TOXICOLOGY AND APPLIED PHARMACOLOGY 17,250261(1970)

The Effect of Hyperbaric Helium-Oxygen on the Acute Toxicity of Several Drugs1

ALFRED SMALL

Pharmacology Division, U.S. Naval Medical Research Institute, Bethesda, Maryland 20014

Received September 26, 1969

The Effect of Hyperbaric Helium-Oxygen on the Acute Toxicity of Several Drugs. SMALL, ALFRED (1970). Toxicol. Appl. Pharmacol. 17, 250- 261. The effect of a hyperbaric helium-oxygen environment on drug toxicity was investigated in the following systems : (1) The intravenous lethal doses of pentobarbital, lidocaine, and ethanol in rats; (2) the intravenous LD50 for morphine in rats; and (3) the oral toxicity of an LD50 dose of aspirin in mice. Except for mice given aspirin, restrained, unanesthetized animals were equilibrated with the atmosphere (19.2 atm abs. helium-O.2 atm abs. oxygen, equivalent to the pressure at a depth of 600 feet) for 45 min in a hyperbaric chamber. Animals were then injected remotely via indwelling jugular vein catheters. The lethal dose of pentobarbital, lidocaine, or ethanol was determined by slow intravenous infusion to the point of respiratory arrest. The LD50 of morphine was determined from the mortality 2 hr after injection. Mice were injected orally with a suspension of aspirin in sodium alginate and pressurized rapidly; the mortality was determined 3 hr later. For each drug, control animals were treated in exactly the same manner as pressurized animals. In the case of every drug examined, the toxicity observed during exposure to the hyperbaric environment did not differ significantly from that under normal pressure.

The establishment of underwater habitats for humans on the continental shelf requires that the occupants live in a hyperbaric environment and breathe artificial gas mixtures. Simulated dives in helium-oxygen atmospheres have shown that no danger to life exists when men are exposed to a pressure equivalent to a depth of 650 feet (Hamilton et al., 1966) or when animals are exposed to a pressure equivalent to 4000 feet (MacInnis et al., 1967).

It is inevitable that contact with the hostile animate and inanimate elements of the underwater environment, as well as spontaneous illness, will require that medical treatment and drug administration be available to the occupants of underwater habi- tats. Yet, at the present time, no data are available to indicate whether the effects of drugs in such an environment are the same as at sea level pressure.

Previous investigators have demonstrated effects of high pressure on physiological 1 From Bureau of Medicine and Surgery, Navy Department, Research Task No. MROO5.01~0093.

The opinions in this paper are those of the author and do not necessarily reflect the views of the Navy Department or the naval service at large. Experiments reported herein were conducted according to the principles enumerated in “Guide for Laboratory Animal Facilities and Care” prepared by the committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research Council.

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TOXICITY IN HYPERBARIC HELIUM-OXYGEN 251

processes in living organisms and isolated systems (Cattell, 1936), but generally the pressures at which such experiments were carried out have been much greater than those to which men will conceivably be exposed. Nevertheless, it is possible that even moderately elevated hydrostatic pressure produces physiologic and biochemical alterations. In addition, helium at high partial pressure should be regarded as a pharmacologic agent. All systems of “inert” gas under pressure, with the exception of helium, have been found to produce some degree of central nervous system depression (Marshall, 1951; Carpenter, 1954), and it has been inferred that helium will also prove to be narcotic at a sufficiently high partial pressure (Carpenter, 1954). Thus, the effects of drugs taken by men living in a hyperbaric helium-oxygen environment may be altered because of modified physiologic or biochemical processes resulting from high hydrostatic pressure per se, or because of superimposition of the pharmacologic effect of helium at high partial pressure. Therefore, it is important to determine, by means of animal studies, if drug dosage schedules used at surface ambient pressure may be used safely by deep diving aquanauts.

The purpose of this study is to determine whether exposure of laboratory animals to a helium-oxygen mixture at elevated ambient pressure changes the acute toxicity of selected drugs from that seen in animals exposed only to air at 1 atmosphere. The drugs should represent the entire spectrum of pharmacologic types. In the present communi- cation, results are reported for representatives of 5 pharmacologic categories: (1) pentobarbital, a barbiturate sedative-hypnotic; (2) ethyl alcohol, a nonbarbiturate sedative-hypnotic; (3) lidocaine, a local anesthetic; (4) acetylsalicylic acid, a non- narcotic analgesic and antipyretic; and (5) morphine, a narcotic analgesic.

METHODS

The hyperbaric chamber. All experiments were carried out inside a steel hyperbaric chamber.2 The internal configuration of the chamber was a cylinder 4 feet long by 18 inches in diameter, and was designed to withstand an internal pressure of 450 psi. The chamber was equipped with three Cinch diameter glass view ports which enabled activi- ties within to be observed from outside the chamber. The wall of the chamber was pro- vided with a number of threaded openings to permit connections to the gas supply, electricity, and to instruments. All high pressure connections were made with l/Cinch nylon pressure tubing fitted with quick-connect adapters. Electrical connections were made via pressure-tight glands3 which allowed electrical conductors to penetrate the chamber wall without gas leaks. Three separate 1 lo-Volt circuits were provided inside the chamber. An end view of the chamber is shown in Fig. 1.

The chamber atmosphere was conditioned to prevent accumulation of carbon dioxide and to properly control temperature. A separate gas scrubber unit4 was con- nected to the chamber with l-inch pipe connections. Chamber gas was forced through cannisters containing barium carbonate limes and chemical adsorbent6 before being returned to the chamber. The adsorbent was used to remove odor and ammonia from

2 Bethlehem Corp., Bethlehem, Pennsylvania. 3 Conax Corp. ; Buffalo, New York. 4 Environmental Control Unit, Model 15-30, Worldwide Development Corp., Columbus, Ohio. 5 Baralyme, National Cylinder Gas Co., Chicago, Illinois. 6 Purafil, York Corp., York, Pennsylvania.

the chamber atmosphere. Temperature was controlled by circulating water of the appropriate temperature through 50 feet of l/4-inch copper tubing coiled against the inside walls of the chamber. A 3-inch electrical fan was used inside the chamber to improve gas mixing.

A flow gauge with needle valve was installed at one of the threaded openings to pro- vide a flow of chamber gas to the oxygen and carbon dioxide monitoring instruments. This flow was maintained at about 100 mljmin during the period of pressurization.

FIG. 1. Rats with indwelling jugular vein catheters, in a pillory-type restrainer, just before being sealed in the hyperbaric chamber. The driven-syringe infusion pump can be seen behind the animals.

Carbon dioxide was read directly from an infrared CO2 analyzer,7 which was set to a full-scale sensitivity of 250 ppm and could detect as little as 2.5 ppm. Oxygen was monitored periodically by sampling the chamber gas with a vapor fractometers that could accurately detect a change of 0.067% oxygen in the gas mixture. The vapor fractometer was also used to measure nitrogen concentration in the chamber atmo- sphere, and could detect as little as 0.1 ‘A nitrogen. Both oxygen and carbon dioxide measuring systems were calibrated with accurately known standard gas mixtures.

Chamber temperature was monitored with a thermistor probe9 suspended within the

7 Model IR-215, Beckman Instruments Co., Fullerton, California. 8 Model 154D, Perkin Elmer Corp., Norwalk, Connecticut. g Model 402, Yellow Springs Instrument Co., Yellow Springs, Ohio.

TOXICITY IN HYPERBARIC HELIUM-OXYGEN 253

chamber and connected via pressure-tight electrical leads to a temperature meter located outside the chamber. Pressure was measured with a gauge connected directly to the chamber. The interior of the chamber was lighted with a single 12-V automotive- type lamp suspended above the animal restrainer.

General experimental procedure. The basic problem which had to be overcome in these experiments was how to administer a drug to an unanesthetized animal that was isolated from the investigator within a high-pressure chamber, and that had equili- brated with the hyperbaric helium of the chamber atmosphere. After a number of unsuccessful attempts to solve this problem, a method was developed by which drug could be administered intravenously to restrained unanesthetized animals via chronic- ally implanted jugular vein catheters. With the exception of toxicity studies with aspirin (described separately below), the experimental procedure was as follows : Male albino rats weighing between 175 and 225 g were used for all experiments. The NMRI : 0 Sprague-Dawley-Derived rat was used in experiments with pentobarbital and lidocaine; the Dublin-Sprague-Dawley-Derived strain was used in experiments with morphine and ethanol.

Rats were anesthetized with sodium pentobarbital*O (40 mg/kg) and surgically pre- pared 24 hr prior to an experimental run. A small incision was made in the skin of the neck directly above the right jugular vein, and the jugular vein was cannulated with an 1 &inch length of PE-50 polyethylene tubing. After the catheter was tied securely in the vein, it was anchored to the neck muscle at several places with 3-O suture, and led via a subcutaneous tunnel through a stab wound in the midline of the back, directly behind the animal’s head. The neck incision was closed with sutures. A 3-inch length of l/4- inch nylon tubing, the end of which had been flanged by heating, was slipped over the free end of the catheter. This tubing was secured to the animal by inserting the flange beneath the stab wound and tying it in place with a purse-string suture. The distal end of the tubing was sutured to the skin on the animal’s back. The catheter was filled with heparinized saline and was then folded and inserted into the lumen of the nylon tubing. This arrangement allowed the animals to recover from surgery and walk around freely without the possibility of their chewing on the catheter.

On the day following surgery, most animals appeared healthy and active. Animals that did not appear normal were never used. Five rats were placed in a specially designed pillorylike restrainer. The hind legs were bound with adhesive tape, and the tails taped to a horizontal bar to minimize struggling movements. The catheter from each animal was straightened, connected to a Touhy tubing adapter, and tested for patency by injecting 0.1 ml Evans Blue in isotonic saline. Evans Blue also served as a drug interface marker. Each catheter was then connected to a syringe containing drug solution, and the syringes mounted in an infusion pump. The infusion pump delivered drug to the animals by driving the syringes at a constant rate.

The pump and restrained animals were kept on a metal tray which could slide into and out of the chamber. When all animals had been prepared, the tray was pushed into the chamber, the hatch closed, and the arrangement inspected to assure that the pump, tubing and animals were visible through the viewing ports. The experimental arrange- ment is shown in Fig. 1.

In control experiments, animals were allowed to remain undisturbed for 45 min. The lo Nembutal Sodium Injection, Abbott Laboratories, North Chicago, Illinois.

9

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infusion pump was then turned on, and the appropriate observations were made (described in detail for each, below). Temperature was maintained at 72°F $- 2” during control runs. In experiments carried out at pressure, nitrogen was flushed from the chamber with a gas mixture of 79 % helium-21 % oxygen. When gas analysis showed residual nitrogen to be less than 0.5x, flushing was stopped, and compression with pure helium was begun at the rate of approximately 27 psi per min (equivalent to a descent rate of 60 feet per min) until the “bottom depth” of 600 feet (270 psig) was reached. Compression was stopped if chamber temperature exceeded lOO”F, and was resumed after a few minutes. After compression, animals remained undisturbed for 45 min. Oxygen partial pressure was maintained between 0.19 and 0.24 atm absolute by adding small amounts of pure oxygen when pOZ fell. Following the equilibration period, the infusion pump was turned on to deliver drug to the rats, and experimental observations were made. Temperature was maintained at 90°F * 2” during all “dives” in order to compensate for increased heat loss in hyperbaric helium atmospheres (Membery and Link, 1964; Raymond, 1967). Animals exposed to high pressure condi- tions were referred to as “dived.” The order in which dived or control conditions were imposed, as well as the sequence in which drug dosages were used, was randomized with the aid of a table of random numbers.

Aspirin toxicity studies. The method used to study the effect of pressure on aspirin toxicity was different from that described above. Because of poor solubility of the drug, it could not be administered as a solution and thus could not be injected remotely by a motor-driven syringe.

Aspirin” was suspended in 0.5 % sodium alginate at a concentration that was varied to maintain injection volume at 0.5 ml, and was administered orally to mice. A pilot study was performed to determine the LD50 for aspirin under the conditions in our laboratory, at normal (surface) pressure, for the 3-hr period following drug adminis- tration. The effect of pressure was investigated by comparing the toxicity of aspirin in dived animals with that seen in control mice, using the LD50 dose determined in the pilot study.

Ten to 21 mice, weighing between 22 and 24 g were injected orally over a period of 5 min, placed in wire mesh cages on the metal tray of the chamber, and dived at a rate of 100 ft/min (45 psi/min) with pure helium until a pressure of 270 psig was reached. Oxygen partial pressure was maintained between 0.19 and 0.24 atm absolute. Animals were observed for 3 hr from the moment final pressure was reached, and the number of deaths was recorded. One dive and 1 control experiment were made each day. Control experiments were performed in exactly the same manner as dives, except that animals were allowed to breathe air at surface pressure. Toxicity difference between the 2 groups was examined statistically using a chi-square analysis (Snedecor, 1956).

Pentobarbital toxicity. Rats, prepared as described above, were infused with a solu- tion containing 20 mg pentobarbital/ml, at a rate of 0.205 ml/min. The solution was prepared by diluting commercially prepared pentobarbital solution10 with isotonic saline. The final solution contained propylene glycol (80 mg/ml) and ethanol (40 mg/ ml), but the quantity of these substances was considered to be negligible. The starting time was taken as the instant that the Evans Blue-drug interface reached the animal. Animals were observed continuously and the time at which respiratory arrest occurred

I1 Acetylsalicylic Acid, USP, Fisher Scientific Co., Fair Lawn, New Jersey.

TOXICITY IN HYPERBARIC HELIUM-OXYGEN 255

was recorded. Using these data, the lethal dose for each animal could be calculated. The lethal doses in the two groups were compared by means of a Student’s t test (Snedecor, 1956).

Ethanol toxicity. Rats were infused with a solution of 50 % (w/v) ethanol in isotonic saline at a rate of 0.104 ml/min. Ethanol solution was prepared by diluting absolute ethanol12 with saline. Starting time was the same point used in the study of pento- barbital toxicity, and respiratory arrest was taken as the end point. A lethal dose of ethanol was calculated for each animal and the mean lethal doses for the 2 groups were compared statistically by means of a Student’s t test.

Lidocaine toxicity. The method for studying the toxicity of lidocaine was similar to that used for pentobarbital and ethanol. Lidocaine solution (9.75 mg/ml in saline) was infused at a rate of 0.205 ml/min. Lidocaine solution was prepared by diluting com- mercial lidocaine solution.13 The final solution contained a small quantity of preserva- tive (methylparaben, 0.485 mg/ml). Respiratory arrest was used as the end point, and the lethal doses were compared statistically with a t test.

Morphine toxicity. The toxic dose of morphine in the rat, administered by constant intravenous infusion, was determined in preliminary experiments. In contrast to the situation with pentobarbital, lidocaine, and ethanol, this dose appeared to be much greater than published values for morphine LD50. This probably occurred because there is a considerable delay between intravenous injection of a minimum lethal dose and death of the animal in the case of morphine. Thus, it was considered that the lethal dose determined by intravenous infusion was not a reliable index of toxicity, and it was decided to study the effect of pressure on the LD50 for morphine. Four doses, plus a saline control, were used for both the dived and control groups, and each group con- sisted of 25 animals. After the 45-min period of equilibration, the pump was turned on to inject the drug. Solution strength was adjusted to keep injection volume about 0.8 ml, and the injection rate was about 0.7 ml/min. Solutions were prepared by dissolving the appropriate weight of morphine sulfate14 in isotonic saline. An excess of the solu- tion equal to the catheter dead space was included in the volume of drug in the syringe. Starting from completion of injection, animals were observed every 10 min for 2 hr, and the mortality was noted at each observation. LD50 values were calculated according to the methods of Berkson (1955) and Litchfield and Wilcoxon (1949). Differences in mortality for the two groups’at each dose level were analyzed statistic- ally with a chi-square test (Snedecor, 1956).

RESULTS

Pentobarbital

Table 1 shows the results of experiments with pentobarbital, given to rats by slow continuous intravenous infusion. There was no significant difference in acute toxicity between the 2 groups. The value for the lethal dose of pentobarbital found in the present experiments is very close to the LD50 reported by Hunt et al. (1946).

I2 Ethanol, analyzed reagent, U.S. Industrial Chemicals Co., New York. l3 Xylocaine hydrochloride, 2 ‘A, Astra, Inc., Worcester, Massachusetts. l4 Morphine Sulfate Merck, USP, Merck and Co., Inc., Rahway, New Jersey.

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TABLE 1 EFFECT OF HYPERBARIC HELIUM ON THE LETHAL INTRAVENOUS DOSE OF

PENTOBARBITAL, ETHANOL, AND LIDOCAINE IN RATS

Lethal dose” (,mg/kg)

Drug Dived animals Control animals P

Pentobarbital 98.9 rt 6.0 n= 12

Ethanol 5513 + 288 n=51

Lidocaine 44.0 + 4.9 n= 10

0 Mean &SE; n = number of animals.

91.6 i 6.5 10.4 n= 12

5886 zt 229 >0.3 n = 50

49.0 rt 3.4 >0.3 n = 10

Ethanol and Lidocaine

The results of studies with ethanol and lidocaine are summarized in Table 1. The mean lethal doses of ethanol in control and dived animals were not significantly different (p > 0.3). Haggard et al. (1940) reported that blood levels of ethanol at the time of respiratory failure in rats was 9.3 mg/ml. If ethanol is distributed in total body water (Haggard and Greenberg, 1934), this would correspond to a dose of 5700 mg/kg, very nearly the same found in the present experiments.

There was no significant effect of high pressure on the mean lethal dose of lidocaine. No report of lidocaine toxicity in the rat could be found in the literature, although an intravenous LD50 value of 25-37 mg/kg was reported for the mouse (Hunter, 1951).

Aspirin

A preliminary toxicity study of aspirin (po in mice) was performed in air at 1 atm to determine the proper dose to be used in later experiments. The mortality ratio was 6/14 at a dose of 1300 mg/kg, 12/15 at 1500 mg/kg, and 26/40 at 2000 mg/kg. From these data, the oral LD50 in mice was calculated to be 1261 mg/kg and a dose of 1260 mg/kg was chosen to test the effect of pressure on aspirin toxicity. This value agrees very well with the value of 1360 mg/kg for oral LD50 in the mouse reported by Brown- lee (1937). Aspirin (1260 mg/kg, po) was administered to 52 control mice and 53 dived mice, and the animals observed for 3 hr. The results of this study are summarized in Table 2. The mortality among dived animals was not significantly different from that observed in the control group.

Morphine

Results of studies with morphine are presented in Table 3. The LD50 for intra- venously administered morphine in rats (120 min after injection) was not affected by hyperbaric helium. Using the method of Berkson (1955), the LD50 was calculated to be 69.5 f 22.5 (SE) mg/kg in control animals and 65.8 * 10.2 mg/kg in dived animals. Comparison of mortality at each dose level tested showed no significant difference between the groups. Using the method of Litchfield and Wilcoxon (1949), which allows estimation of 95 % confidence limits for the LD50, control LD50 was calculated to be 69.9 mg/kg with 95% confidence limits of 30.8-158.7 mg/kg. LD50 for dived

TOXICITY IN HYPERBARIC HELIUM-OXYGEN 257

animals was 66.5 mg/kg with 95 % confidence limits of 43.9-100.6 mg/kg. This calcula- tion also indicated no significant difference between groups, and confirmed that the lines relating probits to log dose for the 2 groups were parallel.

TABLE 2

EFFECT OF A HIGH PRESSURE ENVIRONMENT ON THE TOXICITY OF ASPIRIN IN MICE”

Mortality (dead/total)

Expt. dayb Control Dived

1 7120 6121 2 5116 11/15 3 5116 7117

Total 17152 24153

x2 = 1.26,~ > 0.1

0 Dose of aspirin = 1260 mg/kg, po. b One control and one dived group was tested on each experimental day.

TABLE 3 ACUTE TOXICITY OF MORPHINE SULFATE IN DIVED AND

CONTROL RATS

Dose (m&d

0 20 40 60 80

Mortality (deaths/total number)

Control Dived

O/25 3125 9125 6125 9125 7125

13125 13125 13125 14125

X2 _._____

- 0.381 0.092 0 0

The above values for the iv LD50 are unexplainably much lower than the value of 237 mg/kg previously reported for the rat (Finnegan et al., 1948).

DISCUSSION

The present study was undertaken because it seemed possible that exposure to a hyperbaric helium environment might modify pharmacologic effects of drugs. It has been demonstrated that very high pressures, on the order of several hundred atmo- spheres, inhibit cell division (Pease and Marsland, 1939), increase bioluminescence in bacteria (Johnson et al., 1942), and change the equilibrium between sols and gels (Marsland and Brown, 1942). These pressures are far above any that man will ever experience, and are undoubtedly near the limit that can be tolerated by any form of life. However, the lower pressures that man will encounter may still produce subtle

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changes of the type noted above. Thus, Marsland and Brown (1942) showed that high hydrostatic pressure caused solation of myosin gels, and gelation of gelatin, presum- ably by shifting the sol-gel equilibrium toward the product that occupied the smaller volume. Such an effect, even to a lesser degree at lower pressures, might shift equilibria in biochemical reactions and thus alter drug action, metabolism, or excretion. Cattell and Edwards (1930) have shown that muscle contractility was measurably altered by a pressure of 65 atm, which is not far from pressures that man may encounter.

In addition to the possible effects of hyperbaric pressure per se, the presence of a high partial pressure of helium introduces the possibility of pharmacologic interaction between this gas and administered drugs. Even though no pharmacologic effect of hyperbaric helium has yet been demonstrated, all other inert gases of the helium series, as well as nitrogen, have been shown to be central nervous system depressants at elevated partial pressure (Behnke et al., 1935; Marshall, 1951).

Finally, certain mechanical factors have been shown to alter the normal physiology of the body during exposure to hyperbaric helium-oxygen. Because of the increased gas density at high pressure, the work of breathing increases, alveolar pC02 rises, and the respiratory response to CO2 is decreased (Hamilton, 1967). Thus there are a number of elements in the hyperbaric environment that might modify the toxicity or efficacy of drugs.

The conditions to which “dived” animals were exposed were the same as those encountered by deep-diving aquanauts. Helium was used as the inert gas to avoid the CNS depressant properties of nitrogen, Helium can be substituted for nitrogen at all pressures up to 122 atmospheres without any apparent pharmacologic effect on animals (Carpenter, 1954; MacInnis et al., 1967). In the present study the partial pressure of helium was 19.2 atm absolute (282 psia), pOz was maintained at approximately 0.2 atm absolute, and nitrogen was absent from the mixture.

During dives, chamber temperature was kept at 90°F. Membery and Link (1964) reported that mice at 42 atm pressure in a helium-oxygen atmosphere were comfortable only when temperature was kept above 89”F, and shivered when it fell below this value. This occurs because heat loss in a hyperbaric helium atmosphere is greater than normal as a result of the increased gas density and the greater thermal conductivity of helium (Raymond, 1967).

The results of the present study are very encouraging. They suggest that the acute toxicity of a variety of drugs is not altered by exposure of animals to a hyperbaric helium environment which simulates that of an underwater habitat at a depth of 600 feet. The values for toxic doses of the drugs examined in this work, with the exception of morphine, are in good agreement with the values reported in the literature.

During the course of all experiments, animals were observed either continuously or at intervals of 10 min. Thus, it was possible to observe gross signs associated with the development of toxicity, such as rate and depth of respiration, or struggling move- ments. With all drugs studied, exposure to hyperbaric helium did not appear to alter the character of the toxicity at any stage of its development. In addition, certain animals in the morphine and aspirin studies received a sublethal dose of drug, and survived until the end of the experiment. Again, there appeared to be no differences in the gross behavior or appearance of these animals that could be attributed to exposure to the hyperbaric environment.

TOXICITY IN HYPERBARIC HELIUM-OXYGEN 259

There is a possibility that failure to discover an effect of the hyperbaric environment on toxicity of these drugs occurred because the animals were not completely equili- brated with the helium in the atmosphere. Analyses of whole body helium elimination curves in man (Behnke and Willmon, 1941; Roth, 1967) indicate that 28% of the helium is associated with a half-saturation time of 2.3 min, 48 % of the gas is associated with a half-time of 35 min, and 24 y0 is associated with a half-time of 115 min. These data predict that in a 45-min exposure to helium, the time used in the present experi- ments, those tissues with a half-saturation time of 2.3 min will be more than 99% equilibrated. These most likely include the brain, kidneys, and heart because tissues with high perfusion rates should have shorter time constants (Boycott et al., 1908). Other tissues, those with a half-time of 35 min, will be approximately 60% saturated with helium. The slowest tissues (half-time = 115 min) will be only about 25 % saturated. This last group probably represents very poorly perfused tissues, such as connective tissue.

Unfortunately, corresponding data for the rat are not available, and it would be dangerous to try to apply the foregoing crude estimates of the human tissue saturation profile to this animal. The cardiac index for the rat (cardiac output per square meter of body surface area), 1.6 l/min/m2, is less than in man (2.33 l/min/m2, basal). Liver perfusion in the rat (79 ml/min/lOO g) is also less than in man (100 ml/min/lOO g) (Spector, 1956). Thus, in the rat the relative tissue perfusion may be generally lower and the saturation half-time longer than in man.

In spite of the lack of information on this subject, certain assumptions can be made. The organs most intimately involved in manifestations of acute toxicity of drugs used in the present study are the brain, heart, and lungs. Since these organs all have rela- tively high blood perfusion rates, it seems probable that they would be well saturated with helium within the 45-min period before drug was administered, and that the time- consuming precaution of completely equilibrating the animals would not significantly alter the results.

At best, these experiments represent a compromise in the evaluation of the effect of hyperbaric helium-oxygen on drug toxicity. Some of the shortcomings in this work are : (1) toxicity was evaluated in only one animal species; (2) only a single representative drug of each pharmacologic class was tested; (3) only acute toxicity studies were per- formed, and observations were limited to a few hours after injection; and (4) no data were obtained to determine the effect of exposure on blood levels of drugs after a single dose, or on the biological half-life of the drugs. If metabolism or excretion are de- creased, cumulation might occur when single doses are given repeatedly. Such cumu- lation could alter the acute toxicity of a drug even though actual sensitivity to the drug, as measured in these experiments, is unchanged.

The lack of effect of hyperbaric helium on drug toxicity in the present experiments does not necessarily imply that the efficacy of these agents will be unaffected by such an environment. It will certainly be important to investigate the effect of high pressure helium-oxygen on the therapeutic effect of these compounds in order to determine whether they will be as useful underwater as on the surface. However, it is probably more important to first ascertain that exposure of animals to hyperbaric helium-oxygen does not increase their sensitivity to the toxic effects of drugs.

Because of the great technical difficulties that arise when an investigator is separated

from the experimental animals by a steel wall, it was not possible to avoid these deficiencies and still obtain meaningful data within a reasonable period of time. Such additional information will be very valuable, but its acquisition will have to wait until more pertinent preliminary experiments of the type presented here have been com- pleted, or until it becomes technically feasible to make some of the measurements required.

ACKNOWLEDGMENTS

The author wishes to thank Mr. Howard W. McElroy and Mr. Robert S. Ide for their excellent technical assistance, and Dr. S. L. Friess and Dr. C. E. Brodine for helpful criticisms of the manuscript.

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