Document 44BKrzRm74qvgXR6z9v8GMRpe

FILE NAME: ALCOA (ALC) DATE: 1971 May DOC#: ALC045 DOCUMENT DESCRIPTION: Journal Article - An Experimental Approach to Study the Toxicity of Nonparticulate Air Pollutants 638 : pc ta m fo An Experimental Approach m d to Study the T oxicity of Nonparticulate a VI Air Pollutants el L R ationale and M ethods a Ahmed N . M . Nasr, MD, PhD; Bertram D. Dinman, M D, ScD; and ti 1, A . Bernstein, PhD, Ann Arbor, M ich b c This report describe* an experimental model for on the levels of the coenzyme, nadide phos h studying tire toxicity of Mnpartteutate sir poBu- phate (N A D P), in the tracheal mucosa of f tants, In terms of a `biochemical lesion.' Changes the rat. a In chemical composition of lung tissue homoge Experim ental studies on th e toxicity of air l nate, Mowing Inhalation of a itodous gas, eouM pollutants have been largely lim ited to t neither be traced to a particular tissue component, changes in structure and functions of the ! nor b* regwcM as solitary unrelated Incidents. lungs and bronchial tree, mucous secretion 1 Bseipoachraetmedicabl ocmhanfigees rIenspiaratsoinrygletrakcitndafotefr cethRe, and ciliary motion, deposition and clearance animal's exposure to poisonous gas, may tie more marfMHiifims, mid chemical changes in lung I dhecfy related to the toxic mechanism of the tissue homogenates. i Investigated gas. A technique for Isolating epi- Research tended to focus on those effects which 1 thslum lining the rat trachea has been adapted were easiest to measure, often a t the expense of Im e a w ra f m naAod up^vbVM^ws m id i^ m ris te^ov vuenp wirr oitMiii te*fiehM i md th e m ore delicate and less detectable changes. m bronchial epiflwla from human autopsy material. H ie investigation of respiratory function, for Ozone was chosen as teet gas In this experimental example, has probably been overemphasised in approach because of Its Importance In photo comparison to other physiological and biochem chemical smog and Its presence In certain In ical parameter.1 dustrial enrironmaets. Specific therapy o r prophylaxis for disiwaai caused by trade inhalants (and affecting the T h is IS th e first of two reports dealing with the development of an experim ental model for th e study of th e tenacity of non particulate a ir pollutants. T he second report deals with the effect of inhalation of ozone respiratory system ) is la d in g , in spite of numerous investigations te a t have been con ducted in this field. H ie study of biochemical changes in vari ous tissues has been one m eans of elucidat ing disease mechanisms. In reference to air Submitted for pubtiestioa May 14,1070; scceptad June L From tbs Institut* of Environmental and Indus trial Health, fie University of Michigan. Arm Arbor. Dm. K u r and Dinman are also affiliated w ifi the Department of Internal Mcdktoa, University of Michigan, Arm Arbor. Reprint requests to WG640 University Hoepital, Arm Arbor, Mich 48104 (Dr. Nasr). pollutants, some investigators, eg, Scheel et aP and Buell et al,s have described changes in file chemical composition of lung tissue homogenates following inhalation of ozone. T he interpretation of th eir findings, in terms of the mechanism of toxicity o f ozone, pre sents several difficulties. H ie lung, unlike Arch Environ Health--Vol 22, May 1971 NONPARTICULATE AIR POLLVTANTS--NASR ET AL 539 late iyroe, nadide phosracheal mucosa o! n the toxicity ol air argely lim ited to 1 functions of the , mucous secretion ition and clearance al changes in lung a those effects which en a t the expense of detectable changes, ratory function, for a overemphasized in logical and biochcm- lytaxia for diseases . (and affecting the tdting, in spite of hat have been con- ial changes in vanmeans of elucidatIn reference to air atom, eg, Scheel et s described changes turn of lung tissue lhalation of ozone, r findings, in term s id ty of ozone, pre- The lung, unlike Qte liver, for example, is composed of a more M ethods heterogeneous assortment of cells and tis- T^U-mw, of Tracheal Epithelium.--Thirty- ^goes th at are dissim ilar in structure as well n x young, adult male rata of the CFN train I as function. Thus, a change in the chemical were used in this investigation. The animate I composition of lung tissue homogenates can neither be traced to a particular tissue cosn- ' ponent, nor could it be regarded as a soli tary inddmt I t m ay represent a profile of a m ultitude of changes, a "vector" of several forces operating sim ultaneously. Further more, xml" the chemical changes in the different components of a lung tissue homog enate are additive, they m ay d u d e the in ranged in age from two to four months, and in weight from 200 to 395 gm. The rat* were killed by stunning, and the trachea were im mediately excised through a xnidfine longitudi nal incision. A stream of ice-cold isotonic sucroee-O.OlM edetic arid (EDTA) solution was squeezed into the trachad lumen from a plastic bottle through a fine-tipped nozzle, in order to remove the mucus and blood aspirated by some during death. The esophagus was then vestigator by nullifying each other. excised and the trachea slit open along its T he approach adopted by Spencer4 in the study of the role of smoking in the etiology of bronchial carcinom a has been adapted to the purpose of this investiga tion. H e attem pted to detect precancerous `biochemical lesions' by the correlation of biochemical changes in tracheal and bron dorsal surface. The trachea was then trimmed and further cleaned of mucus in a P etri dish, sing a soft camel-hair brush and ice-cold su crose-edetic acid solution. The technique used for separation of the epithelial cells lining the trachea was adapted from the one reported by Spencer.4 A large dibber stopper 4 inches in diameter^covered chial epithdia with smoking habits and d ire with filter paper, was used (E g 1). The open ical records. Spencer's approach4 required trachea was fixed on the rubber stopper with the development of a m ethod for the remov insect pins, the trough formed facing upward* al of tracheal and bronchial epithelia from human autopsy specimens. In ttus investiga tion his technique has been modified few the preparation of epithelial cell suspensions fnan the trachea of the r a t T his investigation was planned with two goals in mind. T he first goal was to develop an experimental model for the study of the Small volumes of ice-cold sucrose-edefac an d solution, 0.01 to 0.02 ml, were instilled on the mucosal surface through disposable capulaorglass micropipets. T he epithelium was brought into suspension using a No. 2 camel-hair brush, w ith bristles trimmed to a length of approxi m ately 4 mm- The sequential steps of wbllmg gueroee-edetic arid solution, brushing off the epithelium, and pipetting the cell mwpeaMon toxicity of a ir pollutants, in term s of `bio out of the trachea were repeated until a total chemical pathology.' A second goal was to volume of 0.2 to 0.5 m l had accumulated. The try to elucidate sa n e of tire aspects erf the bade mechanism erf ozone toxicity. Ozone was chosen as tire test gas because of ite prevalence under various environm ental cir cumstances, notably its prominence in cer tain kinds erf `smog.' I t also A ares w ith other a ir pollutants, eg, ettides erf nitrogen and organic peroxide compounds, th e two Bid, as well as the bottom, of the tracheal trough were brushed in order to harvest the greatest number of cells. The following procedure was used for cell counting: After repeated passing of the Bie w w m in a microsyringe, 0.08 ml of 0.1% methylene blue in tire isotonic sucrose-edebc acid solution, pH 7.4, was added to an equal volume of epithelial suspension in the same properties of being both an oxidant and an medium. In order to achieve thorough mixing irrita n t Ozone is an im portant component and further cell dispersion, the content* of the erf tire Los Angdes smog.7-8 D uring periods of high photochemical activity, i t accounts for 90% or more of tire total oxidant while nitrogen dioxide is tire m ain component when photochemical activity is low or absent* T he presence of ozone and its ef tube were again passed several times through a microsyringe. Counts of nuclei were made in a hemacytometer chamber in the same fashion as in counting white Mood cells. T he standard error of counting was estimated by the method of Berkson et al-11 Exposure of Animate to Ozone---T he ozone fects on human health in inert-gas-shielded arc welding have been described by several authors.*-10 exposure apparatus is illustrated in Fig 2. Ctamw was generated from pure oxygen (U ltra High P arity Oxygen) obtained from a commercial Arch Environ Health--Voi 22, May 1971 540 NONPARTICULATE A IR POLLUTANTS--NASR E T AL Fig 1.--Apparatus usd for ho. tation of epithelial ceils from the trachea of the rat. Fig 2.--Diagram of the ozone exposure apparatus, showing ozone generator (A), bed jar (B), mixing vessel with glass beads (C). and animal Inhalation chamber (D). manufacturer. Pure oxygen was preferred to air for the purpose of generating ozone in order to avoid simultaneous generation of nitrogen oxides (from the nitrogen in the air). Svirbely and SaKxmanu reported th at substances associated with ozone production from air (including ox ides of nitrogen) are unlikely to influence the toxicity of ozone in laboratory animals. Knee this study was planned to investigate some as pects of the basic mechanism of injury by ozone, and as an experimental model for tox icity studies of other inhalants, the above-men tioned precaution was taken. An ozone generator (Welxone R-2), labeled A in Pig 2, was obtained from a commercial manufacturer. I t was housed in a glaas bell jar (B in Fig 2) tightly fitting over a circular glass {date, on which the ozonator was placed. H ie inflow of pure oxygen to the bell ja r was controlled by a two-stage valve regulator con nected to the oxygen cylinder and measured with a gas flowmeter. Ozonized oxygen flowed through flexible tub ing from the bell ja r to a spherical mixing vessel (C in Fig 2). This vessel is 2,000 ml in capacity and was filled with glass beads 6 mm in diameter. Filtered partially-dried air was introduced Into the mixing vessel through an other in let This arrangement enhanced effi cient mixing of air and ozonized oxygen. A etopoocfced outlet, through which the air-oxy gen-ozone m ixture flowed to the animal inhala tion chamber (D in Fig 2), was built in the mixing vessel a t a level higher than the upper most surface of the glass beads. The air used for diluting ozonized oxygen was supplied from a compressed-air line. It was passed through activated charcoal to remove any mineral oil th at might have contaminated it from tee com pressor, and through silica gel to dry it to a relative humidity of 40% to 50%. The air was then filtered through a membrane filter, 0.8a in pore size. A glass desiccator, 8,000 ml in capacity, was used as an animal inhalation chamber. A thick sheet of aluminum, with several hides drilled ` , enter* gtopct nbalr which placet inhali and 1 Vin matei tire a diami cury 1 taxed indus humn and v were campi (Colli Six the (NA1 (NA1 animi conte while 337 i at ra hour tore, rate minu e e n tr tion < feted S alts Eh becai soon am ir ozora heme effec cono in a ti after venir ed w Arch Environ Health--Vol 22, May 1971 AL --Apparatus ue*d for bo. epithelial cells from the il the ret. > a spherical mixing vessel is 2,000 ml in ito glass heads 6 mm rtially-dried air was ig vessel through anient enhanced effioronized oxygen. A a which the air-oxyto toe anim al tnhala2), was built in toe -gber than the upperbeads. The air used en was supplied from was passed through rove any m ineral oil ated it from toe coin 's gel to dry it to a to 60%. The a ir was snbrane filter, 0.8* in ) ml in capacity, was ion chamber. A thick several holes drilled r NONPARTICULATE A IR POLLUTANTS--N A 8R E T AL 641 Kgto it, was placed on the protruriona in the T he extent of removal of the epithelium of the glass desiccator approximately 4 by this m ethod was studied by examining _i above its bottom. Tins perforated alumi- histological sections of th e trachea before f sheet served as a platform on which toe and after separation of th e cells. Figure 5 (animal was placed during exposure to ozone, pgndi helped to separate toe ra t from its excreta. i-fiie opening in toe lid of the desiccator was pfitted w ith a glass-to-gUies cover through which ' entered inlet and outlet tubes equipped with ; stopcocks. Air flowed from toe outlet of toe ,,halation chamber into a laboratory hood in which toe whole exposure apparatus was placed. The uee of a glass desiccator as an illustrates the appearance of the trachea of a norm al rat. Figures 6 and 7 show toe ap pearance of the trachea after the epithelium bad been removed by th e brush method. T he submucosal layer remained in ta c t T he ab sence of contam ination of toe epithelial cell a igw M m with connective tissue was veri fied by phase-contrast microscopic examina inhalation chamber was adapted from Higgle tion of unstained cell sm ears and examina and Gag-1 Vinyl tubing (Tygon) was used as the tubing material for flexible connections in the en tire apparatus. Temperature in the inhalation chamber was measured with an ordinary mer cury thermometer. Relative humidity was mea sured with an indicator manufactured by an industrial instrum ent company. The relative humidity indicator was calibrated w ith a dry and wet bulb thermometer. All gas flowmeters tion of stained preparations. The appearance of the cells soon after they were separated is illustrated in Fig 8. The brush technique resulted in removal of sheets and clumps of epithelium. Repeated passing of the cell sus pension in a micropipet effected partial sepa ration of the cell dum ps. Repeated passing through toe orifice of a microsyringe resulted in better separation of the cells. Figure 9 s o n obtained from a commercial scientific demonstrates the effect of repeated passing axnpany and calibrated with a spirometer of toe cell suspension through the orifice of (Coffin's) Sixteen animals were used for comparison of the ratio of reduced nadide phosphate (NADPH) to oxidized nadide phosphate (NADP+) in normal animals to th at ratio in Tiiinmln poisoned with ozone, lir e weight of toe control animals (mean SE) was 331 20 gm, while th at of the experimental group was 337 14 gm. Exposure to ozone was assigned a micropipet, while Fig 10 shows the results of using a microsyringe for the same purpose. Considerable breakage of the cells took place when toe microsyringe was used to disperse toe dum ps. The cells w oe ground in tissue horoogenizer jn preparation for the assay of NADP+ and NADPH. Figure 11 illustrates th eir appearance after homogenization. at random. The animals were exposed for 1 T he technique developed for the assay of hour to 33 ppm ozone in an air-oxygen mix coenzyme made a total cell count neither ture, supplied to toe inhalation chamber a t a rate Of 1,000 ml air and SO m l okygen per minute. Samples lor analysis of the ozone con centration were taken a t the inlet of the inhala tion chamber and analyzed by the neutral buffered-potassium iodide method described by Salteman.14 feasible nor essential. However, it was con sidered desirable to obtain an estim ate of the total number of cells isolated from one trachea. Such an estim ate was only approxi m ate, ranee complete separation of the cell dum ps into individual cells was not a t tained. T he num ber of cells th at could be isolated from a trachea was of the order of During the exposure period the anim als became lethargic after a few m inutes, and soon th eir breathing was labored. Gross ex am ination of toe lungs after inhalation of ozone revealed congestion and subpleural hemorrhages. Figures 3 and 4 illustrate toe effect on toe lungs of ozone inhalation a t a concentration of 33 ppm. Histological exam 1.56 X 10* 2% (mean CV). In this study toe coefficient of variation is the standard error, rather than the standard deviation, calculated as percentage of the m am For the purpose of this investigation, we considered tote way at expressing th e coefficient of variation more appropriate for assessment of reproducibility. ination of the trachea, excised immediately after exposure of toe anim al to ozone, re Comment vealed no abnorm ality th at could be detect ed with toe light microscope. The relevance of studying the epithelium Arch Environ Health--Voi 22, May 1971 NONPARTICULATE A IR PO LLU TANTS--N A 8R E T A L 542 Ml 'MUUI MW......." - - pig 3,__Lung of a normal rat (reduced from X 160). n n <j.--Transverse section In trachea of normal rat tar ramoval of the epithelium by brush method. T 'J T r i Z ^ u c w a . fw n which ciliated epithelium w a rn o t r ^ T T s h m - n (reduced from x 400). rjn 7 __Transverse section in trachea o f normal n t after removal of the epithelium by bruUi method. & ^ ! S r t WW I* 1" <*Mly *<* frm x 400). Fig 8.--EpW [ the rat (sBgf Fig 9.--Epit I d the rat. Cel itknes through Fig 4._Lung of a rat which inhaled ozone for 1 hour a t a concentration of 33 ppm (reduced from x 160). pig 5.--Transverse section in the trochee of a nor mal rat (reduced from X 400). of th e trachea could be questioned, sm* serious injury to the respiratory t o r t read from th e pulm onary rather than the tracheal lesions. T racheal epithelium is one of earlier targets of contact w ith a highly reae-1 tive in h alan t I t is biochemically active; L e t mucus and transports through] ciliary motion. Thus, although tracheal ep-1 Arch Environ Health--Vol 22, May 1971 lium 18 fa ta l in i ain st ta x ' th e tr a d oontu 4L NONPARTICULATE A IR PO LLU TANTS-NASR ET AL 543 IS - - In tn d iN of normal Hum by brush method rich dilated epithelium educed from x 400). of L c*** iK>tated from the trachea J" *"* *** W l suspension had been nassed mmimi mes through a mlcrosyrlnge (roduoad from x 400) i s ^ r - a s r , as ^ in trachea of normal Hum by brush method, (slightly reduced from i -5 ? 11.--Epithelial cells Isolated from the trachea lfli:l l S a H S S S S ?*5 S " oTM<* in a S E questioned, since] ratoiy tract results] a-than the I `him is one of with a highly i n ically active; nsports It tough tracheal i menis. Detection of a `biochemical lesion' in such a preparation may thus be more mean ingful with respect to die basic mechanism of injury caused by the inhalant in question. lium is not the site of maximal damage * 1t*tal intoi[ication takes place, it is in w ed m im portant mechanisms of defense m st to n e inhalants. Also, the epithelium 1the trachea can be isolated, relatively free mtamination by other cellular ele- Tbf* * u supported in port by re- 60,0 ta Nati" ut Institutes t " " 1 w&Snt 6-3O1-FR-06447 from NIH to the 8chool of Public Haulfl. the UniTOrity <d Mtehtani. and by American fLicer Sto ty tosbfatiaiMl grant IN-40H. The esperiroanta reported in this investigation are part. "f uws erfunutted by Dr. Near to the Univwroty of Michigan to partial fulfillment of the reqmremwto tor the degree of doctor of philosophy in industrial health. Arch Environ Healthr--Vol 22, May 1971 544 NONPARTICULATE AIR POLLUTANTS--NA8R ET AL References l Progreta in the Prevention and Control of Air Pollution: P int Report of the Secretary of Health, Education, and Welfare to the United Stetee Congreet. Senate document 92, 1968. 2. Scheel ID , Dobeogoudg OJ, Mountain JT , et a t Fbydologic, biochemical, immunologic and pathologic changes following ozone exposure. J Appl Phyriot M7-80, I960. a Buell GC, Tokiwa Y. Mueller PK: Potential croealinking agents in lung tissue: Formation and isolation after in tiw exposure to ozone. Arch Environ Health l(h213-219,1966. 4. Spencer B: A ytotogfcal basis for the bio chemical study of bronchial epithelium. J Hietochem Cytoehem 6:106-111,1968. 5. Speocar B: The biochemistry of epithebe: Hy drolytic enzymes of human bronchial epithelium. Brachem J 71:600-507, I960. 6. Spencer B: The hiochemistry of epitheKa: F.n- xyme levels in bronchial and tracheal epithelia of smokers snd nen-onchen and in bronchial carci noma. Biochem J 7&83-88,196L 7. Haagan-Smit JA, Bradley CE. Fox MM: (te n s fomaOia in the photochemical oxidation of organic substances. Industr Bngin Chem 46:2066- 2089,1953. 8. Xsberahaw IK, Ottoboni F, Cooper WC: Oxj. dents: Air quality criteria based an health effects. J Oeeup Med 10:464-480', 1968. 9. KhrtnMd M, Giel CP: Clinical manifestation, 1 ozone poisoning: Report of a new source of exposure. Amer J Med Sei 231:638-643,1966. 10. Challen P J, Hlckiah DE, Bedford J: Invest!, gstions of some health hazards in an in e rtg s, tungatsu-arc welding shop. Brit J Induetr Med 1&276-282,19SA 11- Berfcacn J , Magath TB, Hum M: The error of asttm ata of the blood call count as m | with the hemocytometer. Amer J Phyaiol 128309323,1940. 12. SriiM y J! Saltzmsn BE: Ozone toxicity ami Huhatanoee associated with its production. Arch Induetr Health 15:111-118,1957. 13. Diggie VIM, Gaga DC: The toxicity of nitro gen pentoxide. Brit J Induetr Med 11:140-144, 1964. 14 Bailsman BE: Determination of oxidants (in cluding ozone): Neutral buffered-potaasium iodide method, in Selected Methods for the Measurement <4 Air Pollutants, Interbrandh Chemical Advisory Committee, publication 999-AP11. Ctacinmti, Pub lic Health Service, 1966, pp D-l--D-5. npari