fe 1301 5 min on Fe 1301 1 min on air
1
(mean with range) 145 (105-194)
212 (178-272) 212 (160-272) 212 (160-270) 212 (155-288)
PR interval (mean with range) (se4
QRS duration (mean with range) (se4
0.08 0.08 0.08 0.08 0.09
0.037 0.050 0.056 0.060 0.059
(0.065-0.09) (0.06-o. 12) (0.07-0.12) (0.06-O. 12) (0.07-O. 12)
(0.035-0.040) (0.04-0.06) (0.04-0.07) (0.04-0.08) (0.04-0.08)
The small increase of QRS duration seen when all 12 cats are treated as a single group can be seen to be entirely accounted for by the aberrancy in ventricular conduction seen in 3 of the 12 animals (Fig. 1). This disturbance in ventricular conduction was associated with frequent nodal beats (+++ in Table 2). In 2 cats, no disturbance in cardiac rhythm was recorded (- in Table 2). In the remaining 7 animals, supraventricular conduction defects, ranging from infrequent premature atria1 contractions to frequent nodal beats, were recorded. In these animals, however, there was no aberrancy in ventricular conduction (-t- to ++ in Table 2). Blood pressure was recorded continuously in 10 of 12 animals. The values during the control period ranged from 140/60 to 180/130 (mean value was 160/l 15), During the exposure to bromotrifluoromethane, the blood pressure range was 90/40 to 166/130 with a mean of 148/96. There were no significant changes in respiratory rate (Table 2) during bromotrifluoromethane exposure, but the arterial carbon dioxide (Pace,) was elevated in 7 of the 10 cats from whom reliable blood gas values were obtained. The blood gas values recorded in Table 2 represent the maximum deviations from the control. The PaoZ decreased in nine of the 10 cats, and the drop in oxygen tension was a slow but steady decline during the bromotrifluoromethane inhalation. The rate of rise of Pa,,, was also slow. The arterial oxygen and carbon dioxide tensions returned to control levels within 2 to 3 min following the return to air breathing.
BROMOTRIFLUOROMETHANE
IN CATS
B.
CAT 119: A)
EKG
CONTROL
6)
EKG
AFTER 4 MIN
PERIOD
AT DEPTH (IGS FT. SW.)-NORMAL
EXPOSURE
INTERVAL WITH CHANGE ABERRENCY c)
EKG AFTER SOSEC SINUS RHYTHM.
FIG. 1. Electrocardiogram at depth.
SINUS RHYTHM
TO 5Y. FREON 1301- FREQUENT
IN PRS CONFIGURATION
BREATHING
recording
AIR (AFTER
NODAL EEATS,DECREASE
SUGGESTIVE
CESSATION
OF VENTRICULAR
OF FREON
IN
PR
COF(DUCTION
1301 EXPOSURE):
during control period and bromotrifluoromethane
NORMAL
exposure
The electrical response of the lateral geniculate neurons to the bromotrifluoromethane exposure was minimal reduction in amplitude and increased latency (Fig. 2). The reductions were variable and for most animals did not exceed 10%. Three animals were sacrificed for detailed examination of pulmonary histology, both by light and electron microscopy. The control animal was prepared in an identical manner to all experimental animals and underwent an identical hyperbaric exposure, with only the elimination of bromotrifluoromethane from the breathing medium. Examination of the pulmonary histology (Fig. 3) revealed normal alveolar capillaries and alveolar walls, with no evidence of acute inflammation. In the one cat that was sacrificed immediately following the bromotrifluoromethane exposure, the alveolar spaces appeared to be of normal size. The alveolar walls were thickened to 1.5 times normal and alveolar capillaries were engorged with erythrocytes
7 9 13 24 36 13 15 19 13 20 20 15
100 101 104 105 106 110 112 113 115 117 118 119
0 See text. b Off scale.
Control
Cat
Respiration Freon
90140 160/130 135180 160/110 166/110 180/100 130185 15OjlOO 150/100 160/100
-
140160 170/140 140/105 170/l 35 -
170/120 185/120 150/85 18Ojl20 180/130 180/130
15 8 16 24 42 18 20 18 9 23 19 17 47 33.5 60 35 62 25.5
43 39.5 46.5 20 -
__- &0, Control
2
705 515 09 660 760 645 700 OS 745 OS
38 45.4 18.5 49 36 68 38.5 69 31
670 695 625 760 735 710
320 790 640 -
625 -
Pao,I____(mm W Control Freon 210 250 272 211 200 200 212 230 186 230 178 182
-I ,. .-; j .: .L. I-I 1~. :. .!
214 197 240 214 255 160 240 198 200
T +-’ j
ECG” abnormalities 217 240 288
Freon
Heart rate
(73 PSIG)
Control
1301 AT 165 FT SEA WATER
48.5 -
Freon
(mm W
TO BROMOTRIFLUOROMETHANE
Control
Blood pressure (mm Hg)
RESPONSES
Freon
rate
PHYSIOLOGICAL
TABLE
BROMOTRIFLUOROMETHANE
7
IN CATS
-
AIR AT 73 PSIG
-----
5% FREON AT
73PSlG
FIG. 2. Electrical response of lateral geniculate neurons (average of 100 responses) during control period and bromotrifluoromethane exposure.
FIG. 3. Pathological specimens of lung tissue from control cat (no bromotrifluoromethane exposure). (A) Essentially normal alveolar capillary. (B) Normal alveolar capillary with normal intraluminal plate let. ~23,100.
8
GREENBAUM, JR. E‘T AL.
and smaller numbers of acute inflammatory cells (Fig. 4A, D). Electron microscopic examination showed the same features, with vacuolization of alveolar capillary endothelium and alveolar epithelium. Numerous neutrophils and platelets were adherent to endothelial surfaces with focal dissolution of opposed unit membranes. The alveolar
FIG. 4. Pathologic specimens of lung tissue from cat sacrificed immediately following bromotrifluoromethane exposure. (A) Alveolar septae are widened. Alveolar capillary engorgement is prominent. Toluidine blue stain. (B) Alveolar capillary showing intraluminal neutrophil with loss of unit membrane integrity of the alveolar capillary endothelial cells. (C) Alveolar wall showing swelling of endothelium and epithelium. A platelet is inside the capillary lumen and adherent to the endothelial cell at a small area of lost integrity of the endothelial unit membrane. (D) Colloidal iron stain of alveolar wall showing irregular acid mucopolysaccharide alveolar lining layer. A, x 1230; B, x5600; C, i: 14,600; D, x18.700.
basement membrane was thickened (Fig. 4B, C). The alveolar surface acid mucopolysaccharide was thinned and occasionally absent (Fig. 4D). One week after exposure, light microscopic examination revealed lung morphology within normal limits. The alveolar wall was much less edematous and there was less vacuolization of alveolar and capillary lining cells (Fig. 5A, B). Occasional margination of platelets still was present (Fig. 32). The alveolar surface acid mucopolysaccharide lining was normal (Fig. 5D).
BROMOTRIFLUOROMETHANE
IN CATS
9
FIG. 5. Pathological specimens of lung tissue from cat sacrificed 1 week after bromotrifluoromethane exposure. (A) Alveolar septum which is engorged. This is the most severely affected area seen; most areas showed no residual abnormality. Toluidine blue stain. (B) Essentially normal alveolar capillary. (C) Margination of platelet against damaged, thickened alveolar endothelial cell. (D) Colloidal iron stain of alveolar wall showing normal amount and distribution of alveolar surface acid mucopolysaccharide. B, ~16,000; C, x19,000; D, ~18,700.
DISCUSSION
The ability of halogenated hydrocarbons to sensitize the myocardium to the arrhythmogenic action of epinephrine is well documented (DiPalma, 1965). The cardiotoxic potential of bromotrifluoromethane in high concentrations (lo-80 %) was demonstrated by Van Stee and Back (1969). They recorded spontaneous cardiac arrhythmias when dogs and monkeys breathed bromotrifluoromethane in concentrations of 40 % or more at atmospheric pressure (14.7 psia). Generally, the higher concentrations evoked the more serious arrhythmias, with ventricular fibrillation noted after the injection of epinephrine (5-10 pug/kg). They also recorded a mild to moderate hypotension and tachycardia which preceded the onset of the arrhythmias. The results of our experiments, utilizing 5 % bromotrifluoromethane at 165 ft (6 atmospheres absolute pressure), show a broad spectrum of effects on cardiac function. In nearly all animals measured (10 of 12), there was a mild-to-moderate hypotension during the bromotrifluoromethane exposure (10-50 mm Hg drop in systolic and/or
10
GREENBAIJM,
JR. ET
AL.
diastolic pressure when compared with control period at depth). There was a mean drop of 12 mm Hg in systolic pressure and a mean drop of 19 mm Hg in diastolic pressure. In 2 of the 12 animals there were no alterations in cardiac rhythm, while various degrees of supraventricular arrhythmias were manifested by the remaining 10 cats. These abnormalities ranged from occasional premature atria1 contractions and/or nodal beats to frequent nodal beats associated with mild aberrancy in ventricular conduction (3 of 12). The control heart rate (mean = 212 beats/min) was higher than that found for normal unanesthetized cats (mean = 145 beats/min). This is suggestive of a relatively high level of sympathetic activity, especially in view of the fact that there was no appreciable increase in the heart rate during the bromotrifluoromethane exposure, despite a drop in blood pressure of 10 to 50 mm Hg (systolic and/or diastolic). This was not confirmed, as no quantitative measurements of circulating catecholamines or levels of sympathetic activity were made. The possible relationship between the observed arrythmias and circulating catecholamines in the presence of bromotrifluoromethane should be evaluated in future experiments. From a cardiovascular standpoint, exposure of cats for 5 min to an atmosphere containing 5% bromotrifluoromethane in air at 165 ft, in the face of the potentially fatal hazard of a chamber flash fire, would seem to be an acceptable risk. The question of use in manned chambers may require further study from a physiological and safety standpoint. There was a tendency for the respiratory rate in cats to increase during the bromotrifluoromethane exposure. Other investigators working with this gas have not reported any significant respiratory effects, even when bromotrifluoromethane concentrations as high as 80 % were employed. Usually, the increase in the respiratory rate paralleled the elevated Pace, (Table 2), but 4 of the animals had rates that did not differ significantly from the control values. The few studies on the central nervous system effects of bromotrifluoromethane which have been reported have been confined to changes in primate performance (Carter et al., 1970). None of these investigations has dealt with alterations in the electrical activity of the nervous system. Fluorinated hydrocarbons are known to produce central nervous system depression sufficient to produce anesthesia (DiPalma, 1965), but in general the concentrations exceed those used in this study. Small increases in response latency and minimal decrements in amplitude of response were recorded during the 5 min of bromotrifluoromethane inhalation. The changes usually did not exceed 10 %. The common denominator for these 2 phenomena, latency and amplitude, is that temporal summation is dependent on the average firing potentials of those elements recorded. During bromotrifluoromethane exposures, we have recorded an increase in latency and a decrease in amplitude. Both of these changes represent manifestations of the same basic phenomenon, a diminution in the responsiveness of the neuron pool. Carter et al. (1970) exposed monkeys to 20-25 % bromotrifluoromethane at the surface and recorded significant decrements in operant behavior. No visible signs of central nervous system depression or analgesia accompanied the loss of ability to perform conditioned performance tasks. They suggested that the mechanism by which bromotrifluoromethane causes impaired performance differs from the central nervous system depression and analgesia produced by the fluorinated anesthetics. The concentrations
BROMOTRIFLUOROMETHANE IN CATS
11
used in our study slightly exceeded those (6 atm x 5.0 % = 228 mm Hg or 30 % surface equivalent) used by Carter et al. (1970). ACKNOWLEDGMENTS We would like to expressour appreciation to Captain Robert D. Workman, MC, USN for scientific support and technical advice and also to the personnel of the Taylor Diving Company, New Orleans, for their discussion of the operational aspectsof fire prevention, It is also a pleasure to acknowledge the excellent technical assistanceof HMl N. L. Everhart, USN, HM3 D. W. Steckel, USN, and Mr. H. C. Langworthy. REFERENCES Report from E. I. duPont DeNemours and Co., Inc. Freon 1301. Wilmington, Delaware. CARTER,V. L., JR., FARRER,D. N., and BACK, K. C. (1970). The effect of bromotrifluoromethane on operant behavior in monkeys. Proc. 9th Ann. Meeting Sot. Toxicol. p. 71. DIPALMA, J. R. (Ed.) (1965). Drill’s Pharmacology in Medicine. McGraw-Hill, New York. ELLIOTT,H. W., and HINE, C. H. (1968). Clinical toxicologic studies of Freon 1301. Hine Laboratory Report, San Francisco, California. HAMLIN, R. L., SMETZER,D. L., and SMITH, C. R. (1963). The electrocardiogram, phonocardiogram and derived ventricular activation process in domestic cats. Amer. J. Vet. Res.
241,792-802. HAUN, C. C., VERNOT, E. H., MACEWEN, J. D., GEIGER,D. L., MCNERNEY,J. M., and GECKLER,R. P. (1967). Inhalation toxicity of pyrolysis products of monobromomonochloromethane (CB) and monobromomonotritluoromethane (CBrF,). Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, Ohio. Rept. TR-66-240. U.S. Navy. (1969). Navy Diving Program Review. Fire protection in hyperbaric chambers. Report 3-70, U.S. Navy Supervisor of Diving, Washington, D.C. VAN STEER,E. W., and BACK,K. C. (1969). Short-term inhalation exposure to bromotrifluoromethane. Toxicol. Appl. Pharmacol. 15, 164174.