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Clearance of sulfuric acid-introduced 35S from the respiratory tracts of rats, guinea pigs and dogs following inhalation or instillation
FUNDAMENTAL AND APPLIED TOXICOLOGY 3:293-297 (lot~3)
Clearance of Sulfuric Acid-Introduced 35S from the Respiratory Tracts of Rats, Guinea Pigs and Dogs Following Inhalation or Instillation ALAN R. DAHL, SHARON A. FELICETTI and BRUCE A. MUGGENBURG Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, NM 87185
ABSTRACT
Clearance of Sulfuric Acid-Introduced :~:'Sfrom the Resp i r a t o r y Tracts of Rats, Guinea Pigs and Dogs Following inhalation or Instillation. Dahl, A.R., Felicelti, S.A., and Muggenburg, B.A. (1983). Fundanz. AppL ToxicoL 3:293297. The clearance o f sulfuric acid-introduced :~"S from the upper and lower respiratory tracts o f rats, guinea pigs and dogs was measured. Sulfuric acid was administered by instillation and by inhalation for each species. Clearance into the blood and gastrointestinal tract was measured along with determination of:~S remaining at the site of administration at sacrifice. Different rates of clearance from different sites within the dog lung were indicated with rates of clearance increasing with decreasing airway diameter. Half-times of clearance from all sites in the hmg and for all species were from 2-9 min. There appeared to be some species differences, with clearance for dogs being slower than for guinea pigs, which was slower than for rats. Upper respiratory tract clearance was much slower than for lung and may not have been primarily by way of the blood. The data indicate that the clearance of sulfuric acid-introduced :~r'sin vivo is faster than previous studies in isolated perfused lungs had indicated. The results may be general for water soluble, ionized chemical species. INTRODUCTION Sulfur oxidation products occur in the atmosphere largely as a result of the burning of fossil fuels. Sulfuric acid appears to be the most acutely toxic of the many sulfur oxidation products w h i c h have been tested for inhalation toxicity (Amdur, 1971; Amdur, et aL, 1975). The toxicity of sulfuric acid mists in guinea pigs appears to be related to droplet diameter (Wolff, et aL, 1979) and the guinea pig is one of the most sensitive species for acute toxicity of sulfuric acid by inhalation (Treon, et aL, 1950). It has been suggested that the sensitivity of guinea pigs to sulfate ion may be due to release of histamine in the presence of sulfate (Charles and Menzel, 1975). The concentration of sulfate ion in the tissues of the lungs and other portions of the respiratory tract will depend upon both the rate of deposition and the rate of clearance. The clearance of sulfate ion in the presence of a variety of co-administered ions has been studied in isolated perfused rat lungs (Charles, etal., 1977a and 1977b). However, study of the in vivo respiratory clearance" rate of sulfate has not been reported, ,nor have species other than the rat been used for measurements of clearance of sulfuric acid or sulfate.
The purpose of the experiments reported here was to determine if species differences in clearances of sulfuric acidintroduced sulfate occurred w h i c h might influence relative toxicity. In addition, we wanted to determine the rates of clearance of sulfate from different parts of the respiratory tract and to determine if clearance following inhalation was different than clearance following instillation. METHODS Aerosol generation Sulfuric acid aerosols were produced using a compressed air nebulizer w i t h glass and Teflon~"J parts. The aerosols were heated to 220°C and diluted w i t h mixing air. The flow rate of aerosols past the noses of the animals was 20 L/rain w h i c h corresponded to a linear velocity of 2 m/rain. Concentration of sulfuric acid in the nebulizer was from 0 . 0 3 5 - 0 . 8 5 M depending on the mass median aerodynamic diameter (MMAD) desired. The sulfuric acid was radiolabeted by the addition of 1 mL of water containing 50-1 O0 mCi of :~'S-labeled sulfuric acid (New England Nuclear) to 4-5 mL of unlabeled sulfuric acid (Ventron, ultra-pure) diluted with water distilled in glass. Aerosol sizes and air concentrations were from 0.4-1.2/~m M M A D and 1-20 mg H.~SO.a/m~},respectively. Detailed aerosol characteristics, sizing of aerosols and air concentrations used in these experiments have been described elsewhere (Dahl, 1979 and 1980). Animals Male Beagle dogs ranging in age from 917-1834 days and weighing from 9.2-14 kg were from the Inhalation Toxicology Research Institute (ITRI) colony. Each dog was given a physical examination. Blood cell counts and serum chemical determinations were made to verify the dogs' health status. During the pre-exposure period the dogs were housed in kennel buildings with indoor and outdoor runs. They were fed Wayne® Lab Blox dog food (Allied Mills, Chicago, IL) daily and had free-access to water. The dogs were not fed on the day of exposure or instillation. Male, laboratory-reared, specific-pathogen-free Fischer-344 rats from the ITRI colony, aged 12-18 weeks and weighing from 180-282 grams were used. They were housed in polycarbonate shoe-box type cages in a temperature and humidity controlled room (approximately 23 °C and 30-50% R.H.) and fed a rat pelleted diet (Allied Mills, Chicago, IL). Bedding material was ash wood shavings. Only animals that appeared to be healthy were used. Hartley guinea pigs [CRL:COBS (HA)] rang-
Copyright 1983, Society of Toxicology
I:undanlental and Applied Toxicolol~y
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DA! iL. I:EI.ICE l T! A N D MUGGENBURG
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serum estimated for the whole dog (Cuddihy, et al., unpublished data). From this calculation, the percentage appearing in the blood was determined for Figure 1. For intranasal instillation of 'r'S-labeled sulfuric acid in rats and guinea pigs the animals were held and 5 pL of the carrierfree acid (5.7-9.5 uCi) was placed 5-6 mm w i t h i n one nostril using a syringe and catheter. The animals' heads were held upright to insure dispersion of the droplet. After 5 rain the animals were sacrificed by intraperitoneal injection of T-61 euthanasia solution (National Laboratories, Sommerville, NJ) and were dissected and further treated as described below for inhalation experiments.
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I l(, I I:!l,.'~ t ~,t dct,o.',ili~m ~=tt' ¢,n Ir,..ti~,n of instilled r'S-lab,.,,led sultur'l~ .1~=d .q,pearHL~; HI the" bh,od ~t the l:~t'agle dog. l..)~,g A had 2 0 , , l . ~ t _,,ultur,: a c i d ( 3 . 5 • l O : d p m , ~ a r r i e r - f r e e ) deposited I to 2 t i n past the nart.s. Dog I~ had 25 pl, ( 1.8 ;( lO"dpm, carrivr-freel dt.po.~iled in .I .-,ecl~nd guneratii,n brl~nchtls. 12)~g C had I00 /~L (3 5 " 10' dpm c a r r i e r - t r v v ) ~utlurw,lcid deposiIed in a s e v e n t h
!,wneratit,n bronchu.,,. I{r~or bar.~ rt.presen! Mandard deviations for two dvtvrn',inations, whole blood and pla.,,,na. No error bar ind, atv.., a pl.,,ma s.m~ph, only. ing |rein 4 5 5 - 6 0 0 grams, were obtained from Charles River. They were housed as described above for rats except that they were fed a pelleted guinea pig diet (Allied Mills, Chicago, IL) supplemented with lettuce. Animals were quarantined 14 days before use,
Instillation experiments Data in Figure 1 were obtained from dogs as follows. Dogs were anesthetized with halothane (Fluothane, Ayerst, NY) gas in 100% oxygen and arterial or venous catheters were inserted via the femoral artery or vein by vessel cutdown and a urinary catheter was inserted, as described previously (Muggenburg and Mauderly, 1974). The dogs were allowed to recover from anesthesia, placed in slings and were kept sedated w i t h triflupromazine (0,5 mg per kg body weight). Instillation of 20-100 ,uL of distilled water containing 200t O00,u Ci of ¢'S-labeled sulfuric acid, carrier-free, was accomplished by use of a fiberoptic bronchoscope or, for nasal instillation, 20,uL was placed 1-2 cm posterior to the nares using a syringe and a 2 cm |ong catheter. The so|utior~s ranged from pH 4.5-5.9. Blood specimens, 0.5 to 1.5 mL were d r a w n from the catheter at 15- to 60-second intervals and placed immediately into evacuated vials containing EDTA. After 30 minutes, the blood vessel catheters were removed, the vessels were repaired and the incision was sutured. For dogs A and C, Figure 1, blood samples were divided. One portion was centrifuged to separate plasma and blood cells. Because it was found that all the :~'S was plasma associated, for dog B, Figure 1, only plasma samples were used. One hundred microl;,ters of blood or plasma was placed in a liquid scintillation vial and decolorized by addition of H202 prior to liquid scintillation counting. After initial counting, blood samples were spiked w i t h known amounts of ;¢r'S activity five to ten times the original activity and were recounted to determine the quench factor. The activity was then factored by the,total volume of blood or 294
Prior to exposure, dogs were anesthetized with halothane (Fluothane~, Ayerst, NY) in oxygen and a catheter was placed in the femoral vein using a surgical cut down procedure and advanced to the posterior vena cava. The dog was allowed to recover from anesthesia and was tranquilized with Vetame® (E.R. Squibb and Sons) intravenously at 0.55 mg/kg body weight. The dogs were exposed to aerosols as has been described elsewhere (Dahl, t 979 and 1980). Inhalation exposures of l-rain duration were conducted in an apparatus similar to that described by Boecker, et al., (1964), This apparatus allows nose-only exposure of the dog while the animal is suspended in a sling and completely enclosed in a containment box. The venous catheter was passed through a hole in the containment box w h i c h was made air-tight by means of a split, bored cork. The catheter permitted immediate sacrifice of the dogs by injection of T-61 euthanasia solution (National Laboratories, Sommerville, NJ) using 0.3 mL/kg body weight. Following sacrifice, 5 mL of a solution of saturated potassium chloride solution was injected through the catheter to arrest heartbeat. The carcass was removed from the exposure apparatus and immediately dissected. The thoracic cavity was opened and the following samples and tissues were removed: 1) blood from the right ventricle of the heart, 2) trachea, larynx and lungs, 3) head, 4) stomach and 5) small intestines. The blood sample was placed in a vial containing heparin. The lower
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FIG. Z. C l e a r a n c e of s u l f u r i c acid-introduced a:'S f r o m rat l u n g s to blood. Sulfuric acid was a d m i n i s t e r e d as aerosols (0.4-1.2. #m, M M A D ) at 1-20 m g / m :l for 30 sec. Each data point is f r o m a n individual rat killed a f t e r a d m i n i s t r a t i o n of sulfuric acid. Data are e x p r e s s e d as fraction of :~r'5 deposited in lung which r e m a i n e d at sacrifice (cessation of h e a r t beat). Best fit by least s q u a r e s m e t h o d gives tl,~ 170 sec. (Y i n t e r c e p t 1.2, slope - 4 X 10 :~, r = -0.93). Fundam. AppL Toxicol. (3)
July~August, 1983
CLEARANCE FROM THE RESPIRATORY TRACT
respiratory tract was dissected into the individual lung lobes, portions of bronchial tree containing the carina and 2-3 cm of each bronchus and the trachea {arynx. The head was frozen in liquid nitrogen and the remaining portions of the carcass were stored at - 10 °C. The sections of the gastrointestinal tract and the respiratory tract were dissolved in concentrated nitric acid containing 0.1-0.3 % hydrogen peroxide. The frozen head was partially thawed and the nares and head skin were removed. The upper snout, anterior to the eyes, was separated from the remaining skull. The individual portions of the head were then dissolved in nitric acid/hydrogen peroxide solution. The frozen carcass was thawed, depelted and the pelts and carcasses were dissolved in nitric acid/hydrogen peroxide solution. Blood samples were centrifuged and a 250 or 500/~L aliquot of the plasma was decolorized w i t h hydrogen peroxide and analyzed for radioactivity by liquid scintillation counting. A 100- or 250-/~L aliquot of each tissue sample was analyzed for :¢'S activity by liquid scintillation counting in Aquasol® 11 (New England Nuclear). Each sample was then spiked with 10-20 times the original activity and recounted to determine the amount of quench. Procedures for the inhalation exposures of rats and guinea pigs were identical. The animals were exposed individually by the nose-only route (Raabe, et al., 1973). The exposure apparatus used by Raabe, et al. was modified to facilitate the rapid removal of the animal following exposure. The modification consisted of building a single animal nose-only exposure containment box made of Plexiglas with a hinged top. Exposure was for 30 seconds. After the exposure, the animals were killed with intraperitoneal injection of T-61 euthanasia solution (National Laboratories, Sommerville, NJ) and induction of a pneumothorax. A blood sample was drawn immediately from the heart. Time elapsed from the initiation of exposure to the drawing of the blood sample was recorded. The carcass was then dissected by first depelting the head and removing the mandible (snout) anterior to the eyes. The remaining portion of the head, the GI tract, the trachea and the lungs were removed. The carcass was then depetted. Each sample was placed separately into a labeled glass vial. Blood samples were centrifuged and a 250-/zL aliquot of the plasma was decolorized w i t h hydrogen peroxide and analyzed for radioactivity by liquid scintillation counting as described above for the dog. Individual portions of the carcass were dissolved in concentrated nitric acid/hydrogen peroxide solution. Aliquots of these solutions were analyzed by liquid scintillation counting as described above. Data treatment All calculations took into account the radioactive decay of":'S w h i c h has a half-life of 87.5 days. Tissues were assigned to the upper or lower respiratory tract as follows: The upper respiratory tract included sulfuric acid deposited in the snout, the skull and the GI tract. The lower respiratory tract included the larynx-trachea, lungs, blood samples and the rest of the carcass. Radioactivity from the carcass was assumed to have deposited originally in the lungs. Radioactivity in the gastrointestinal tract was assumed to have originally deposited in the nasopharyngeal region and to have been swallowed following mucociliary clearance. Percent clearance into the blood was calculated from the amount of label found in the blood and carcass divided by the sum of the amount found in the blood and carcass and the amount found in the lower respiratory tract. All calculations of clearance halftimes assumed first order kinetics (dN/dt = AN, w h e r e No is the total amount of Fundamental and Applied Toxicology
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FIG. 3. C l e a r a n c e of sulfuric acid f r o m g u i n e a pig l u n g s to blood. Sulfuric acid was a d m i n i s t e r e d as aerosols (0.4-1.2 ,urn, M M A D ) at 1-20 m g / m :~for 30 sec. Each data point is from an individual guinea pig killed a f t e r a d m i n i s t r a t i o n of.sulfuric acid. Data are expressed as fraction of :¢'S deposited in lung which remaii~ed at sacrifice. Best fit line by least s q u a r e s m e t h o d gives tl .., 230 sec. (Y i n t e r c e p t 1.I, slope - 3.0 X 10 '*, r : -0.81).
sulfate deposited, d N / d t is the clearance rate and ,k is tile first order rate constant). For the data in Table 1, the origin was taken as a data point. In Figures 2 and 3, a linear regression on the data was not forced through the origin. R E S UL TS
The results are recorded in Table 1 and 2 and Figures 1-3. Table 1 contains clearance data for dogs along with pertinent data regarding exposure conditions and aerosol characteristics. A plot of fraction :~;'Scleared versus time using these data gave an erratic curve indicating that there is considerble individual variability in clearance rates. As explained below, this may in part be due to differences in regional deposition of the aerosol w i t h i n the lungs. However, a calculation of clearance half-time for each dog allows establishment of a mean clearance half-time of 261 + 108 (SD.) sec for clearance of sulfuric acid-introduced :~:'S from dog lungs. Looking at Figure 1, it is observed that, apparently, clearance from the second generation bronchi is less complete and slightly less rapid than clearance from the seventh generation bronchi. This difference in rates may merely reflect individual variability between dogs B and C or may indicate a real difference in rates of clearance for different parts of the lung. In the latter case, it may, in part, be the cause of the variability of the calculated clearance half-times in Table I if the individual dogs deposited sulfuric acid to variable extents in different regions of the lung. Figures 2 and 3 show the rates of clearance of sulfuric acid for rats and guinea pigs following inhalation exposure. For these animals, the variation among individuals appears to be less than that observed with dogs. A plot of percentage remaining versus time can be made and a positive correlation is found. One observation made by comparing Figures 2 and 3 is that guinea pigs appear to clear sulfuric acid more slowly from the respiratory tract than do rats. From the data for clearance of ~}r'Sfollowing inhalation (i.e., Table 1 and Figures 2 and 3) clearance half-times of 230, 281 and 170 seconds, respectively, are calculated for guinea pigs, dogs and rats. For the pulmonary instillation experiments in 295
DA111., FLLICETTI AND MUC.;GI2NBURG
Clearance
"FABLE 1 o f I n h a l e d S u l f u r i c A c i d f r o m D o g Lungs" Time Allowed for Clearance From Lungs'
Calc. t~ (sec)"
Dog Number
MMAD" (~am
Percent Cleared From L u n g s '
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m~et te~t on c[eara+w('. }:rtm~ 7o-eL~'~,,t~ the total de[~L,,itedactivity deposited m lhe Illn~. "NIMAI) i~ ma.~.~median aert~dyrhHllicdiameter ot the aerosol. Ge~mwtric ~.{.mdard deviatum~ were 1.2-I .-1. 'l)ata are percent uf radiua~tivity originally depo~iled in lung~ which had Lh.ared into du, blood at time ot death (cessation of heart beatL I.M~ost res were }(~r v0 seconds. T h e s e times are from m i d - e x p o s u r e (/.('. 30 .,,w+mds a l t e r mitiatiun) to time ot c e s s a t i o n of heart heal.
dogs, Figure 1, clearance half-times for+e'S of 110sec and 200 sec can be calculated from the initially straight lines for the seventh and second generation bronchi, respectively. Taking all of the data presented into account, including the data for each of the three species and for administration of sulfuric acid by inhalation or instillation, the range of halftimes for clearance of sulfuric acid from the pulmonary region was 110-510 seconds (2-9 minutes). Qn the other hand, clearance from the upper respiratory tract, that is to say the nasal region, is insiginficant in this length of time. This is clear trom the data in Table 2 and Figure 1. Again, it is true for all three species examined. DISCUSSION The subject of clearance of soluble materials, either water soluble or lipid soluble, has recently been discussed at length (Schanker, 1978). In this commentary it was noted that absorption from the lungs can occur rapidly but the relative rates depend on the physicochemical properties of the compound and on whether absorption occurs by diffusion or carrier mediated transport, tn another paper (Schanker and Less, 1977) it was shown that, for weak acids and weak bases, absorption from the lungs was fastest at pH values at which the compounds were least ienized. This consideration is not important for sulfuric acid since under physiological conditions it would exist only as sulfate or, possibly, bisulfate ions. Studies in isolated perfused lungs of rats have shown that sulfate is cleared by a simple diffusive process (Charles, et al., 1977a). In those studies the half-times for clearance of sulfate administered as either sodium sulfate or ammonium sulfate ranged from 8.8-34.6 minutes and were apparently dependent on the dose and the cations which were co-administered. This effect of cations on the rate of diffusion of sulfate ion through isolated perfused rat lungs was further demonstrated in another study (Charles, et aL, 1977b). In both of the studies
296
with isolated perfused rat lungs, the half-times for clearance of sulfate were around 8-35 min+Thisis in contrast to what we observe f o r / n v/vo systems, where the half-times fall in the range of 2-9 min for sulfuric acid. The difference between 2-9 rain and the half-times observed by Charles, eta/. possibly may be an effect of the different cations co-administerd. The observed differences may also be due to differences in clearance rates between in vivo and isolated perfused lungs. Since in the present experiments sulfuric acid was administered as aerosols or in small volumes of dilute solutions at near-neutral pH, no effect from non-physiological cations would be expected to influence clearance. From the data in Figure 1, it is possible to conclude that for dogs there is some difference in clearance of the second generation bronchi compared to the seventh generat+on bronchi in intratracheal instillations. That this might be the case was also suggested by Schanker (1978). The difference in clearance rates may explain the variability for the data for the dog in Table 1 where the half-times for clearance from the lung varied from 110 seconds to 520 seconds, with a rnean of 261.
Distribution
TABLE 2 of Nasally Instilled
Sulfuric Acid-introduced :¢'S in Rats and Guinea P~gs 5 Minutes After Instillation" (Percent of Total) Animal Species G u i n e a Pigs
Rats
Head
Region
90.8 ± 1.3
97.1 ± 0
Body
2,7 :t: 0.5
2.9 :[: 0
" M e a n o f v a l u e s for t w o a n i m a l s per e x p e r i m e n t + S.D. Five microliters of c a r r i e r - f r e e s u l f u r i c acid c o n t a i n i n g 5.79.5 ~Ci of s u l f u r - 3 5 was instilled.
Fundam. AppL ToxicoL (3)
July/August, 1983
CLEARANCE F[~.OM THE RESPIRATORYTRACT tt should be noted that half-times of 110 and 520 seconds are the extreme points and are outlyers, since seven of the nine data points lie in the tight range of 215-268 seconds for the half-times of clearance. Variability might be expected due to individual differences in the dogs for the deposition of the sulfuric acid in the various portions of the respiratory tract distal to the trachea. Comparing the clearance rates for rats and guinea pigs in Figures 2 and 3, the outstanding feature is the somewhat slower clearance of sulfuric acid from the lungs of the guinea pigs compared to the rats. Guinea pigs are more sensitive to the acute toxic effects of inhaled sulfuric acid than are rats (Wolff, et aL, 1980), and it is conceivable that part of this difference in sensitivity may be related to the slower clearance rate from the lungs of guinea pigs, A slower clearance rate, all other things being equal, would lead to a greater effective dose per gram tissue weight when integrated over time. In conclusion, we have found that sulfuric acid-introduced :~:'Sdeposited in the lungs of guinea pigs, rats or dogs by either inhalation or intratracheal instillation is cleared w i t h a halftime of about 2-9 rain. This is a shorter half-time than was observed in previous studies with isolated perfused lungs. It appears that the smaller airways clear sulfuric acid-introduced '~:'S faster than larger airways. All portions of the lung clear sulfuric acid-introduced :*r'S much faster than does the nasal region. There appears to be some species difference in the rate of clearance of sulfuric acid-introduced :¢'S; the guinea pig being somewhat slower than the rat. It is not clear, however,
that this interspecies difference is large enough to have toxicological significance under ordinary inhalation conditions. A CKN 0 WL ED GEMEN TS We thank D. Esparza and C. Headrick for their technical assistance and M.B. Snipes, J.L. Mauderly, R.K. Wolff, S.A. Silbaugh and R.F. Henderson for their advice and comments throughout this work. Research supported inpart by the Environmental Protection Agency via InteragencyAgreement Number EPA-81 -D-X0533 under U.S. Department of Energy Contract Number DE-AC0476EV01013 and in part by the U.S. Department of Energy under Contract No. DE-AC04-76EV01013 and conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.
Fundamental and Applied Toxicology
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REFERENCES Amdur, M.O. (1971). Aerosols Formed by Oxidation of Sulfur Dioxide - Review of Their Toxicology. Arch. Envircm. Iteahh 23:459-408. Amdur, M.O., Bayles, J., Urgo, V., Dubrie|, M., and Underhi|l, D.W. (1975). Respiratory Response of Guinea Pigs to Sulfuric Acid and Sulfate Salts. Paper presented at the Symposium on Sulfur Pollution and Research Approaches, May 27-28, Duke University Medical Center. Boecker, B.B., Aguilar, F.L., and Mercer, T.T. (1904). A Canine Inhalation Exposure Apparatus Utilizing a Whole-body Plethysmograph. Ileahh Ph.rs. 10:1077-1089. Charles, J.M. and Menzel, D.B. (1975). Ammonium and Sulfate Ion Release of Histamine from Lung Fragments, ,'lrch. Environ. Heahh 30:314-316. Charles, I,M., Anderson, W.G., and Menzel, D.B. (1977a). Sulfa te Absorption from the Airways of the Isolated Perfused Rat Lung. ToxicoL AppL PharmaeoL 41:91-99. Charles, I.M., Gardner, D.E., Coffin, D.L., and Menzel, D.B. (1977b). Augmentation of Sulfate Ion Absorption from the Rat Lung by Heavy Metals. ToxieoL AppL PharmaeoL 42:531-538. Dahl, A.R. (1979). Deposition and Clearance of Sulfuric Acid Mist in Rats, Guinea Pigs and Dogs. Inhalation Toxicology Research Institute Annual Report, 1978-1979, LF-69, p. 462-468. Available from the National Technical Information Service, U.S. Department of Commerce, Springfield, VA. Dahi, A.R. (1980). Deposition and Clearance tff Inhaled Sulfuric Acid in Dogs, Guinea Pigs and Rats. Inhalation Toxicology Research Institute Annual Report, 1979-1980, LMF-34, p. 401-4u5. Available from the National Technic01 hfformation Service, U.S. Department of Commerce, Springfield, VA. Muggenburg, B.A. and Mauderly, J.L. (1974). Cardiopulmonary Function of Awake, Sedated and Anesthetized Beagle Dogs. J. AppL Ph.vsiol. 37:152-157. Schanker, L.S. (1978). Drug Absorption from the Lung. Bio('hem. PharmacoL 27:381-385. Schanker, L.S. and Less, M.J. (1977). Lung pH and Puhnonary Absorption of Nonvolatile Drugs in the Rat. Drug Metah. Di.ff;os. 5:174-178. Treon, ].F., Dutra, F.R., Cappel, J., Sigmon, H., and Younker, W. (1950). Toxicity of Sulfuric Acid Mist. ,,trctt. huL Ilyg. Occlq~. ,tletL 2:710-734. Wolff, R.K., Siibaugh, S.A., Brownstein, D.G., Carpenter, R.L., and Mauderly, J.L. (1979). Toxicity of 0.4- and 0.8-# Sulfuric Acid Aerosol in the Guinea Pig. d. To.~'icol. Environ. Iteahh 5:1037-10,17.
297
Clearance of Sulfuric Acid-Introduced 35S from the Respiratory Tracts of Rats, Guinea Pigs and Dogs Following Inhalation or Instillation ALAN R. DAHL, SHARON A. FELICETTI and BRUCE A. MUGGENBURG Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, NM 87185
ABSTRACT
Clearance of Sulfuric Acid-Introduced :~:'Sfrom the Resp i r a t o r y Tracts of Rats, Guinea Pigs and Dogs Following inhalation or Instillation. Dahl, A.R., Felicelti, S.A., and Muggenburg, B.A. (1983). Fundanz. AppL ToxicoL 3:293297. The clearance o f sulfuric acid-introduced :~"S from the upper and lower respiratory tracts o f rats, guinea pigs and dogs was measured. Sulfuric acid was administered by instillation and by inhalation for each species. Clearance into the blood and gastrointestinal tract was measured along with determination of:~S remaining at the site of administration at sacrifice. Different rates of clearance from different sites within the dog lung were indicated with rates of clearance increasing with decreasing airway diameter. Half-times of clearance from all sites in the hmg and for all species were from 2-9 min. There appeared to be some species differences, with clearance for dogs being slower than for guinea pigs, which was slower than for rats. Upper respiratory tract clearance was much slower than for lung and may not have been primarily by way of the blood. The data indicate that the clearance of sulfuric acid-introduced :~r'sin vivo is faster than previous studies in isolated perfused lungs had indicated. The results may be general for water soluble, ionized chemical species. INTRODUCTION Sulfur oxidation products occur in the atmosphere largely as a result of the burning of fossil fuels. Sulfuric acid appears to be the most acutely toxic of the many sulfur oxidation products w h i c h have been tested for inhalation toxicity (Amdur, 1971; Amdur, et aL, 1975). The toxicity of sulfuric acid mists in guinea pigs appears to be related to droplet diameter (Wolff, et aL, 1979) and the guinea pig is one of the most sensitive species for acute toxicity of sulfuric acid by inhalation (Treon, et aL, 1950). It has been suggested that the sensitivity of guinea pigs to sulfate ion may be due to release of histamine in the presence of sulfate (Charles and Menzel, 1975). The concentration of sulfate ion in the tissues of the lungs and other portions of the respiratory tract will depend upon both the rate of deposition and the rate of clearance. The clearance of sulfate ion in the presence of a variety of co-administered ions has been studied in isolated perfused rat lungs (Charles, etal., 1977a and 1977b). However, study of the in vivo respiratory clearance" rate of sulfate has not been reported, ,nor have species other than the rat been used for measurements of clearance of sulfuric acid or sulfate.
The purpose of the experiments reported here was to determine if species differences in clearances of sulfuric acidintroduced sulfate occurred w h i c h might influence relative toxicity. In addition, we wanted to determine the rates of clearance of sulfate from different parts of the respiratory tract and to determine if clearance following inhalation was different than clearance following instillation. METHODS Aerosol generation Sulfuric acid aerosols were produced using a compressed air nebulizer w i t h glass and Teflon~"J parts. The aerosols were heated to 220°C and diluted w i t h mixing air. The flow rate of aerosols past the noses of the animals was 20 L/rain w h i c h corresponded to a linear velocity of 2 m/rain. Concentration of sulfuric acid in the nebulizer was from 0 . 0 3 5 - 0 . 8 5 M depending on the mass median aerodynamic diameter (MMAD) desired. The sulfuric acid was radiolabeted by the addition of 1 mL of water containing 50-1 O0 mCi of :~'S-labeled sulfuric acid (New England Nuclear) to 4-5 mL of unlabeled sulfuric acid (Ventron, ultra-pure) diluted with water distilled in glass. Aerosol sizes and air concentrations were from 0.4-1.2/~m M M A D and 1-20 mg H.~SO.a/m~},respectively. Detailed aerosol characteristics, sizing of aerosols and air concentrations used in these experiments have been described elsewhere (Dahl, 1979 and 1980). Animals Male Beagle dogs ranging in age from 917-1834 days and weighing from 9.2-14 kg were from the Inhalation Toxicology Research Institute (ITRI) colony. Each dog was given a physical examination. Blood cell counts and serum chemical determinations were made to verify the dogs' health status. During the pre-exposure period the dogs were housed in kennel buildings with indoor and outdoor runs. They were fed Wayne® Lab Blox dog food (Allied Mills, Chicago, IL) daily and had free-access to water. The dogs were not fed on the day of exposure or instillation. Male, laboratory-reared, specific-pathogen-free Fischer-344 rats from the ITRI colony, aged 12-18 weeks and weighing from 180-282 grams were used. They were housed in polycarbonate shoe-box type cages in a temperature and humidity controlled room (approximately 23 °C and 30-50% R.H.) and fed a rat pelleted diet (Allied Mills, Chicago, IL). Bedding material was ash wood shavings. Only animals that appeared to be healthy were used. Hartley guinea pigs [CRL:COBS (HA)] rang-
Copyright 1983, Society of Toxicology
I:undanlental and Applied Toxicolol~y
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DA! iL. I:EI.ICE l T! A N D MUGGENBURG
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serum estimated for the whole dog (Cuddihy, et al., unpublished data). From this calculation, the percentage appearing in the blood was determined for Figure 1. For intranasal instillation of 'r'S-labeled sulfuric acid in rats and guinea pigs the animals were held and 5 pL of the carrierfree acid (5.7-9.5 uCi) was placed 5-6 mm w i t h i n one nostril using a syringe and catheter. The animals' heads were held upright to insure dispersion of the droplet. After 5 rain the animals were sacrificed by intraperitoneal injection of T-61 euthanasia solution (National Laboratories, Sommerville, NJ) and were dissected and further treated as described below for inhalation experiments.
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I l(, I I:!l,.'~ t ~,t dct,o.',ili~m ~=tt' ¢,n Ir,..ti~,n of instilled r'S-lab,.,,led sultur'l~ .1~=d .q,pearHL~; HI the" bh,od ~t the l:~t'agle dog. l..)~,g A had 2 0 , , l . ~ t _,,ultur,: a c i d ( 3 . 5 • l O : d p m , ~ a r r i e r - f r e e ) deposited I to 2 t i n past the nart.s. Dog I~ had 25 pl, ( 1.8 ;( lO"dpm, carrivr-freel dt.po.~iled in .I .-,ecl~nd guneratii,n brl~nchtls. 12)~g C had I00 /~L (3 5 " 10' dpm c a r r i e r - t r v v ) ~utlurw,lcid deposiIed in a s e v e n t h
!,wneratit,n bronchu.,,. I{r~or bar.~ rt.presen! Mandard deviations for two dvtvrn',inations, whole blood and pla.,,,na. No error bar ind, atv.., a pl.,,ma s.m~ph, only. ing |rein 4 5 5 - 6 0 0 grams, were obtained from Charles River. They were housed as described above for rats except that they were fed a pelleted guinea pig diet (Allied Mills, Chicago, IL) supplemented with lettuce. Animals were quarantined 14 days before use,
Instillation experiments Data in Figure 1 were obtained from dogs as follows. Dogs were anesthetized with halothane (Fluothane, Ayerst, NY) gas in 100% oxygen and arterial or venous catheters were inserted via the femoral artery or vein by vessel cutdown and a urinary catheter was inserted, as described previously (Muggenburg and Mauderly, 1974). The dogs were allowed to recover from anesthesia, placed in slings and were kept sedated w i t h triflupromazine (0,5 mg per kg body weight). Instillation of 20-100 ,uL of distilled water containing 200t O00,u Ci of ¢'S-labeled sulfuric acid, carrier-free, was accomplished by use of a fiberoptic bronchoscope or, for nasal instillation, 20,uL was placed 1-2 cm posterior to the nares using a syringe and a 2 cm |ong catheter. The so|utior~s ranged from pH 4.5-5.9. Blood specimens, 0.5 to 1.5 mL were d r a w n from the catheter at 15- to 60-second intervals and placed immediately into evacuated vials containing EDTA. After 30 minutes, the blood vessel catheters were removed, the vessels were repaired and the incision was sutured. For dogs A and C, Figure 1, blood samples were divided. One portion was centrifuged to separate plasma and blood cells. Because it was found that all the :~'S was plasma associated, for dog B, Figure 1, only plasma samples were used. One hundred microl;,ters of blood or plasma was placed in a liquid scintillation vial and decolorized by addition of H202 prior to liquid scintillation counting. After initial counting, blood samples were spiked w i t h known amounts of ;¢r'S activity five to ten times the original activity and were recounted to determine the quench factor. The activity was then factored by the,total volume of blood or 294
Prior to exposure, dogs were anesthetized with halothane (Fluothane~, Ayerst, NY) in oxygen and a catheter was placed in the femoral vein using a surgical cut down procedure and advanced to the posterior vena cava. The dog was allowed to recover from anesthesia and was tranquilized with Vetame® (E.R. Squibb and Sons) intravenously at 0.55 mg/kg body weight. The dogs were exposed to aerosols as has been described elsewhere (Dahl, t 979 and 1980). Inhalation exposures of l-rain duration were conducted in an apparatus similar to that described by Boecker, et al., (1964), This apparatus allows nose-only exposure of the dog while the animal is suspended in a sling and completely enclosed in a containment box. The venous catheter was passed through a hole in the containment box w h i c h was made air-tight by means of a split, bored cork. The catheter permitted immediate sacrifice of the dogs by injection of T-61 euthanasia solution (National Laboratories, Sommerville, NJ) using 0.3 mL/kg body weight. Following sacrifice, 5 mL of a solution of saturated potassium chloride solution was injected through the catheter to arrest heartbeat. The carcass was removed from the exposure apparatus and immediately dissected. The thoracic cavity was opened and the following samples and tissues were removed: 1) blood from the right ventricle of the heart, 2) trachea, larynx and lungs, 3) head, 4) stomach and 5) small intestines. The blood sample was placed in a vial containing heparin. The lower
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FIG. Z. C l e a r a n c e of s u l f u r i c acid-introduced a:'S f r o m rat l u n g s to blood. Sulfuric acid was a d m i n i s t e r e d as aerosols (0.4-1.2. #m, M M A D ) at 1-20 m g / m :l for 30 sec. Each data point is f r o m a n individual rat killed a f t e r a d m i n i s t r a t i o n of sulfuric acid. Data are e x p r e s s e d as fraction of :~r'5 deposited in lung which r e m a i n e d at sacrifice (cessation of h e a r t beat). Best fit by least s q u a r e s m e t h o d gives tl,~ 170 sec. (Y i n t e r c e p t 1.2, slope - 4 X 10 :~, r = -0.93). Fundam. AppL Toxicol. (3)
July~August, 1983
CLEARANCE FROM THE RESPIRATORY TRACT
respiratory tract was dissected into the individual lung lobes, portions of bronchial tree containing the carina and 2-3 cm of each bronchus and the trachea {arynx. The head was frozen in liquid nitrogen and the remaining portions of the carcass were stored at - 10 °C. The sections of the gastrointestinal tract and the respiratory tract were dissolved in concentrated nitric acid containing 0.1-0.3 % hydrogen peroxide. The frozen head was partially thawed and the nares and head skin were removed. The upper snout, anterior to the eyes, was separated from the remaining skull. The individual portions of the head were then dissolved in nitric acid/hydrogen peroxide solution. The frozen carcass was thawed, depelted and the pelts and carcasses were dissolved in nitric acid/hydrogen peroxide solution. Blood samples were centrifuged and a 250 or 500/~L aliquot of the plasma was decolorized w i t h hydrogen peroxide and analyzed for radioactivity by liquid scintillation counting. A 100- or 250-/~L aliquot of each tissue sample was analyzed for :¢'S activity by liquid scintillation counting in Aquasol® 11 (New England Nuclear). Each sample was then spiked with 10-20 times the original activity and recounted to determine the amount of quench. Procedures for the inhalation exposures of rats and guinea pigs were identical. The animals were exposed individually by the nose-only route (Raabe, et al., 1973). The exposure apparatus used by Raabe, et al. was modified to facilitate the rapid removal of the animal following exposure. The modification consisted of building a single animal nose-only exposure containment box made of Plexiglas with a hinged top. Exposure was for 30 seconds. After the exposure, the animals were killed with intraperitoneal injection of T-61 euthanasia solution (National Laboratories, Sommerville, NJ) and induction of a pneumothorax. A blood sample was drawn immediately from the heart. Time elapsed from the initiation of exposure to the drawing of the blood sample was recorded. The carcass was then dissected by first depelting the head and removing the mandible (snout) anterior to the eyes. The remaining portion of the head, the GI tract, the trachea and the lungs were removed. The carcass was then depetted. Each sample was placed separately into a labeled glass vial. Blood samples were centrifuged and a 250-/zL aliquot of the plasma was decolorized w i t h hydrogen peroxide and analyzed for radioactivity by liquid scintillation counting as described above for the dog. Individual portions of the carcass were dissolved in concentrated nitric acid/hydrogen peroxide solution. Aliquots of these solutions were analyzed by liquid scintillation counting as described above. Data treatment All calculations took into account the radioactive decay of":'S w h i c h has a half-life of 87.5 days. Tissues were assigned to the upper or lower respiratory tract as follows: The upper respiratory tract included sulfuric acid deposited in the snout, the skull and the GI tract. The lower respiratory tract included the larynx-trachea, lungs, blood samples and the rest of the carcass. Radioactivity from the carcass was assumed to have deposited originally in the lungs. Radioactivity in the gastrointestinal tract was assumed to have originally deposited in the nasopharyngeal region and to have been swallowed following mucociliary clearance. Percent clearance into the blood was calculated from the amount of label found in the blood and carcass divided by the sum of the amount found in the blood and carcass and the amount found in the lower respiratory tract. All calculations of clearance halftimes assumed first order kinetics (dN/dt = AN, w h e r e No is the total amount of Fundamental and Applied Toxicology
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FIG. 3. C l e a r a n c e of sulfuric acid f r o m g u i n e a pig l u n g s to blood. Sulfuric acid was a d m i n i s t e r e d as aerosols (0.4-1.2 ,urn, M M A D ) at 1-20 m g / m :~for 30 sec. Each data point is from an individual guinea pig killed a f t e r a d m i n i s t r a t i o n of.sulfuric acid. Data are expressed as fraction of :¢'S deposited in lung which remaii~ed at sacrifice. Best fit line by least s q u a r e s m e t h o d gives tl .., 230 sec. (Y i n t e r c e p t 1.I, slope - 3.0 X 10 '*, r : -0.81).
sulfate deposited, d N / d t is the clearance rate and ,k is tile first order rate constant). For the data in Table 1, the origin was taken as a data point. In Figures 2 and 3, a linear regression on the data was not forced through the origin. R E S UL TS
The results are recorded in Table 1 and 2 and Figures 1-3. Table 1 contains clearance data for dogs along with pertinent data regarding exposure conditions and aerosol characteristics. A plot of fraction :~;'Scleared versus time using these data gave an erratic curve indicating that there is considerble individual variability in clearance rates. As explained below, this may in part be due to differences in regional deposition of the aerosol w i t h i n the lungs. However, a calculation of clearance half-time for each dog allows establishment of a mean clearance half-time of 261 + 108 (SD.) sec for clearance of sulfuric acid-introduced :~:'S from dog lungs. Looking at Figure 1, it is observed that, apparently, clearance from the second generation bronchi is less complete and slightly less rapid than clearance from the seventh generation bronchi. This difference in rates may merely reflect individual variability between dogs B and C or may indicate a real difference in rates of clearance for different parts of the lung. In the latter case, it may, in part, be the cause of the variability of the calculated clearance half-times in Table I if the individual dogs deposited sulfuric acid to variable extents in different regions of the lung. Figures 2 and 3 show the rates of clearance of sulfuric acid for rats and guinea pigs following inhalation exposure. For these animals, the variation among individuals appears to be less than that observed with dogs. A plot of percentage remaining versus time can be made and a positive correlation is found. One observation made by comparing Figures 2 and 3 is that guinea pigs appear to clear sulfuric acid more slowly from the respiratory tract than do rats. From the data for clearance of ~}r'Sfollowing inhalation (i.e., Table 1 and Figures 2 and 3) clearance half-times of 230, 281 and 170 seconds, respectively, are calculated for guinea pigs, dogs and rats. For the pulmonary instillation experiments in 295
DA111., FLLICETTI AND MUC.;GI2NBURG
Clearance
"FABLE 1 o f I n h a l e d S u l f u r i c A c i d f r o m D o g Lungs" Time Allowed for Clearance From Lungs'
Calc. t~ (sec)"
Dog Number
MMAD" (~am
Percent Cleared From L u n g s '
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m~et te~t on c[eara+w('. }:rtm~ 7o-eL~'~,,t~ the total de[~L,,itedactivity deposited m lhe Illn~. "NIMAI) i~ ma.~.~median aert~dyrhHllicdiameter ot the aerosol. Ge~mwtric ~.{.mdard deviatum~ were 1.2-I .-1. 'l)ata are percent uf radiua~tivity originally depo~iled in lung~ which had Lh.ared into du, blood at time ot death (cessation of heart beatL I.M~ost res were }(~r v0 seconds. T h e s e times are from m i d - e x p o s u r e (/.('. 30 .,,w+mds a l t e r mitiatiun) to time ot c e s s a t i o n of heart heal.
dogs, Figure 1, clearance half-times for+e'S of 110sec and 200 sec can be calculated from the initially straight lines for the seventh and second generation bronchi, respectively. Taking all of the data presented into account, including the data for each of the three species and for administration of sulfuric acid by inhalation or instillation, the range of halftimes for clearance of sulfuric acid from the pulmonary region was 110-510 seconds (2-9 minutes). Qn the other hand, clearance from the upper respiratory tract, that is to say the nasal region, is insiginficant in this length of time. This is clear trom the data in Table 2 and Figure 1. Again, it is true for all three species examined. DISCUSSION The subject of clearance of soluble materials, either water soluble or lipid soluble, has recently been discussed at length (Schanker, 1978). In this commentary it was noted that absorption from the lungs can occur rapidly but the relative rates depend on the physicochemical properties of the compound and on whether absorption occurs by diffusion or carrier mediated transport, tn another paper (Schanker and Less, 1977) it was shown that, for weak acids and weak bases, absorption from the lungs was fastest at pH values at which the compounds were least ienized. This consideration is not important for sulfuric acid since under physiological conditions it would exist only as sulfate or, possibly, bisulfate ions. Studies in isolated perfused lungs of rats have shown that sulfate is cleared by a simple diffusive process (Charles, et al., 1977a). In those studies the half-times for clearance of sulfate administered as either sodium sulfate or ammonium sulfate ranged from 8.8-34.6 minutes and were apparently dependent on the dose and the cations which were co-administered. This effect of cations on the rate of diffusion of sulfate ion through isolated perfused rat lungs was further demonstrated in another study (Charles, et aL, 1977b). In both of the studies
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with isolated perfused rat lungs, the half-times for clearance of sulfate were around 8-35 min+Thisis in contrast to what we observe f o r / n v/vo systems, where the half-times fall in the range of 2-9 min for sulfuric acid. The difference between 2-9 rain and the half-times observed by Charles, eta/. possibly may be an effect of the different cations co-administerd. The observed differences may also be due to differences in clearance rates between in vivo and isolated perfused lungs. Since in the present experiments sulfuric acid was administered as aerosols or in small volumes of dilute solutions at near-neutral pH, no effect from non-physiological cations would be expected to influence clearance. From the data in Figure 1, it is possible to conclude that for dogs there is some difference in clearance of the second generation bronchi compared to the seventh generat+on bronchi in intratracheal instillations. That this might be the case was also suggested by Schanker (1978). The difference in clearance rates may explain the variability for the data for the dog in Table 1 where the half-times for clearance from the lung varied from 110 seconds to 520 seconds, with a rnean of 261.
Distribution
TABLE 2 of Nasally Instilled
Sulfuric Acid-introduced :¢'S in Rats and Guinea P~gs 5 Minutes After Instillation" (Percent of Total) Animal Species G u i n e a Pigs
Rats
Head
Region
90.8 ± 1.3
97.1 ± 0
Body
2,7 :t: 0.5
2.9 :[: 0
" M e a n o f v a l u e s for t w o a n i m a l s per e x p e r i m e n t + S.D. Five microliters of c a r r i e r - f r e e s u l f u r i c acid c o n t a i n i n g 5.79.5 ~Ci of s u l f u r - 3 5 was instilled.
Fundam. AppL ToxicoL (3)
July/August, 1983
CLEARANCE F[~.OM THE RESPIRATORYTRACT tt should be noted that half-times of 110 and 520 seconds are the extreme points and are outlyers, since seven of the nine data points lie in the tight range of 215-268 seconds for the half-times of clearance. Variability might be expected due to individual differences in the dogs for the deposition of the sulfuric acid in the various portions of the respiratory tract distal to the trachea. Comparing the clearance rates for rats and guinea pigs in Figures 2 and 3, the outstanding feature is the somewhat slower clearance of sulfuric acid from the lungs of the guinea pigs compared to the rats. Guinea pigs are more sensitive to the acute toxic effects of inhaled sulfuric acid than are rats (Wolff, et aL, 1980), and it is conceivable that part of this difference in sensitivity may be related to the slower clearance rate from the lungs of guinea pigs, A slower clearance rate, all other things being equal, would lead to a greater effective dose per gram tissue weight when integrated over time. In conclusion, we have found that sulfuric acid-introduced :~:'Sdeposited in the lungs of guinea pigs, rats or dogs by either inhalation or intratracheal instillation is cleared w i t h a halftime of about 2-9 rain. This is a shorter half-time than was observed in previous studies with isolated perfused lungs. It appears that the smaller airways clear sulfuric acid-introduced '~:'S faster than larger airways. All portions of the lung clear sulfuric acid-introduced :*r'S much faster than does the nasal region. There appears to be some species difference in the rate of clearance of sulfuric acid-introduced :¢'S; the guinea pig being somewhat slower than the rat. It is not clear, however,
that this interspecies difference is large enough to have toxicological significance under ordinary inhalation conditions. A CKN 0 WL ED GEMEN TS We thank D. Esparza and C. Headrick for their technical assistance and M.B. Snipes, J.L. Mauderly, R.K. Wolff, S.A. Silbaugh and R.F. Henderson for their advice and comments throughout this work. Research supported inpart by the Environmental Protection Agency via InteragencyAgreement Number EPA-81 -D-X0533 under U.S. Department of Energy Contract Number DE-AC0476EV01013 and in part by the U.S. Department of Energy under Contract No. DE-AC04-76EV01013 and conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.
Fundamental and Applied Toxicology
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REFERENCES Amdur, M.O. (1971). Aerosols Formed by Oxidation of Sulfur Dioxide - Review of Their Toxicology. Arch. Envircm. Iteahh 23:459-408. Amdur, M.O., Bayles, J., Urgo, V., Dubrie|, M., and Underhi|l, D.W. (1975). Respiratory Response of Guinea Pigs to Sulfuric Acid and Sulfate Salts. Paper presented at the Symposium on Sulfur Pollution and Research Approaches, May 27-28, Duke University Medical Center. Boecker, B.B., Aguilar, F.L., and Mercer, T.T. (1904). A Canine Inhalation Exposure Apparatus Utilizing a Whole-body Plethysmograph. Ileahh Ph.rs. 10:1077-1089. Charles, J.M. and Menzel, D.B. (1975). Ammonium and Sulfate Ion Release of Histamine from Lung Fragments, ,'lrch. Environ. Heahh 30:314-316. Charles, I,M., Anderson, W.G., and Menzel, D.B. (1977a). Sulfa te Absorption from the Airways of the Isolated Perfused Rat Lung. ToxicoL AppL PharmaeoL 41:91-99. Charles, I.M., Gardner, D.E., Coffin, D.L., and Menzel, D.B. (1977b). Augmentation of Sulfate Ion Absorption from the Rat Lung by Heavy Metals. ToxieoL AppL PharmaeoL 42:531-538. Dahl, A.R. (1979). Deposition and Clearance of Sulfuric Acid Mist in Rats, Guinea Pigs and Dogs. Inhalation Toxicology Research Institute Annual Report, 1978-1979, LF-69, p. 462-468. Available from the National Technical Information Service, U.S. Department of Commerce, Springfield, VA. Dahi, A.R. (1980). Deposition and Clearance tff Inhaled Sulfuric Acid in Dogs, Guinea Pigs and Rats. Inhalation Toxicology Research Institute Annual Report, 1979-1980, LMF-34, p. 401-4u5. Available from the National Technic01 hfformation Service, U.S. Department of Commerce, Springfield, VA. Muggenburg, B.A. and Mauderly, J.L. (1974). Cardiopulmonary Function of Awake, Sedated and Anesthetized Beagle Dogs. J. AppL Ph.vsiol. 37:152-157. Schanker, L.S. (1978). Drug Absorption from the Lung. Bio('hem. PharmacoL 27:381-385. Schanker, L.S. and Less, M.J. (1977). Lung pH and Puhnonary Absorption of Nonvolatile Drugs in the Rat. Drug Metah. Di.ff;os. 5:174-178. Treon, ].F., Dutra, F.R., Cappel, J., Sigmon, H., and Younker, W. (1950). Toxicity of Sulfuric Acid Mist. ,,trctt. huL Ilyg. Occlq~. ,tletL 2:710-734. Wolff, R.K., Siibaugh, S.A., Brownstein, D.G., Carpenter, R.L., and Mauderly, J.L. (1979). Toxicity of 0.4- and 0.8-# Sulfuric Acid Aerosol in the Guinea Pig. d. To.~'icol. Environ. Iteahh 5:1037-10,17.
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