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Hematological responses to arsine exposure: Quantitation of exposure response in mice
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
5, 499-505
(1985)
Hematological Responses to Arsine Exposure: Quantitation of Exposure Response in Mice’ DAVID
P. PETERSON~
AND MARYRA
H. BHATTACHARYYA
Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 Hematological Responses to Amine Exposure: Quantitation of Exposure Response in Mice.. D. P., AND BHAITACHARYYA, M. H. (1985) Fundam. Appl. Toxicol. 5,499-505. Hematological responses of mice to amine exposures for 1 hr at 5 to 26 part per million volume (ppmv) are described. Exposure concentrations ranged from a no-effect level for the endpoints studied (5 ppmv) to a concentration lethal to all mice in 4 days (26 ppmv). Hematocrit values at 24 hr after exposure decreased linearly with increasing amine concentration in the range 5 to 26 ppmv; the hematocrit of the 26-ppmv group reached 10.5% at 24 hr, compared to 48.4% for control mice. Hematocrits of mice from all surviving groups were at or slightly above control values by I1 days after exposure. Changes in numbers of erythrocytes paralleled changes in hematocrit. Significant increases in circulating reticulocytes occurred at 1 and 5 days aher exposure; reticulocyte values returned to control levels by 11 days after exposure. Changes in erythrocyte osmotic fragility were observed in mice exposed to 15 and 26 ppmv amine. PETERSON,
The hemolytic property of the gaseous metal hydride arsine (AsH3) has been known for more than a century. Human exposures to arsine have occurred mainly in industrial settings, where arsine can be generated following accidental contact between arseniccontaining alloys and acid or water (Spolyar and Harger, 1950). Arsine can also be generated during charging of lead-acid batteries containing arsenic (Dudley, 19 19; Varma and Yao, 1978). Many articles have been published concerning human exposures to amine. They describe the origin of the gas, the clinical features of poisoning, and treatment (Fowler and Weissberg, 1974; Hocken and Bradshaw, 1970; Jenkins et al., 1965; Levinsky et al., 1970; Uldall et al., 1970; Wilkinson et al., 1975; Williams et al., 1981). A threshold limit value (TLV) for amine in ’ This work supported by the Department of Energy under Contract W-3I-109-ENG-38. The U.S. Govemment’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for govemmental purposes, is acknowledged. ’ To whom all correspondence should be addressed. 499
the occupational setting (0.05 ppm, 0.2 mg/ m3) was adopted by the American Conference of Governmental Industrial Hygienists (ACGIH) in 1977. Documentation for this TLV provides little human exposure-response information (ACGIH, 197 1). This is in part due to the fact that (1) arsine is a colorless, nonirritating, almost odorless gas and (2) the hemolytic response to arsine exposure is not immediate, with onset of symptoms appearing as long as 24 hr after exposure. As a result, human exposures often are not sensed at the time of exposure, and exposure levels remain undetermined. Even in animals, little quantitative doseresponse information on the hemolytic effects of arsine is available. One quite extensive study focuses on lethality (medium lethal exposures) in mice exposed to arsine concentrations ranging from 8 to 800 ppm (Lewy, 1947). Another report focuses on responses to at-sine at lower concentrations (0.5 to 2 ppm), but it presents little detail concerning exposure conditions or animal responses (Nau, 1948). The present investigation was undertaken to provide quantitative hemato0272-0590/85 $3.00
500
PETERSON
AND BHATTACHARYYA
Air -
FIG. 1. Schematic of hydride generator and exposure apparatus. Flowmeter (A), servo-flow control (B), microvalve (C), mass transducer (D), hydride generator (E), magnetic stirrer (F), syringe pump 0, fluorocarbon tubing (H), manifold (I), exposure chamber (J), absorber solutions (K).
logical exposure-response information following arsine exposure in mice. Arsine concentrations ranged from a no-effect level for the endpoints studied to a concentration lethal to all mice in 4 days. Hematological endpoints determined were hematocrit; erythrocyte, leukocyte, and reticulocyte counts; and erythrocyte osmotic fragility. METHODS Hydride Generation The hydride generator and accessories used are illustrated schematically in Fig. 1. A chemical reduction procedure, described in a study conducted for the National Institute of Occupational Safety and Health concerning collection and analysis of stibine from work place atmospheres, was used for the continuous generation of amine at controlled rates3 A 2.1 N KOH solution containing arsenic trioxide (reagent grade, Merck) and sodium borohydride (0.37 M) was placed in a syringe (G) and pumped into a reaction flask (E) containing 4 N H2S04. A carrier gas (medical-grade air) was passed through the reaction flask (E) then through fluorocarbon tubing (H) either (1) directly into an absorber solution (K) or (2) into a 4-liter ErIenmeyer flask (J) used for animal exposures and then into an absorber solution (K). (Fhmrocarbon tubing was used to prevent oxidation of the amine.) The absorber solution was 3 N Hz!Q containing 0.04 M 12 and 0.48 M KI. Flow of the carrier gas was controlled at two points: (1) at the two-stage regulator attached to a tank of compressed, medic&grade air and 3 The stibine generator design used in the NIOSH study was obtained by personal communication with Itamar B&k, Arthur D. Little, Inc., Acorn Park, Cambridge, Mass 02 140.
(2) at the microvalve (C) connected to a reversible electronic motor. Nearly constant gas flow (500 cm’/ min) was obtained by attaching a custom-built servoflow control (B) to the flow meter (A). Changes in gas flow as monitored by the mass transducer (D) signaled the servo-flow control to open or close the microvalve (C) in the appropriate direction. Arsine concentrations produced in the exposure flask (J) were changed by varying the concentrations of the alkaline arsenic solution in the syringe and/or by varying the syringe pumping rates. Concentrations of amine generated were analyzed by passing the carrier gas for a measured time at a controlled rate (500 cm3/min) through the reaction flask (E) into the strongly oxidizing absorber solution (K). Arsenic concentrations in the absorber solution were measured using flameless atomic absorption spectrophotometry on an IL 951 atomic absorption spectrophotometer with an IL555 controlled-tempemture furnace atomizer (Instrumentation Laboratories, Wilmington, Mass.). Prior to exposure of mice, the hydride generator was characterized with respect to (I) variability of hydride concentrations with time and (2) reaction yields. Working with amine poses a significant potential biological hazard. As mentioned earlier, amine is colorless, nonirritating and without significant warning properties that would alert potential exposure victims to its presence. Under all circumstances, the generation of amine was performed in a properly functioning fume hood. A source of supplied air was also available during the hydride generations to handle potential emergency situations arising from an accidental uncontrolled release of amine outside the fume hood. Exposure of Animals B6CF,/Anl mice (Argonne National Laboratory, Argonne, Ill.) were maintained on Wayne Lab Blox rodent chow and water (pH = 2.3) ad libitum. (The drinking water was acidified to pH = 2.3 to control the growth of Pseudomonus) Five groups of female mice (eight mice
ARSINE TOXICITY per group; 90 to 120 days old; 20 -C3 g) were exposed to arsine for 1 hr. Each group was exposed to one of the following five amine concentrations: 5, 9, 11, 15, or 26 parts per million volume (ppmv). An additional group of eight control mice was sham exposed for 1 hr to an identical gas phase excluding the arsine; the control gas phase was generated by omitting arsenic trioxide from the sodium borohydride solution in the syringe (G). The mice were exposed to arsine by placing them in groups of eight in a 4-liter Erlenmeyer flask (Fig. 1, J). Pre- and postexposure amine concentrations were monitored by directing the gas flow into a different absorber solution before and after the exposure period. Arsine concentrations in the flask during exposures of the mice were also monitored.
501
IN MICE
of reticulocytes (Deiss and Kurth, 1970). On the 1st day after exposure, following retroorbital blood sampling, three mice from each group were anesthetized with sodium pentobarbital and exsanguinated by cardiac puncture. Erythrocyte and leukocyte counts were determined electronically using a Coulter counter (Model ZBI). The osmotic fragility of the erythrocytes was determined on blood taken by cardiac puncture according to the method of Parpart et al. (1947). On the 1 ltb day after exposure, the remaining five mice in each group were anesthetized and exsanguinated by cardiac puncture. Heparinized whole blood was used for red and white blood cell determinations. Thme of the five blood samples were assayed for erythrocyte osmotic fragility. Statistical analyses of the data were conducted by oneway analysis of variance (ANOVA), followed by Duncan’s multiple range test (Zar, 1974).
Hematological Evaluation From each experimental and each control mouse, serial blood samples were taken at 1, 5, and I 1 days after exposure to amine. Blood was taken by rupturing the retroorbital venous plexus with heparinized microhematocrit capillary tubes (100 ~1) and allowing the tubes to fill by capillary action. After whole blood smears were prepared, the tubes were sealed and centrifuged, and hematocrits were determined. The capillary tubes were then broken near the seal at the bottom and the blood was transferred to a small test tube. Two drops of new methylene blue solution were added, and the mixture was incubated at room temperature for 5 to 10 min, then examined microscopically to determine the number
RESULTS Hydride
Generator Characterization
The results of the hydride generator characterization are presented in Table 1. Results demonstrate that a reproducible concentration of arsine was generated by our generator system. Concentrations of arsine varied little from one experiment to another. In addition, within a given period of generation, concentrations were maintained at a constant level,
TABLE 1 HYDRIDE GENERATOR CHARACTERIZATION’
Experiment I. AsHx II. AsH3 III. ASH, A. Exposure chamber in line’ B. Exposure chamber bypassed
Total collection time/interval b (min)
Hydride concentrationc (PPmv)
Yieldd (%)
160/10 160/10
5.3 + 0.3 (16) 4.2 + 0.4 (16)
89.8 86.4
2OJlO 30110
4.5 f 0.3 (2) 4.7 + 0.5 (3)
93.6 97.5
’ A 2.1 N KOH solution containing A%03 (2.25 mM) and NaBI& (0.37 M) was pumped at a flow rate of 1.76 ml/hr into the reaction flask containing 4 N H$O+ Medical-grade air was used as the carrier gas at a flow rate of 500 cm3/min. b Aliquots of absorber solution were taken at lO-min intervals for the total collection time specified. c Hydride concentrations are expressed as 2 + SE, with the number of measurements in parentheses. dYield = 100X [As recovered in absorber solution + As added to flask (E)). ‘The 44iter exposure chamber (J) was equilibrated for -20 min prior to taking samples for the 20-min collection period reported in this table.
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PETERSON
AND BHATTACHARYYA 50
as indicated by the small standard errors for hydride concentrations determined at 1Omin intervals over 160 min of generation in Experiments I and II. The yield of arsine was consistently high. In Experiment III AsH3, the effect on hydride concentrations and on reaction yield of passing the arsine through the animal exposure chamber was shown to be slight, indicating that the glass surface of the exposure chamber did not provide a surface for oxidation of the arsine and adsorption during its passage through the chamber. Hematological
Effects
By 24 hr after exposure to amine, the hematocrits of mice exposed to the higher arsine concentrations had decreased significantly (Table 2). The decrease in hematocrit at 24 hr was linear with increasing exposure concentration in the range 5 to 26 ppmv arsine (Fig. 2). Hematocrit values were 98.8, 80.2, 79.7, 6 1.4, and 2 1.7% of sham-exposed control values for the 5-, 9-, 1l-, 15-, and 26-ppmv AsHs exposure levels, respectively. For mice exposed to intermediate arsine concentrations (9, 11, 15 ppmv), the hematocrits increased by Day 5 after exposure but were still significantly lower than the sham-exposed controls (Table 3). Hematocrit values 5 days after exposure were 96.8, 91.5, 92.1, and 79.6% of control values for the 5-, 9-, 1 l-,
40
0
I
I
I
I
I
1
5
IO
15
20
25
Xl
ARSINE
CONCENTRATION
(ppmvl
FIG. 2. Hematocrit values in mice 24 hr after amine exposure. Mice were exposed to indicated concentrations of amine for 1 hr as described in text. A, sham exposed, 0, amine exposed. Sham-exposed mice were exposed to the same atmosphere minus amine. Values are X ? SE for eight mice per group.
and 15-ppmv arsine exposure levels, respectively. At the 26-ppmv arsine exposure level, the five mice remaining beyond the 24-hr sampling period died within 4 days. By the 1 lth day after exposure, the arsine-exposed mice (excluding the 26-ppmv level) had hematocrits that were similar to those of the sham-exposed controls (Table 3). Hematocrit values 11 days after exposure were 104.5, 102.1, 99.2, and 106.4% of control values for the 5-, 9-, 1 I-, and 15-ppmv AsH3 exposure levels, respectively. The hematocrits of the
TABLE 2 HEMATOLOGICAL EFFECTSIN MICE AT 24 hr AFTER A I-hr EXFTXURE TO ARSINE Exposure Sham exposed 5 ppmv AsH3 9 ppmv AsH3 11 ppmv AsH3 15 ppmv AsH3 26 ppmv AsH3
Hematocrit 48.4 47.8 38.8 38.6 29.7 10.5
+ + + + + +
0.7” 0.3” 0.5’ l.lb 1.2c l.5d
Etythrocytes/mm3 (X 10-6) 7.8 8.0 6.1 6.2 4.0 2.2
+ + + k f +
0.2” 0.2” O.lb 0.2’ 0.3’ 0.2d
Leukocytes/mm3 (x10-3) 2.7 2.4 4.8 3.9 6.2 4.2
f + + f + +
0.3” 0.3b O.24b 0.7”b 0.9” 1.0’~~
Reticulocytes (S) 0.6 + 0.1’ 1.0 + 0.3”’ 3.2 + 0.5’ 0.8 3~ 0.5’ 6.7 + 1.1” 4.0 + 2.O”b
Note. Values presented are X f SE for eight mice per group (hematocrit and reticulocyte) or three mice per group (erythrocytes and leukocytes). Means followed by the same superscript letter are not significantly different from each other according to ANOVA followed by Duncan’s multiple range test.
ARSINE TOXICITY
503
IN MICE
TABLE 3 HEMATOLCGICAL EFFECTSIN MICE AT 5 Day
after
Exposure
exposure
Hematocrit
Sham exposed 5 ppmv ASH, 9 ppmv AsH3 11 ppmv ASH, 15 ppmv ASH,
5 5 5 5 5
47.1 45.6 43.1 43.4 37.5
+ f f + f
0.7” 0.8”sb 0.56 0.5b 1.4’
Sham exposed 5 ppmv AsH3 9 ppmv AsH3 1 I ppmv AsH3 15 ppmv ASH,
11 11 I1 I1 II
48.5 50.7 49.5 48.1 51.6
+ + k + k
0.3’ 0.8”b 0.8”~~ 0.8c 0.5”
AND
11
DAYS AVER
A
I-hr
EXPOSURE
TO ARSINE
Erythrocytes/mm3 (x10-6)
Leukocytes/mm3 (x10-3)
Reticulocytes PJ)
-
-
1.1 + 0.4d 3.5 I!z 0.3”d 25.0 f 5.3b 12.9 f 1.6’ 34.6 + 4.1’
8.2 6.3 7.1 7.8 7.6
k 0.1’ + 0.5c f 0.1” + O.l”b zk O.larb
2.7 2.8 3.2 3.5 2.4
+ 0.3”~~ + 0.3”~~ f 0.4”,b * 0.2” -+ 0.3b
1.9 f 2.8 f 2.9 + 2.0 f 4.0 f
1.0” 0.4” 0.2” 0.3” 0.9”
Nofe. Values presented are the X ? SE, five mice per group. Means followed by the same superscript letter are not statistically different from each other, p < .05, according to ANOVA followed by Duncan’s multiple range test.
sham-exposed controls decreased only slightly from Day 1 to 5 (~3%) and returned to Day 1 values by the 11th day after sham exposure. Decreases in the number of erythrocytes seen at the intermediate at-sine exposure levels paralleled changes observed in the hematocrits (Tables 2 and 3). Low hematocrit values on Day 1 after exposure were accompanied by low erythrocyte values. The erythrocyte counts per cubic millimeter 1 day after exposure were 101.7, 78.4, 79.5, 51.4, and 28.8% of sham-exposed control values for the 5-, 9-, 1 l-, 15-, and 26-ppmv arsine exposure levels, respectively. Day 11 erythrocyte values indicated a dramatic recovery, similar to the recovery seen in hematocrit values. Erythrocyte counts of mice exposed to 5 and 9 ppmv arsine were significantly lower on Day 11 after exposure compared to sham-exposed controls. These erythrocyte counts were 76.9, 87.0, 94.6, and 92.9% of sham-exposed control values for the 5-, 9-, 1 l-, and 15-ppmv arsine exposure levels, respectively. The arsine-exposed mice exhibited a pronounced reticulocytosis by 5 days after exposure at all exposure levels (excluding the lethal 26-ppmv-arsine level) (Tables 2 and 3). Reticulocyte counts in peripheral blood
on Day 5 after exposure were 3.2-, 22.7-, 11.7-, and 31.4-fold higher than those observed in sham-exposed controls for the 5-, 9-, 1 l-, and 15-ppmv arsine exposures, respectively (Table 3). By the 1 lth day after exposure, reticulocyte counts in peripheral blood of arsine-exposed mice were not significantly different than levels observed in the sham-exposed controls. A transient leukocytosis was seen in the mice exposed to the intermediate dose levels of arsine. For the 9- and 15-ppmv groups, the concentration of leukocytes was 1.8- and 2.3-fold higher, respectively, in the arsineexposed mice than in the sham-exposed controls on the first day after exposure (Table 2). Erythrocyte osmotic fragility was altered relative to controls on the first day after the 15- and 26-ppmv-arsine exposures. Osmotic fragility curves for the sham-exposed controls and the 15-ppmv arsine-exposed mice can be compared in Fig. 3. The osmotic fragility curve of the sham-exposed mice is sigmoidal with nearly 100% of the erythrocyte lysis occurring between 0.6% and 0.4% NaCl. The osmotic fragility curve of the 15-ppmv arsineexposed mice is greatly flattened with only 40% of the erythrocyte lysis occurring between
504
PETERSON
AND BHATTACHARYYA
FIG. 3. Erythrocyte osmotic fragility curves 24 hr after arsine exposure. Values are zi + SE for three mice per group. 0, sham exposed; and A, 15 ppmv amine-exposed. Error bars which are omitted indicate that the SE was smaller than the height of the symbols used.
0.6 and 0.4% NaCl. Of the erythrocyte population in the 15-ppmv amine-exposed mice 28% had an increased osmotic fragility and 32% had a decreased osmotic fragility. By Day 11 after exposure, osmotic fragility was normal for all exposure groups. DISCUSSION The hematological response of mice to arsine exposure has not hitherto been quantitatively investigated. Previous work on the toxicity of this hydride focused on lethality (Kensler et al., 1946; Levvy, 1947). Hematological responses have been reported as ancillary information but little detail has been presented for methods or quantification (Nau, 1948). The results of the investigation reported here indicate that the hemolytic response of mice to a 1-hr exposure to arsine is dramatic. The concentration range from a no-effect level (5 ppmv) to a lethal concentration (26 ppmv) is narrow, less than lo-fold for the lhr exposure studied. In our study, the lowest hematocrit was observed at 24 hr. Hematologic recovery of the surviving mice was gradual but nearly complete within 11 days after exposure. The hemolytic response of humans accidently exposed to arsine is also very dramatic (Wilkinson et al., 1975) but
the time course of human recovery appears to be slower than that observed in mice. The data in humans are complicated because severe amine poisonings resulting in extensive hemolysis are normally treated with whole blood transfusions. In vitro exposure of erythrocytes to arsine in concentrations of 1 to 5 X 10e4 M resulted in hemolysis with a latency period of 20 to 40 min (Pernis and Magistretti, 1960). The extent of hemolysis was proportional to the arsine concentration, as was the case for our in vivo exposures in mice (Fig. 2). The investigators also found a strong inverse correlation between the level of reduced glutathione and the extent of hemolysis in arsine-exposed erythrocytes. Reduced glutathione has been implicated as being essential for maintaining the structural integrity of the erythrocyte membrane (Fegler, 1952; Kosower et al., 1969). Altered erythrocyte osmotic fragility was evident only at the higher amine exposure levels in the present investigation (15 and 26 ppmv). Determinations of erythrocyte osmotic fragility might therefore provide insight into the severity of an arsine exposure and could become an integral clinical test in suspected arsine poisonings. Recently our laboratory has initiated investigations into the toxicity of a similar hydride, stibine, the hydride of antimony. Our preliminary study and others (Webster, 1946; Smith et al., 1948) have shown stibine to have hemolytic properties similar to amine. Further studies testing additional concentrations of stibine are anticipated. The results from these studies will allow comparison of the hematological responses in mice presented here for arsine exposure with responses to another hemolytic metal hydride. REFERENCES American Conference of Governmental Industrial Hygienists (ACGIH) (1971). Documentation of the Threshold Limit Value for Substances in Workroom Air, 3rd ed., Cincinnati. DEB, A., AND KURTH, D. (1970). Circulating reticulo-
ARSINE TOXICITY cytes in normal adults as determined by the new methylene blue method. Amer. J. Cfin. Pathol. 53, 481-484. DUDLEY, S. F. (1919). Toxemic anemia from arseniuretted hydrogen gas in submarines. J. Ind. Hyg. 1, 2 U-232.
FEGLER, G. (1952). Relationship between reduced glutathione content and spontaneous haemolysis in shed blood. Nature (London) 170, 624-625. FOWLER, B. A., AND WEISSBERG, J. B. (1974). Amine Poisoning. N. Engl. J. Med. 291, 1171-1174. HOCKEN, A. G., AND BRADSHAW, G. (1970). Amine poisoning. Brit. J. Ind. Med. 27, 56-60. JENKINS, G. C., IND, J. E., KAZANTZIS, G., AND OWEN, R. (1965). Amine poisoning: Massive haemolysis with minimal impairment of renal function. Brit. Med. J. 2, 78-80.
KENSLER, C. J., ABELS, J. C., AND RHOADS, C. P. ( 1946). Amine poisoning, mode of action and treatment. J. Pharmacol. Exp. Ther. 88, S&108. KOSOWER, N. S., SONG, K., AND KOSOWER, E. M. (1969). Glutathione. IV. Intracellular oxidation and membrane injury. Biochim. Biophys. Acta 192, 2328.
LEVINSKY, W. J., SMALLEY, R. V., HILLYER, P. N., AND SHINDLER, R. L. (1970). Amine hemolysis. Arch. Environ. Health 20, 436-440. LEVVY, G. A. (1947). A study of amine poisoning. Quart. J. Exp. Physiol. 34, 47-67. NAU, C. A. (1948). The accidental generation of amine gas in an industry. Southern Med. J. 41, 341-344. PARPART, A. K., LORENZ, P. B., PARPART, E. R., GREGG, J. R., AND CHASE, A. M. (1947). The osmotic
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resistance (fragility) of human red cells. J. C/in. Invest. 26,636. PERNIS, B., AND MAGISTRETTI, M. (1960). A study of the mechanism of acute hemolytic anemia from amine. Med. Lav. 51, 3741. SMITH, R. E., STEELE, J. M., EAIUN, R. E., AND COWIE, D. G. (1948). The tissue distribution of radio-antimony inhaled as stibine. J. Lab. Clin. Med. 33, 635-643. SPOLYAR, L. W., AND HARGER, R. N. (1950). Amine poisoning: Epidemiological studies of outbreak following exposure to gases from metallic dross. Arch. Ind. Hyg. L419-436. ULDALL, P. R., KHAN, H. A., ENNIS, J. E., MCCALLUM, R. I., AND GRIMSON, T. A. (1970). Renal damage from industrial amine poisoning. Brit. J. Ind. Med. 27, 372-377.
VARMA, R., AND YAO, N. P. (1978). Stibine and Arsine Generation from a Lead-Acid Cell during Charging Modes under a Utility Load-Leveling Duty Cycle, Argonne National Laboratory Report ANL/OEPM77-5.
WEBSTER, S. H. (1946). Volatile hydrides of toxicological importance. J. Ind. Hyg. Toxicol. 28, 167-I 82. WILKINSON, S. P., MCHUGH, P., HORSELEY, S., TIJB~S, H., LEWIS, M., THOULD, SONS, V., AND WILLIAMS,
A., WINTERTON,
M.,
PAR-
R. (1975). Amine toxicity aboard the Asiafreighter. Brit. Med. J. 3, 559-563. WILLIAMS, P. L., SPAIN, W. H., AND RUBENSTEIN, M. (1981). Suspected amine poisoning during the restoration of a large cyclorama painting. Amer. Ind. Hyg. Assoc. J. 42,9 11-9 13. ZAR, J. H. (1974). Biostatistical Analysis. Prentice Hall, Englewood Cliffs, N.J.
AND
APPLIED
TOXICOLOGY
5, 499-505
(1985)
Hematological Responses to Arsine Exposure: Quantitation of Exposure Response in Mice’ DAVID
P. PETERSON~
AND MARYRA
H. BHATTACHARYYA
Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 Hematological Responses to Amine Exposure: Quantitation of Exposure Response in Mice.. D. P., AND BHAITACHARYYA, M. H. (1985) Fundam. Appl. Toxicol. 5,499-505. Hematological responses of mice to amine exposures for 1 hr at 5 to 26 part per million volume (ppmv) are described. Exposure concentrations ranged from a no-effect level for the endpoints studied (5 ppmv) to a concentration lethal to all mice in 4 days (26 ppmv). Hematocrit values at 24 hr after exposure decreased linearly with increasing amine concentration in the range 5 to 26 ppmv; the hematocrit of the 26-ppmv group reached 10.5% at 24 hr, compared to 48.4% for control mice. Hematocrits of mice from all surviving groups were at or slightly above control values by I1 days after exposure. Changes in numbers of erythrocytes paralleled changes in hematocrit. Significant increases in circulating reticulocytes occurred at 1 and 5 days aher exposure; reticulocyte values returned to control levels by 11 days after exposure. Changes in erythrocyte osmotic fragility were observed in mice exposed to 15 and 26 ppmv amine. PETERSON,
The hemolytic property of the gaseous metal hydride arsine (AsH3) has been known for more than a century. Human exposures to arsine have occurred mainly in industrial settings, where arsine can be generated following accidental contact between arseniccontaining alloys and acid or water (Spolyar and Harger, 1950). Arsine can also be generated during charging of lead-acid batteries containing arsenic (Dudley, 19 19; Varma and Yao, 1978). Many articles have been published concerning human exposures to amine. They describe the origin of the gas, the clinical features of poisoning, and treatment (Fowler and Weissberg, 1974; Hocken and Bradshaw, 1970; Jenkins et al., 1965; Levinsky et al., 1970; Uldall et al., 1970; Wilkinson et al., 1975; Williams et al., 1981). A threshold limit value (TLV) for amine in ’ This work supported by the Department of Energy under Contract W-3I-109-ENG-38. The U.S. Govemment’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for govemmental purposes, is acknowledged. ’ To whom all correspondence should be addressed. 499
the occupational setting (0.05 ppm, 0.2 mg/ m3) was adopted by the American Conference of Governmental Industrial Hygienists (ACGIH) in 1977. Documentation for this TLV provides little human exposure-response information (ACGIH, 197 1). This is in part due to the fact that (1) arsine is a colorless, nonirritating, almost odorless gas and (2) the hemolytic response to arsine exposure is not immediate, with onset of symptoms appearing as long as 24 hr after exposure. As a result, human exposures often are not sensed at the time of exposure, and exposure levels remain undetermined. Even in animals, little quantitative doseresponse information on the hemolytic effects of arsine is available. One quite extensive study focuses on lethality (medium lethal exposures) in mice exposed to arsine concentrations ranging from 8 to 800 ppm (Lewy, 1947). Another report focuses on responses to at-sine at lower concentrations (0.5 to 2 ppm), but it presents little detail concerning exposure conditions or animal responses (Nau, 1948). The present investigation was undertaken to provide quantitative hemato0272-0590/85 $3.00
500
PETERSON
AND BHATTACHARYYA
Air -
FIG. 1. Schematic of hydride generator and exposure apparatus. Flowmeter (A), servo-flow control (B), microvalve (C), mass transducer (D), hydride generator (E), magnetic stirrer (F), syringe pump 0, fluorocarbon tubing (H), manifold (I), exposure chamber (J), absorber solutions (K).
logical exposure-response information following arsine exposure in mice. Arsine concentrations ranged from a no-effect level for the endpoints studied to a concentration lethal to all mice in 4 days. Hematological endpoints determined were hematocrit; erythrocyte, leukocyte, and reticulocyte counts; and erythrocyte osmotic fragility. METHODS Hydride Generation The hydride generator and accessories used are illustrated schematically in Fig. 1. A chemical reduction procedure, described in a study conducted for the National Institute of Occupational Safety and Health concerning collection and analysis of stibine from work place atmospheres, was used for the continuous generation of amine at controlled rates3 A 2.1 N KOH solution containing arsenic trioxide (reagent grade, Merck) and sodium borohydride (0.37 M) was placed in a syringe (G) and pumped into a reaction flask (E) containing 4 N H2S04. A carrier gas (medical-grade air) was passed through the reaction flask (E) then through fluorocarbon tubing (H) either (1) directly into an absorber solution (K) or (2) into a 4-liter ErIenmeyer flask (J) used for animal exposures and then into an absorber solution (K). (Fhmrocarbon tubing was used to prevent oxidation of the amine.) The absorber solution was 3 N Hz!Q containing 0.04 M 12 and 0.48 M KI. Flow of the carrier gas was controlled at two points: (1) at the two-stage regulator attached to a tank of compressed, medic&grade air and 3 The stibine generator design used in the NIOSH study was obtained by personal communication with Itamar B&k, Arthur D. Little, Inc., Acorn Park, Cambridge, Mass 02 140.
(2) at the microvalve (C) connected to a reversible electronic motor. Nearly constant gas flow (500 cm’/ min) was obtained by attaching a custom-built servoflow control (B) to the flow meter (A). Changes in gas flow as monitored by the mass transducer (D) signaled the servo-flow control to open or close the microvalve (C) in the appropriate direction. Arsine concentrations produced in the exposure flask (J) were changed by varying the concentrations of the alkaline arsenic solution in the syringe and/or by varying the syringe pumping rates. Concentrations of amine generated were analyzed by passing the carrier gas for a measured time at a controlled rate (500 cm3/min) through the reaction flask (E) into the strongly oxidizing absorber solution (K). Arsenic concentrations in the absorber solution were measured using flameless atomic absorption spectrophotometry on an IL 951 atomic absorption spectrophotometer with an IL555 controlled-tempemture furnace atomizer (Instrumentation Laboratories, Wilmington, Mass.). Prior to exposure of mice, the hydride generator was characterized with respect to (I) variability of hydride concentrations with time and (2) reaction yields. Working with amine poses a significant potential biological hazard. As mentioned earlier, amine is colorless, nonirritating and without significant warning properties that would alert potential exposure victims to its presence. Under all circumstances, the generation of amine was performed in a properly functioning fume hood. A source of supplied air was also available during the hydride generations to handle potential emergency situations arising from an accidental uncontrolled release of amine outside the fume hood. Exposure of Animals B6CF,/Anl mice (Argonne National Laboratory, Argonne, Ill.) were maintained on Wayne Lab Blox rodent chow and water (pH = 2.3) ad libitum. (The drinking water was acidified to pH = 2.3 to control the growth of Pseudomonus) Five groups of female mice (eight mice
ARSINE TOXICITY per group; 90 to 120 days old; 20 -C3 g) were exposed to arsine for 1 hr. Each group was exposed to one of the following five amine concentrations: 5, 9, 11, 15, or 26 parts per million volume (ppmv). An additional group of eight control mice was sham exposed for 1 hr to an identical gas phase excluding the arsine; the control gas phase was generated by omitting arsenic trioxide from the sodium borohydride solution in the syringe (G). The mice were exposed to arsine by placing them in groups of eight in a 4-liter Erlenmeyer flask (Fig. 1, J). Pre- and postexposure amine concentrations were monitored by directing the gas flow into a different absorber solution before and after the exposure period. Arsine concentrations in the flask during exposures of the mice were also monitored.
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of reticulocytes (Deiss and Kurth, 1970). On the 1st day after exposure, following retroorbital blood sampling, three mice from each group were anesthetized with sodium pentobarbital and exsanguinated by cardiac puncture. Erythrocyte and leukocyte counts were determined electronically using a Coulter counter (Model ZBI). The osmotic fragility of the erythrocytes was determined on blood taken by cardiac puncture according to the method of Parpart et al. (1947). On the 1 ltb day after exposure, the remaining five mice in each group were anesthetized and exsanguinated by cardiac puncture. Heparinized whole blood was used for red and white blood cell determinations. Thme of the five blood samples were assayed for erythrocyte osmotic fragility. Statistical analyses of the data were conducted by oneway analysis of variance (ANOVA), followed by Duncan’s multiple range test (Zar, 1974).
Hematological Evaluation From each experimental and each control mouse, serial blood samples were taken at 1, 5, and I 1 days after exposure to amine. Blood was taken by rupturing the retroorbital venous plexus with heparinized microhematocrit capillary tubes (100 ~1) and allowing the tubes to fill by capillary action. After whole blood smears were prepared, the tubes were sealed and centrifuged, and hematocrits were determined. The capillary tubes were then broken near the seal at the bottom and the blood was transferred to a small test tube. Two drops of new methylene blue solution were added, and the mixture was incubated at room temperature for 5 to 10 min, then examined microscopically to determine the number
RESULTS Hydride
Generator Characterization
The results of the hydride generator characterization are presented in Table 1. Results demonstrate that a reproducible concentration of arsine was generated by our generator system. Concentrations of arsine varied little from one experiment to another. In addition, within a given period of generation, concentrations were maintained at a constant level,
TABLE 1 HYDRIDE GENERATOR CHARACTERIZATION’
Experiment I. AsHx II. AsH3 III. ASH, A. Exposure chamber in line’ B. Exposure chamber bypassed
Total collection time/interval b (min)
Hydride concentrationc (PPmv)
Yieldd (%)
160/10 160/10
5.3 + 0.3 (16) 4.2 + 0.4 (16)
89.8 86.4
2OJlO 30110
4.5 f 0.3 (2) 4.7 + 0.5 (3)
93.6 97.5
’ A 2.1 N KOH solution containing A%03 (2.25 mM) and NaBI& (0.37 M) was pumped at a flow rate of 1.76 ml/hr into the reaction flask containing 4 N H$O+ Medical-grade air was used as the carrier gas at a flow rate of 500 cm3/min. b Aliquots of absorber solution were taken at lO-min intervals for the total collection time specified. c Hydride concentrations are expressed as 2 + SE, with the number of measurements in parentheses. dYield = 100X [As recovered in absorber solution + As added to flask (E)). ‘The 44iter exposure chamber (J) was equilibrated for -20 min prior to taking samples for the 20-min collection period reported in this table.
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PETERSON
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as indicated by the small standard errors for hydride concentrations determined at 1Omin intervals over 160 min of generation in Experiments I and II. The yield of arsine was consistently high. In Experiment III AsH3, the effect on hydride concentrations and on reaction yield of passing the arsine through the animal exposure chamber was shown to be slight, indicating that the glass surface of the exposure chamber did not provide a surface for oxidation of the arsine and adsorption during its passage through the chamber. Hematological
Effects
By 24 hr after exposure to amine, the hematocrits of mice exposed to the higher arsine concentrations had decreased significantly (Table 2). The decrease in hematocrit at 24 hr was linear with increasing exposure concentration in the range 5 to 26 ppmv arsine (Fig. 2). Hematocrit values were 98.8, 80.2, 79.7, 6 1.4, and 2 1.7% of sham-exposed control values for the 5-, 9-, 1l-, 15-, and 26-ppmv AsHs exposure levels, respectively. For mice exposed to intermediate arsine concentrations (9, 11, 15 ppmv), the hematocrits increased by Day 5 after exposure but were still significantly lower than the sham-exposed controls (Table 3). Hematocrit values 5 days after exposure were 96.8, 91.5, 92.1, and 79.6% of control values for the 5-, 9-, 1 l-,
40
0
I
I
I
I
I
1
5
IO
15
20
25
Xl
ARSINE
CONCENTRATION
(ppmvl
FIG. 2. Hematocrit values in mice 24 hr after amine exposure. Mice were exposed to indicated concentrations of amine for 1 hr as described in text. A, sham exposed, 0, amine exposed. Sham-exposed mice were exposed to the same atmosphere minus amine. Values are X ? SE for eight mice per group.
and 15-ppmv arsine exposure levels, respectively. At the 26-ppmv arsine exposure level, the five mice remaining beyond the 24-hr sampling period died within 4 days. By the 1 lth day after exposure, the arsine-exposed mice (excluding the 26-ppmv level) had hematocrits that were similar to those of the sham-exposed controls (Table 3). Hematocrit values 11 days after exposure were 104.5, 102.1, 99.2, and 106.4% of control values for the 5-, 9-, 1 I-, and 15-ppmv AsH3 exposure levels, respectively. The hematocrits of the
TABLE 2 HEMATOLOGICAL EFFECTSIN MICE AT 24 hr AFTER A I-hr EXFTXURE TO ARSINE Exposure Sham exposed 5 ppmv AsH3 9 ppmv AsH3 11 ppmv AsH3 15 ppmv AsH3 26 ppmv AsH3
Hematocrit 48.4 47.8 38.8 38.6 29.7 10.5
+ + + + + +
0.7” 0.3” 0.5’ l.lb 1.2c l.5d
Etythrocytes/mm3 (X 10-6) 7.8 8.0 6.1 6.2 4.0 2.2
+ + + k f +
0.2” 0.2” O.lb 0.2’ 0.3’ 0.2d
Leukocytes/mm3 (x10-3) 2.7 2.4 4.8 3.9 6.2 4.2
f + + f + +
0.3” 0.3b O.24b 0.7”b 0.9” 1.0’~~
Reticulocytes (S) 0.6 + 0.1’ 1.0 + 0.3”’ 3.2 + 0.5’ 0.8 3~ 0.5’ 6.7 + 1.1” 4.0 + 2.O”b
Note. Values presented are X f SE for eight mice per group (hematocrit and reticulocyte) or three mice per group (erythrocytes and leukocytes). Means followed by the same superscript letter are not significantly different from each other according to ANOVA followed by Duncan’s multiple range test.
ARSINE TOXICITY
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TABLE 3 HEMATOLCGICAL EFFECTSIN MICE AT 5 Day
after
Exposure
exposure
Hematocrit
Sham exposed 5 ppmv ASH, 9 ppmv AsH3 11 ppmv ASH, 15 ppmv ASH,
5 5 5 5 5
47.1 45.6 43.1 43.4 37.5
+ f f + f
0.7” 0.8”sb 0.56 0.5b 1.4’
Sham exposed 5 ppmv AsH3 9 ppmv AsH3 1 I ppmv AsH3 15 ppmv ASH,
11 11 I1 I1 II
48.5 50.7 49.5 48.1 51.6
+ + k + k
0.3’ 0.8”b 0.8”~~ 0.8c 0.5”
AND
11
DAYS AVER
A
I-hr
EXPOSURE
TO ARSINE
Erythrocytes/mm3 (x10-6)
Leukocytes/mm3 (x10-3)
Reticulocytes PJ)
-
-
1.1 + 0.4d 3.5 I!z 0.3”d 25.0 f 5.3b 12.9 f 1.6’ 34.6 + 4.1’
8.2 6.3 7.1 7.8 7.6
k 0.1’ + 0.5c f 0.1” + O.l”b zk O.larb
2.7 2.8 3.2 3.5 2.4
+ 0.3”~~ + 0.3”~~ f 0.4”,b * 0.2” -+ 0.3b
1.9 f 2.8 f 2.9 + 2.0 f 4.0 f
1.0” 0.4” 0.2” 0.3” 0.9”
Nofe. Values presented are the X ? SE, five mice per group. Means followed by the same superscript letter are not statistically different from each other, p < .05, according to ANOVA followed by Duncan’s multiple range test.
sham-exposed controls decreased only slightly from Day 1 to 5 (~3%) and returned to Day 1 values by the 11th day after sham exposure. Decreases in the number of erythrocytes seen at the intermediate at-sine exposure levels paralleled changes observed in the hematocrits (Tables 2 and 3). Low hematocrit values on Day 1 after exposure were accompanied by low erythrocyte values. The erythrocyte counts per cubic millimeter 1 day after exposure were 101.7, 78.4, 79.5, 51.4, and 28.8% of sham-exposed control values for the 5-, 9-, 1 l-, 15-, and 26-ppmv arsine exposure levels, respectively. Day 11 erythrocyte values indicated a dramatic recovery, similar to the recovery seen in hematocrit values. Erythrocyte counts of mice exposed to 5 and 9 ppmv arsine were significantly lower on Day 11 after exposure compared to sham-exposed controls. These erythrocyte counts were 76.9, 87.0, 94.6, and 92.9% of sham-exposed control values for the 5-, 9-, 1 l-, and 15-ppmv arsine exposure levels, respectively. The arsine-exposed mice exhibited a pronounced reticulocytosis by 5 days after exposure at all exposure levels (excluding the lethal 26-ppmv-arsine level) (Tables 2 and 3). Reticulocyte counts in peripheral blood
on Day 5 after exposure were 3.2-, 22.7-, 11.7-, and 31.4-fold higher than those observed in sham-exposed controls for the 5-, 9-, 1 l-, and 15-ppmv arsine exposures, respectively (Table 3). By the 1 lth day after exposure, reticulocyte counts in peripheral blood of arsine-exposed mice were not significantly different than levels observed in the sham-exposed controls. A transient leukocytosis was seen in the mice exposed to the intermediate dose levels of arsine. For the 9- and 15-ppmv groups, the concentration of leukocytes was 1.8- and 2.3-fold higher, respectively, in the arsineexposed mice than in the sham-exposed controls on the first day after exposure (Table 2). Erythrocyte osmotic fragility was altered relative to controls on the first day after the 15- and 26-ppmv-arsine exposures. Osmotic fragility curves for the sham-exposed controls and the 15-ppmv arsine-exposed mice can be compared in Fig. 3. The osmotic fragility curve of the sham-exposed mice is sigmoidal with nearly 100% of the erythrocyte lysis occurring between 0.6% and 0.4% NaCl. The osmotic fragility curve of the 15-ppmv arsineexposed mice is greatly flattened with only 40% of the erythrocyte lysis occurring between
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FIG. 3. Erythrocyte osmotic fragility curves 24 hr after arsine exposure. Values are zi + SE for three mice per group. 0, sham exposed; and A, 15 ppmv amine-exposed. Error bars which are omitted indicate that the SE was smaller than the height of the symbols used.
0.6 and 0.4% NaCl. Of the erythrocyte population in the 15-ppmv amine-exposed mice 28% had an increased osmotic fragility and 32% had a decreased osmotic fragility. By Day 11 after exposure, osmotic fragility was normal for all exposure groups. DISCUSSION The hematological response of mice to arsine exposure has not hitherto been quantitatively investigated. Previous work on the toxicity of this hydride focused on lethality (Kensler et al., 1946; Levvy, 1947). Hematological responses have been reported as ancillary information but little detail has been presented for methods or quantification (Nau, 1948). The results of the investigation reported here indicate that the hemolytic response of mice to a 1-hr exposure to arsine is dramatic. The concentration range from a no-effect level (5 ppmv) to a lethal concentration (26 ppmv) is narrow, less than lo-fold for the lhr exposure studied. In our study, the lowest hematocrit was observed at 24 hr. Hematologic recovery of the surviving mice was gradual but nearly complete within 11 days after exposure. The hemolytic response of humans accidently exposed to arsine is also very dramatic (Wilkinson et al., 1975) but
the time course of human recovery appears to be slower than that observed in mice. The data in humans are complicated because severe amine poisonings resulting in extensive hemolysis are normally treated with whole blood transfusions. In vitro exposure of erythrocytes to arsine in concentrations of 1 to 5 X 10e4 M resulted in hemolysis with a latency period of 20 to 40 min (Pernis and Magistretti, 1960). The extent of hemolysis was proportional to the arsine concentration, as was the case for our in vivo exposures in mice (Fig. 2). The investigators also found a strong inverse correlation between the level of reduced glutathione and the extent of hemolysis in arsine-exposed erythrocytes. Reduced glutathione has been implicated as being essential for maintaining the structural integrity of the erythrocyte membrane (Fegler, 1952; Kosower et al., 1969). Altered erythrocyte osmotic fragility was evident only at the higher amine exposure levels in the present investigation (15 and 26 ppmv). Determinations of erythrocyte osmotic fragility might therefore provide insight into the severity of an arsine exposure and could become an integral clinical test in suspected arsine poisonings. Recently our laboratory has initiated investigations into the toxicity of a similar hydride, stibine, the hydride of antimony. Our preliminary study and others (Webster, 1946; Smith et al., 1948) have shown stibine to have hemolytic properties similar to amine. Further studies testing additional concentrations of stibine are anticipated. The results from these studies will allow comparison of the hematological responses in mice presented here for arsine exposure with responses to another hemolytic metal hydride. REFERENCES American Conference of Governmental Industrial Hygienists (ACGIH) (1971). Documentation of the Threshold Limit Value for Substances in Workroom Air, 3rd ed., Cincinnati. DEB, A., AND KURTH, D. (1970). Circulating reticulo-
ARSINE TOXICITY cytes in normal adults as determined by the new methylene blue method. Amer. J. Cfin. Pathol. 53, 481-484. DUDLEY, S. F. (1919). Toxemic anemia from arseniuretted hydrogen gas in submarines. J. Ind. Hyg. 1, 2 U-232.
FEGLER, G. (1952). Relationship between reduced glutathione content and spontaneous haemolysis in shed blood. Nature (London) 170, 624-625. FOWLER, B. A., AND WEISSBERG, J. B. (1974). Amine Poisoning. N. Engl. J. Med. 291, 1171-1174. HOCKEN, A. G., AND BRADSHAW, G. (1970). Amine poisoning. Brit. J. Ind. Med. 27, 56-60. JENKINS, G. C., IND, J. E., KAZANTZIS, G., AND OWEN, R. (1965). Amine poisoning: Massive haemolysis with minimal impairment of renal function. Brit. Med. J. 2, 78-80.
KENSLER, C. J., ABELS, J. C., AND RHOADS, C. P. ( 1946). Amine poisoning, mode of action and treatment. J. Pharmacol. Exp. Ther. 88, S&108. KOSOWER, N. S., SONG, K., AND KOSOWER, E. M. (1969). Glutathione. IV. Intracellular oxidation and membrane injury. Biochim. Biophys. Acta 192, 2328.
LEVINSKY, W. J., SMALLEY, R. V., HILLYER, P. N., AND SHINDLER, R. L. (1970). Amine hemolysis. Arch. Environ. Health 20, 436-440. LEVVY, G. A. (1947). A study of amine poisoning. Quart. J. Exp. Physiol. 34, 47-67. NAU, C. A. (1948). The accidental generation of amine gas in an industry. Southern Med. J. 41, 341-344. PARPART, A. K., LORENZ, P. B., PARPART, E. R., GREGG, J. R., AND CHASE, A. M. (1947). The osmotic
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resistance (fragility) of human red cells. J. C/in. Invest. 26,636. PERNIS, B., AND MAGISTRETTI, M. (1960). A study of the mechanism of acute hemolytic anemia from amine. Med. Lav. 51, 3741. SMITH, R. E., STEELE, J. M., EAIUN, R. E., AND COWIE, D. G. (1948). The tissue distribution of radio-antimony inhaled as stibine. J. Lab. Clin. Med. 33, 635-643. SPOLYAR, L. W., AND HARGER, R. N. (1950). Amine poisoning: Epidemiological studies of outbreak following exposure to gases from metallic dross. Arch. Ind. Hyg. L419-436. ULDALL, P. R., KHAN, H. A., ENNIS, J. E., MCCALLUM, R. I., AND GRIMSON, T. A. (1970). Renal damage from industrial amine poisoning. Brit. J. Ind. Med. 27, 372-377.
VARMA, R., AND YAO, N. P. (1978). Stibine and Arsine Generation from a Lead-Acid Cell during Charging Modes under a Utility Load-Leveling Duty Cycle, Argonne National Laboratory Report ANL/OEPM77-5.
WEBSTER, S. H. (1946). Volatile hydrides of toxicological importance. J. Ind. Hyg. Toxicol. 28, 167-I 82. WILKINSON, S. P., MCHUGH, P., HORSELEY, S., TIJB~S, H., LEWIS, M., THOULD, SONS, V., AND WILLIAMS,
A., WINTERTON,
M.,
PAR-
R. (1975). Amine toxicity aboard the Asiafreighter. Brit. Med. J. 3, 559-563. WILLIAMS, P. L., SPAIN, W. H., AND RUBENSTEIN, M. (1981). Suspected amine poisoning during the restoration of a large cyclorama painting. Amer. Ind. Hyg. Assoc. J. 42,9 11-9 13. ZAR, J. H. (1974). Biostatistical Analysis. Prentice Hall, Englewood Cliffs, N.J.