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Apparatus for administration of gaseous 14CO2 to small animals
International Journal of Applied Radiation and Isotopes, 1969, Vol. 20, pp. 157-165. Pergamon Press. Printed in Northern Ireland
Apparatus for Administration of Gaseous '*CO to Small Animals* D A V I D L. J O F T E S Cancer Research Institute, New England Deaconess Hospital, Boston, Massachusetts
(Received in revisedform 16 June 1968) It is well known that x4C-labeled inorganic compounds acutely administered have a very short biological half-life. Nearly all the 14C is breathed off as laCO~ in a few hours. I n order to study the possible effects of the increased availability of laC in the biosphere on spermatogenesis, two controlled environment chambers have been constructed. Using these chambers it has been possible to circumvent the short residence time and to expose mice for long periods to an atmosphere containing x4CO2 in one chamber, and to a control atmosphere containing the same amount ofl~CO~ in the other chamber. The 12C enters the organism via the blood carbonate pool and under these conditions remains long enough to become available for synthesis by known (and perhaps some unknown) pathways. The construction of the chambers and their control systems as well as the necessary safety precautions are described. Increases in the 14C content of tissues of exposed animals versus the tissues of control animals have been detected by liquid scintillation counting. Other radioactive gases or aerosols could be substituted for the ~aCO2 if they are available in pressure tanks or could be generated within the chambers for use in other investigations. APPAREIL P O U R L ' A D M I N I S T R A T I O N DU 14COz G A Z E U X A U X PETITS A N I M A U X I1 est bien connu que les compos4s inorganiques marquds de 14C aigu~ment administrds ont une demi-p4riode tr6s courte. La respiration 6te presque tout le 14C en la forme de ~4CO~ ea quelques heures. /kiln d'dtudier les effets possibles de la plus forte disponibilit5 de 14(3 darts la biosph6re sur la spermatogen6se, on a construit deux chambres d'environnement contr61d. En utilisant ces chambres on a pu circonvenlr la courte p4riode de r6sidence et d'exposer des souris pour de longues p4riodes ~t une atmosphSre contenant du ~4CO2 daus une chambre, at ~ une atmosph6re-tdmoin contenant la m~me quantitd de 12COz dans l'autre chambre. Le 14C entre dans l'organisme par le r~ervoir de carbonate dans le sang et sous ces conditions y reste assez longtemps pour devenir disponible ~tla synth6se par des chemins connns (et peut~tre par quelques chemins inconnus). O n d4crit la construction des chambres et de leurs syst6mes de contr61e, ainsi que des prdcautions de sdcurit6 n4cessaires. O n a observ~ par le comptage de scintillation liquide des augmentations du contenu en 14C des tissus des animaux expos6s compar5s aux tissus des animaux t4moins. D'autres gaz radioactifs ou des aerosols pourraient remplacer le 14CO2 pourvu qu'ils soient disponibles en r~servoirs sous pression ou qu'on pnisse les g4n6rer darts les ehambres afin qu'ils servent ~t d'autres recherches. AIIIIAPAT ~JIH HPHMEHEHHH FA3OOBPA3HOFO 1~CO2 HA MEJIHIIX H(HBOTHbIX Xopomo H3BeCTHO, tITO MeqeH~e x4C ~eopraHHueeR~e cOeRHHeH~ npH peaRo~ npHMeHeHHH OTJIHqaIOTCH oqeHb I~OpOTKHM nepHo~oM HoJIyBbIBe~eHHH Ha opraH~aMa. IIo~T~ Bee c0e~HHeHHH 1~(~ BLI~hlXalOTCH B BH~e 1 ~ 0 2 qepea HecHoJI/~K0 qaC0B. ~ } ~ HsyqeHHH BJ/HHHHH Ha cnepMaToreHe3Hc B o 3 p a c T a m ~ e r o B 6 H o c ~ e p e HOYlHqecTBa 14(], ~blJIH CHOHCTp y H p o B a n ~ 7~Be HaMep~,I C peryzmpyeMoi~ cpe~oi~. YIpI4MeHenne 9THX HaMep ;iaJio SOaM0h~-
HOCT~ y;~JmHHT~, aTOT nepno~ n ~epmaT~, M~mei~ ;~om,me B O~HOtt ~aMepe c aTMoceepoi~, co~ep~ameli uCO2, a SOHTpO~bHHX--B ~pyroiI, c ~on~po~,HOfl aTMoceep01L co~ep~a~el~ * This investigation was supported by U.S. Public Health Service Grant RH-243 with the New England Deaconess Hospital. 157
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David L. Joftes TaKoe me HOnH'-IeCTBO1~(~02. 14C nocTynaeT B opraHnsM qepe3 yroae~Hcaoe pycao HpOBH, ocTasagc~, B ~THX yCaOBH.X R0CTaTOqHO K0aro R~IA CHHTeea HSBeCTHI,IMH (a MO~eT HeRoTOBIpMH eme HeHsBeCTHMMH)HyTHMH. OHIICMBaeTCHROHCTpyRI~HHH CHOTeMI,IperyaHpOBRH HaMep, a TaRme Heo6xo~nMI,ie MepM Hpo~OCTOpOH~HOCTH. Y[OBt~HIeHHecoRepmaHHH xaC B TI-~aHHXHcnt,ITyeMI,IX ~nHBOTHIxIXno cpaBHeHHm C coKep~aHneM ero B TRaHHX ROHTpOaBHhIX ;~ICHBOTHLIX onpeRea~eTca H'~H~I(OCTHI)IMC~HHTHJIaln~HOHHIaIM ctIeTtIHI~OM. BMeCTO ~a(~Oz MOH(HO Mcn0aI,3OBaTt, 7~pyrne pa3~oaRTnBHHe rae~,~ Man aap03oaH, ecan OHH MMeIOTCH B 6aRax no~ RaBneHneM, nnH nx MOP,nO renepHpoBaT5 BHyTpH KaMep n npMMeHHTB RJIH ~pyrnx nccaeKoBaHni~. E I N R I C H T U N G Z U R V E R A B R E I C H U N G V O N 14CO~ ALS GAS AN K L E I N T I E R E Es ist bekannt, dass laC-markicrtc unorganischc Vcrbindungcn bci kurzzeitiger Vcrabreichung cine schr kurze biologische Halbwcrtzeit haben. Fast das ganzc 14C wird als 14COz in wenigert Sturtden ausgeatmet. U m die m6gllchen Wirkungen einer gr6sseren Verffigbarkeit 14G in tier Biosphiire auf die Spermatogenese zu studieren, wurden zwei regelbare Gaskammern gebaut. Mit diesen Kammern wurde es m6glich die kurze Verweiheit zu umgehen und M~iuse auf liingere Zeit einer Atmosphiire mit einem Gehalt von riCO2 in einer Kammer und einer geregelten Atmosphiire mit demselben Gehah vo~ l'COn in der anderen Kammer auszusetzen. Das xaC erreicht deu Orgarfismus fiber den Blutkarbonatvorrat und verweilt unter diesen Bedingtmgen lartge genug, um zur Synthese durch bekannte (und vielleicht einige urtbekannt) Pfade verftigbax zu sein. Die Ausffihrung der Kammern und ihre Regelsysteme und auch die notwendigen Sicherheitsmassnahmen werden beschrieben. Ein Ansteigen des xaC Gehahes im Gewebe der bestrahltert Tiere gegenfiber dem Gewebe von Vergleichstieren wurde durch Fltissigkeits-Szintillationszahlung entdeckt. Andere radioaktive Gase ocler Aerosole kSnnten anstelle von x4COa verwendet werden, falls sic in Druckzylindern erhiiltlich sind oder in den Kammern zur Verwendung in anderen Uutersuchungea erzeugt werden k6rmten.
IN ORDER to s t u d y some o f t h e effects on future generations o f t h e increased a v a i l a b i l i t y o f r a d i o c a r b o n in t h e biosphere, (1-3) a p a i r o f controlled environment chambers have been b u i l t a n d a r e n o w in o p e r a t i o n . W h i l e t h e r e has b e e n m u c h s p e c u l a t i o n on t h e effects o f fallout r a d i a t i o n on future g e n e r a t i o n s b y C R O W (4), PAULING (5), LEIPUNSKY (6), a n d others, it is v e r y difficult to do definitive research in this area, a n d t h e r e is v e r y little definite inf o r m a t i o n . O n e a r e a in w h i c h i n f o r m a t i o n c a n b e a c c u m u l a t e d r e l a t i v e l y q u i c k l y is the cytogenetic effect o f 14C i n c o r p o r a t i o n into mouse testicular tissue. T h e mouse testis was selected b e c a u s e a g o o d d e a l o f cytogenetic c h a r a c t e r i z a t i o n has a l r e a d y been d o n e on it. T h e c h a m b e r s w h i c h h a v e b e e n c o n s t r u c t e d m a k e it possible to expose mice in a n e n v i r o n m e n t cont r o l l e d w i t h respect to t e m p e r a t u r e , h u m i d i t y . a n d gas c o n t e n t as well as r a d i o a c t i v i t y . O n e c h a m b e r is used to p r o v i d e t h e e x p e r i m e n t a l e n v i r o n m e n t , w h i c h consists o f a n a t m o s p h e r e to w h i c h a m e a s u r e d a m o u n t o f 14CO2 is a d d e d as a gas, a n d t h e a t m o s p h e r e o f the second c h a m b e r has a d d e d to it r i C O 2 in similar quantities, to serve for the c o n t r o l animals. I t is
well established t h a t air C O 2 (as distinguished from m e t a b o l i c CO2) c a n e n t e r t h e b l o o d b i c a r b o n a t e p o o l v i a t h e lungs despite t h e n e t outflow (7's) a n d b e fixed in t h e tissues b y m a n y b i o c h e m i c a l reactions. ( a - m A c c o r d i n g to BIANCO, GIUSTINA a n d LAZAVINI (12), C O 2 is a d i r e c t p r e c u r s o r o f D N A . I t was t h o u g h t t h a t it w o u l d b e m o r e c o n v e n i e n t to a d m i n i s t e r 14C as gaseous 14CO2, for c h r o n i c e x p e r i m e n t s w h i c h w o u l d s i m u l a t e n a t u r a l conditions m o r e closely than other methods of administration, and e l i m i n a t e the p r o b l e m o f a4CO2 b e i n g b r e a t h e d off r a p i d l y as occurs w i t h o t h e r m e t h o d s of administration. V e r y little w o r k has b e e n d o n e a d m i n i s t e r i n g 14CO~ v i a the lung, b u t m a n y studies h a v e b e e n p e r f o r m e d in w h i c h l a b e l e d i n o r g a n i c c a r b o n c o m p o u n d s were i n j e c t e d or fed. T h e results a r e p r e t t y m u c h t h e same b y either route. I n all cases v e r y r a p i d loss o f n e a r l y all 14CO~ v i a t h e lungs was observed. B U C H A N A N (7), w h o d i d the o n l y r e l a t i v e l y l o n g - t e r m studies i n v o l v i n g x4CO2 i n h a l a t i o n , c a l c u l a t e d t h a t o n l y 10-4-3 × 10 -5 o f tissue c a r b o n was fixed from air C O 2 o v e r a 3 4 - d a y p e r i o d . H e o b s e r v e d v a r i a t i o n s from
FIG. 1. View of experimental chamber showing the air lock on left end, glove ports and covers, sampling chamber and gas scrubbing towers. The tank of 14CO~ contaminated 12CO~, the control panel and clock, on the back of which panel are mounted the control and safety relays, and the freezer are all visible at the right. Pressure gauge is visible between the glove ports.
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FIG. 2. View of control c h a m b e r showing freezer a n d humidifier system to left. O n the floor are seen the air compressor a n d tank a n d the n a t u r a l C O 2 supply tanks a n d manifold. T h e heat exchanger is fastened u n d e r n e a t h the table above the compressor.
Apparatusfor administration of gaseous 14G0 2 t0 small animals
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directs the air over a reservoir of water kept at a constant level by means of a float valve connected to the building cold water supply. The temperature of the water is maintained at approximately 35°C by DESIGN OF EXPOSURE APPARATUS means of an electric immersion heater under thermoSince long exposure intervals were anticipated, it static control. Each chamber has its own humidifier was thought that neither a closed system nor a gas recycling system would be suitable for the studies system. A lithium chloride gold grid humidity contemplated. It was known that animals would sensor [22] in each chamber operates a relay system need to be exposed to t4CO~ for periods of many [18] which actuates the control valve to direct the weeks to achieve significant gonadal radiation doses, air stream over the surface of the water when the and a closed or a recycling system which could work relative humidity of the chamber falls below a set for very long periods would be extremely complex. point, or bypasses the reservoir when the relative The continuous flow system decided upon most humidity exceeds another set point. Signals from the easily permits a fresh supply of oxygen, sweeps out humidity sensor cart be temporarily diverted manumetabolic COa as well as ammonia fumes, and allows ally from the relay system to an external meter by means of self-restoring switches so that visual checks addition of 14CO2 at a controlled known rate. The apparatus which has been constructed consists on the relative humidity can be made at will. The of two 320 1. rectilinear tanks made of 25 mm thick air, now conditioned so that it will maintain 45-52 Plexiglas, with sealable glove ports, an air lock sys- per cent relative humidity within each chamber, tem, and internal drinking water and electricity then passes through a check valve [6] intended to supplies (Figs. 1 and 2). The description will be to prevent a reversal of direction, and continues past more easily followed with the block diagram (Fig. the orifice of a branch line (this line will be described 3). * All plastic joints are stepped and have a bead later) through which a mixture of 12CO2 and x4CO2 or I2COz alone is added and into a mixing chamber which is triangular in cross-section cemented inside all corners, as well as 6 mm strips of Plexiglas [7] designed to mix the gases thoroughly by turbucemented over the raw edges, so that leaking gas lence. The turbulence is created by dividing the would have to traverse at ieast 6.3 cm of cemented mixing chamber into three compartments by two seam before escaping (see Item 34 of Fig. 3). I n plastic plates containing staggered 6 mm dia. holes. operation a heavy-duty air compressor [1] with a A needle valve [12] in the last compartment of the capacity of more than 100 1. per mln pumps air mixing chamber permits samples of gas to be drawn successively through a copper coil heat exchanger [2] off for testing. When the gas leaves the mixer, it (to dissipate the heat resulting from compression) and passes through a flowmeter [13] and then a main two stainless steel boxes baffled with stainless steel shut-off valve [14]. This valve opens into the end wool, which are contained in a freezer [3] at --25°C of a 3.1 cm i.d. Plexiglas cylinder which runs the where the moisture is frozen out of the air. An addi- length of the interior rear upper corner of the main tional pair of boxes are available to be substituted at chamber. This cylinder is sealed at its opposite end weekly intervals for those in use by quick-connect and has a narrow slit running its whole length, which fittings, while the ones previously in use are being slit is directed downwards and to the rear of the thawed, emptied, and dried. The quick-connect chamber [15], forcing the air (which at this point fittings are defrosted twice daily by heating tapes still has considerable velocity) against the rear wall which are wrapped around the fittings and controlled and floor of the chamber and diffusing it thoroughly. by automatic timers. During the summer when the The air leaves via an orifice placed in a front upper relative humidity is high, the air entering the com- corner of the chamber, through another shut-off pressor is drawn through an ordinary room air valve [25], and enters a sampling chamber [27] where dehumidifier. Upon leaving the freezer, the gas line another needle valve [28] makes it possible to sample divides into two essentially identical lines, one to the effluent gas mixture. From the sample chamber carry the control gas supply and the other the radio- the gas is directed through a second ftowmeter [26] active gas supply. A vacuum breaker installed just to measure outflow and assure that input and Output before the gas line division insures that should the are in the desired relationship. The gas leaving the compressor fail while the exhaust pump is working, flowmeter then passes through a two-way valve [29] which allows the gas to he passed to either of two no high vacuum will develop in the system. The air next enters a humidifying system [5] con- identical gas scrubbing tower systems [30, 30a]. Each sisting of a two-way control valve which bypasses or scrubbing tower system consists of three Plexiglas cylinders. Each cylinder can contain 1.5 1. of con• Numbers in square brackets refer to numbered centrated K O H solution. The effluent gas bubbles items in Fig. 3. through each cylinder in turn by means of four
tissue to tissue i n both the final fraction a n d the time at w h i c h e q u i l i b r i u m was achieved.
David L. Joftes
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33 Fro. 3. Block diagram of 14CO s and control inhalation chambers ("a" indicates control chamber items) 1 Air compressor 19, 19a Thermostats 2 Heat exchanger 20, 20a Water supply with automatic valves 21 Air lock with purge valves 3 Freezer 22, 22a Lithium chloride gold grid humidity 4, 4a Heaters 5, 5a Humidifiers sensors 23, 23a Thermometers 6, 6a Check valves 7, 7a Mixing chambers 24, 24a Pressure gauges 25, 25a Main shut off valves, output 8, 8a Flow meters 26, 26a Flow meters showing bypass lines and 9, 9a Solenoid safety control valves valves 10, 10a Alarm relays controlling solenoids 11 14CO2 contaminated CO= supply 27, 27a Sampling chambers 28, 28a Valves for sampling output gas mixture 1l a Natural CO S supply 29 Three-way valve 12, 12a Valves for sampling input gas mixture 30 Triple gas scrubbing towers (double system) (from mixing chambers) 30a Triple gas scrubbing towers (single system) 13, 13a Flow meters 31, 31 a Vacuum line valves 14, 14a Main shut off valves, input 32 Vacuum pump 15 Chamber containing radioactive CO S 33 Vent to fume hood flue 16 Control chamber 34 Detail--cross-section through cemented plastic 17, 17a Glove port covers with purge valves joint showing how 6-5 cm leak path is achieved (gloves not indicated) in joining 2.5 cm stock. 18, 18a Humidistats
Apparatusfor administration of gaseous J-4C02 to small animals polyethylene filter candles attached to a stainless steel manifold inside the cylinder. All the cylinders are provided with individual stainless steel valves at the bottom for drawing off exhausted K O H solution and have stainless steel plugs at the top which can be removed to add fresh K O H solution. As noted above there are two systems so that one can be cleaned and recharged while the other is absorbing the CO 2 from the effluent gas mixture without shutting down. The gas leaving the scrubbing towers passes through a needle valve [31] used to control the rate of outflow and is exhausted through a vacuum pump [32] into a pipeline [33] carried up through a fume hood flue and out of the building, so that the minuscule amounts of 14CO2 which might evade K O H absorption will be safely diluted and diffused in the atmosphere. The exhaust line is wrapped in a thermostated heating cable so that condensed moisture does not freeze in it when the temperature drops to freezing. The room air and the effluent air are regularly monitored so that unsafe levels of x4CO2 will be detected should they occur. The 14CO2 branch llne, mentioned but not described above, leads from a tank of x~CO~ [11] which has been contaminated with the desired amount of a4CO2 (specific activity O.0485pc/mM) through the usual reduction valve to a normally closed solenoid valve [9] which is actuated to remain open unless a variety of unsafe conditions develop, in which case the current is cut offby means of a relay system [10], and the valve automatically closes to stop the flow of x4CO2. Under normal operating conditions, the gas flows from the solenoid valve through a flowmeter [ 13], which constantly monitors the rate of flow, and then it enters the main air stream just before the mixing chamber and continues as described above. An essentially identical branch line carries ordinary CO 2 from tanks [ 11a] to the control chamber air line. For ease in manipulating animals and supplies inside the chamber, glove ports are provided which are sealed by removable 2.5 cm Plexiglas covers [17] with neoprene " O " ring seals. Bolts fixed in the chamber walls and wing nuts every two inches around the perimeter of the covers insure a tight seal of the " O " rings which are let into the covers. Valves through the Plexiglas port covers make it possible to blow out and absorb any 14CO2 which passes through the neoprene gloves and collects inside them. This flushing is done routinely before unsealing the ports. An air lock [21] is used for passing animals and materials in and out of the chamber without permitting radioactive gas to escape into the room. The air lock is also provided with purge valves so that the radioactive gas may be blown out and absorbed before the outer door is opened. Both inner and outer air lock doors are sealed with neoprene
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" O " rings and are also fitted with bolts every 2 in. around their perimeters. All bolts are 1/4 in.-20 and have an individual neoprene " O " ring mounted under the bolt head where it bears against the plastic wall. Another chamber [16] identical with the one described, except that it has no air lock, and is provided with only a single set of gas scrubbing towers, is used as a control chamber. In this case the scrubbing towers function primarily to provide the same resistance to air flow as the experimental chamber scrubbing towers. It has been so designed that an air lock and additional scrubbing towers can be added should they prove to be necessary at a later date. The control chamber is supplied with ordinary CO 2 with only then aturally occurring amount of CO~ in it, and the rate of addition is the same as that of the radioactive gas, so that the total CO 2 content is as nearly the same as possible in both chambers. The chambers are warmed by external electric radiators placed close to the rear wall of each chamber. Each radiator is controlled by a thermostat inside its own chamber. Cooling is accomplished by maintaining the ambient room temperature a few degrees below the desired chamber temperature. In practice it has been found that the animals provide enough heat to maintain a higher temperature than the desired 22-23°C, so that cooling is necessary nearly all year round. Both chambers have a volume of 320 1., and the air flow through each is adjusted to 32 l./min so that a complete exchange of air occurs every I0 min. In order to overcome the reduction in flow rate due to constrictions in the lines, such as the small apertures of the flowmeters and the filter candies used as air bubblers, input air is pumped at a pressure of about 1½ to 2 p.s.i. The pressure inside the chambers is reduced by pumping air out at a slightly greater rate than that at which it is entering. Chamber pressure is kept between 1-4 mm Hg pressure below atmospheric so that if any leaks should occur, air is drawn into the chambers instead of escaping from them. Mercury manometers [24] installed through the front wall of each chamber monitor the internal pressure. SAFETY W h e n the construction o f these c h a m b e r s was first c o n t e m p l a t e d , the i d e a o f using Plexiglas was rejected since it is k n o w n t h a t this plastic p er m i t s C 0 2 to diffuse t h r o u g h it. H o w ever, o t h e r m at er i al s p r esen t ed m a n y o t h e r d r a w b a c k s w i t h o u t t h e t r a n s p a r e n c y a n d insulative v a l u e o f Plexiglas. F i n a l l y the a m o u n t w h i c h c o u l d diffuse t h r o u g h the plastic was c a l c u l a t e d (using a f o r m u l a supplied b y t h e
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David L. Joftes
manufacturer) for a chamber of the dimensions which it was proposed to build. I t turned out that for every 1 cm H g positive differential pressure, 9 mg per day of C O s could diffuse out of the chamber if C O 2 concentrations are the same inside and out. T h e chamber is intended to operate under fairly large increases of internal C O s concentration over that occurring naturally, and at a slight negative pressure. If, for argument's sake, a pressure of 1 cm H g and a five-fold higher internal concentration of C O s are assumed, a m a x i m u m of 50/~c 14C/day would be released into the room air along with the nonradioactive C O S. AEC regulations governing the permissible levels of 14C in this room (which has an 1100 cfrn air exchange) allow a release of 28 t~c/min. It was thought that while the 50 tic/day would certainly be acceptable, it would not be necessary to tolerate even this exposure if the chamber pressure were kept slightly negative, since the diffusion outward would be much decreased under negative pressure. I n practice only a few cpm/1, attributable to 14C were ever detected in the room air. It is necessary to provide a variety of safety devices to guard against several contingencies in order to assure the safety of personnel as well as the well-being of the animals in the chambers. T h e method selected to prevent excursion of gas from the chamber into the atmosphere depends upon control of a solenoid valve which is installed in each of the C O s branch lines immediately after the gas tank reduction valves. These solenoids are normally closed, and require electricity to remain open. A relay system operates to stop the flow of current to the solenoid, and thereby shut off the flow of COs: if there is failure of the room ventilation system; if there is an increase in pressure in the chamber to 1 cm of mercury; if there is failure of the x4CO2 tank reduction value; or if either the compressor or exhaust p u m p fails to function. Failure of the room ventilation is sensed by a flap in the exhaust flue of the room, which flap falls of its own weight when insufficient air is being drawn through the flue, and activates a microswitch. An increase in the chamber pressure is signalled by the rising mercury column of the manometer closing a circuit which consists of a pair of insulated platinum wires, the
uninsulated tips of which are positioned half a centimeter above the normal position of the mercury column on the up side of the m a n o m eter. Failure of the reduction valve on the 14CO 2 tank is sensed by a pressure switch which is set to activate the relay system if there is an increase in pressure in the line above 20 psi. Normal operating pressure is 7-9 psi in this line. I f the exhaust p u m p fails to operate, chamber pressure will increase and activate the relay system via the manometer. Failure of the compressor is monitored by a pressure switch which responds to the loss of air pressure. However, a vacuum breaker is installed in the main air line before it bifurcates, so that if the compressor fails but the exhaust p u m p continues to operate, air will be drawn into the system in suffÉcient quantity to prevent a v a c u u m of greater than 2 cm mercury from developing. U n d e r these conditions the animals would receive fresh air but the addition of C O 2 would be stopped. A general power failure would prevent operation of the relay system, but will also result in the closing of the solenoid valves, and so continued addition of C O s to the chambers will be prevented. T h e relay system is so arranged that if any one of the conditions mentioned above develops, both the 14CO2 and a~CO 2 solenoid valves will be closed and an electric clock will be stopped simultaneously. This is done so that control and experimental animals will be treated as m u c h alike as possible, even in the event of malfunction. T h e stoppage of the electric clock indicates how long the malfunction has continued. Should the malfunction correct itself, the clock starts again, but its discrepany with correct time will indicate the length of time of the malfunction. Diagnostic signal lights indicate proper function (amber) or malfunction (red) of various component circuits including the radiators, the freezer, and the humidifier systems. W h e n a malfunction occurs, a specific red signal lights and a buzzer sounds. These signals continue even if self-repair occurs, until a clear switch is operated manually. OPERATING CONDITIONS T h e experimental and control chambers have been in satisfactory operation since December, 1965. Each chamber holds 4 individual cages
Apparatusfor administration of gaseous a4C02 to small animals containing 4 or 5 mice each, or a total of 16 or 20 mice in each chamber. T h e r e have been some minor breakdowns which caused involuntary shutdowns of less than 2 hr each, and it is thought that in no case were these shutdowns significant to the results. During operation, rate of air input and output from the chambers and radioactive and normal C O 2 input are continually monitored by predictably flow meters. T h e degree of v a c u u m m a y be read at will from mercury manometers; relative humidity m a y be read at will as described above, and temperature m a y be read on dual thermometers installed in each chamber. All measurements are recorded twice daily on check sheets. Total C O s content of the input and effluent air are determined every other day by means of infrared absorption using a 1-m folded p a t h cell. At the same time 14C content is measured by absorption of C O S from samples of the input and effluent air into hyamine, aliquots of which are then submitted to scintillation counting. T h e room air before start-up was at background with respect to 14C content and contained approximately 0.04 per cent C O 2. T e n days after start-up it contained 7 dpm/1 above background; and 6 months later, after almost continuous operation, it had reached only 33 dpm/l. This level has never been exceeded. Rather, the level has dropped considerably to average 11 dpm/1. T h e m a x i m u m permissible level has been calculated to be 2,220 dpm/1 for the room in which the chambers are housed ~13). This room has a nominal volume of approximately 1300 ft 3 and an air exchange of approximately I I00 cfm. T h e chambers are operated at a temperature of 23-25°C. Pressure is maintained at approximately - - 4 m m Hg. T h e nominal chamber volume is 320 1. T h e input volume is 32.6 1./ min which allows for a total air exchange at least 6 times every hour in both the experimental and control chamber. O u t p u t volume is approximately 33.5 1/min. This removal at a greater rate than input allows us to maintain the negative pressure. In terms of the radioactivity input the experimental chamber receives 3,500 dpm/1 on the average during a normal run under present conditions. T h e control chamber gas is at
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background. T h e output from the experimental c h a m b e r is approximately the same, 3,500 dpm/l, indicating that the amount taken out by the animals is less than can be detected b y our method of air sampling. T h e control output is at background. These values have been obtained by scintillation counting and are within 10 per cent of the values calculated on a theoretical basis. Both the experimental and control chambers receive approximately 34 ml per min. of C O 2. This includes both the naturally occurring C O s in the air being p u m p e d in and the additional C O , containing the 1~C being added in the case of the experimental chamber or additional natural C O s being added in the case of the control chamber. I n this run the C O 2 input in both chambers averaged approximately 0.12 per cent and the output averaged 0.18 per cent from both chambers. T h e additional C O 2 in the output air is metabolic C O 2 breathed off by the animals. This means that the animals are living in an atmosphere where the C O , content is approximately 4½ times that of the free atmosphere in the Boston area. T h e 4½-fold increase in C O s concentration does not yield an appreciable increase in p C O 2. While this m a y seem surprising, H A L D ~ . and PRIESTLY(14) showed in 1905 with h u m a n subjects that an approximately 26-fold increase in inspired CO~ concentration from 0.03 to 0.79 per cent did not change the alveolar p C O a within their experimental error of 1 m g Hg, although there was a slight increase in tidal air and pulmonary ventilation without an increase in respiratory rate. Alveolar C O 2 concentration did not change. It seems reasonable that for this physiological response we m a y extrapolate from m a n to mouse under these conditions. BUCm~AN t~) has indicated and we have confirmed that only a negligible fraction of the isotope is removed by the animal from the throughput air. BUCHANANc7~ also indicated that total C O 2 concentration in the range under discussion had no effect on the amount of 14C retained. U n d e r these conditions an animal breathing a concentration of 0.04 per cent CO~ therefore will still have an alveolar concentration of 5-5 per cent which is an increase in concentration of the ambiant to alveolar air C O S of a factor of 137.5. O n the basis of HALDAN~ and
164
David L. Joftes
PRIESTLY’S(~~) work, we can assume that the mice in the chambers are maintaining an alveolar CO, concentration of 5.5 per cent, or a factor of increase of approximately 30.5 over the CO, at O-18 per cent which they are breathing. With respect to the amount of 14C in the chambers, the stock supply of 14C0, contained very close to 0.002 &ml. The input per min of 2 1 ml CO, would, therefore, contain O-042 ,UC. The total 14C in the chamber at any instant after the first 10 min of gas addition would then be 0.42 ,UC. The specific activity in the 14C supply is O-0485 ,uc/mM. If effect is given to the dilution from the input air and the animal metabolic contribution, the chamber content has a specific activity of 0.03 pc/mM carbon. In the alveoli the CO, of the inspired air is further diluted by alveolar CO, to a specific activity of O*OOl ,uc/mM (computed). Based on the increase of CO, in the output gas it has been computed that the animals are putting out 3.04 ml (STP)/g/hr CO,. This compares favorably with BUCHANAN’S(~)figures of 3-3.3 ml (STP)/g/hr and thus adds confidence that our infrared measurements of CO, are accurate. ANIMAL
MAINTENANCE
Animals have been kept alive and in good health in the chambers for periods of 8-10 weeks. In the first test run when animals were kept for a period of 3 months without changing the cages in the chambers many of them developed a Pseudomonas infection of the urinary tract which resulted in urinary retention as evidenced by bladder distention. To prevent recurrence of this condition it is now routine to change the animals’ cages at two-week intervals or more frequently if indicated. The usual type of water bottle is used with a special stainless steel spout which contains 2 ball bearings, the lower of which fits closely against the in-curved opening of the spout. The upper bearing simply adds weight to insure a tight fit. This reduces dripping in response to the minor pressure changes which occur in the chambers. There is a cafeteria style water valve [20] installed in each of the chambers so that, using the gloves, the water bottles can be refilled whenever required. The animals are fed a commercial pelleted
dry food with no supplements of any kind. No gross evidence of vitamin deficiency has been observed even in the animals which remained in the chambers for 3 months. A large supply of the food is placed in a container in each of the chambers with the animals at the start of a run and more food is brought in through the airlock as needed. Food is added to the hoppers in the stainless steel cage tops as necessary, by means of the gloves. The cages used are of a solid bottom, clear plastic, nesting type. The bottom of one cage is sawed off and fitted with galvanized hardware cloth screen having approximately 4 wires per in. in both directions, in place of the original bottom. This modified cage is then placed in an intact cage which contains a commercially available absorbant bedding material which is treated to minimize urinary ammonia formation and reduce odor. The animals live on the screen and do not come in contact with the bedding. In practice either the top or bottom of the double cage unit is changed as required. The fresh and used cages enter and leave the experimental chamber via the air-lock so operation need not be suspended. The control chamber door is removed when necessary without stopping operations. REFERENCES 1. LIBBY W. F. Science, N.Y. 122, 57 (1958). 2. TOTTERJ. R., ZELLE M. R. and HOLLISTERH. Science, N. Y. 128, 1490 ( 1958). 3. LIBBY W. F. Statement delivered before the Federation of American Scientists, Washington, D.C. (1958). 4. CROW J. F. Hearings before the Special Subcommittee on Radiation of the Joint Committee on Atomic Energy, Congress of the U.S., 85th Congress, First Session on The Nature of Radioactive Fallout and Its Effects on Man, June 4, 1957, pp. lOG9-1028. 5. PAULINGL. Science, N.Y. 128, 1183 (1958). 6. LEIPUNSKY 0. I. Quoted in TOTTERet al. (Ref. 2). 7. BUCHANAN D. L. J. gen. Physiol. 34, 737 (1951). 8. BRUES A. M. and BUCHANAN D. L. Cold Spring Harb. Symp. pant. Biol. 13, 52 (1948). 9. KREBS H. A. Symposium, Sot. exptl Biol. 5, 1 (1951). 10. EVANSE. A. JR. Harvey Lectures 39, 273 (1944). 11. COHEN S. Exptl Cell Res. 29, 207 (1963). 12. BIANCOL., GIUSTINAG. and LAZAVINIE.Nature, Lond. 194 (4825), 289( 1962).
Apparatus for administration of gaseous 1~C0~ to small animals 13. Code of Fed. Reg, USAEC Title 10, Part 20. Standards for Protection Against Radiation. Appendix B, Table II. 14. I~LDANE J. S. and PRmSa'LEYJ. G. 2". Physiol. 32, 225 (1905).
ADDENDUM--PARTS LIST No attempt has been made in this description to specify or designate precisely the manufacturer and dimensions of the many parts used in building the
165
chambers. However, all parts used were commercially available from electrical, plumbing, or air conditioning supply houses. The humidity controls were purchased from the Honeywell Corporation of Minneapolis, Minnesota. The dimensions of the air supply line and valves should be as large as possible consistent with economy. On the input side we used 1/4 in. (6 ram) plastic tubing of 2 in. (5 cm) i.d. The copper tubing coil used as a heat exchanger has a diameter of 3/4in. (18 ram) i.d. Valve apertures were at least 1/4 in. (6 ram). On the output side we used 3/4 in. soft copper tubing throughout.
Apparatus for Administration of Gaseous '*CO to Small Animals* D A V I D L. J O F T E S Cancer Research Institute, New England Deaconess Hospital, Boston, Massachusetts
(Received in revisedform 16 June 1968) It is well known that x4C-labeled inorganic compounds acutely administered have a very short biological half-life. Nearly all the 14C is breathed off as laCO~ in a few hours. I n order to study the possible effects of the increased availability of laC in the biosphere on spermatogenesis, two controlled environment chambers have been constructed. Using these chambers it has been possible to circumvent the short residence time and to expose mice for long periods to an atmosphere containing x4CO2 in one chamber, and to a control atmosphere containing the same amount ofl~CO~ in the other chamber. The 12C enters the organism via the blood carbonate pool and under these conditions remains long enough to become available for synthesis by known (and perhaps some unknown) pathways. The construction of the chambers and their control systems as well as the necessary safety precautions are described. Increases in the 14C content of tissues of exposed animals versus the tissues of control animals have been detected by liquid scintillation counting. Other radioactive gases or aerosols could be substituted for the ~aCO2 if they are available in pressure tanks or could be generated within the chambers for use in other investigations. APPAREIL P O U R L ' A D M I N I S T R A T I O N DU 14COz G A Z E U X A U X PETITS A N I M A U X I1 est bien connu que les compos4s inorganiques marquds de 14C aigu~ment administrds ont une demi-p4riode tr6s courte. La respiration 6te presque tout le 14C en la forme de ~4CO~ ea quelques heures. /kiln d'dtudier les effets possibles de la plus forte disponibilit5 de 14(3 darts la biosph6re sur la spermatogen6se, on a construit deux chambres d'environnement contr61d. En utilisant ces chambres on a pu circonvenlr la courte p4riode de r6sidence et d'exposer des souris pour de longues p4riodes ~t une atmosphSre contenant du ~4CO2 daus une chambre, at ~ une atmosph6re-tdmoin contenant la m~me quantitd de 12COz dans l'autre chambre. Le 14C entre dans l'organisme par le r~ervoir de carbonate dans le sang et sous ces conditions y reste assez longtemps pour devenir disponible ~tla synth6se par des chemins connns (et peut~tre par quelques chemins inconnus). O n d4crit la construction des chambres et de leurs syst6mes de contr61e, ainsi que des prdcautions de sdcurit6 n4cessaires. O n a observ~ par le comptage de scintillation liquide des augmentations du contenu en 14C des tissus des animaux expos6s compar5s aux tissus des animaux t4moins. D'autres gaz radioactifs ou des aerosols pourraient remplacer le 14CO2 pourvu qu'ils soient disponibles en r~servoirs sous pression ou qu'on pnisse les g4n6rer darts les ehambres afin qu'ils servent ~t d'autres recherches. AIIIIAPAT ~JIH HPHMEHEHHH FA3OOBPA3HOFO 1~CO2 HA MEJIHIIX H(HBOTHbIX Xopomo H3BeCTHO, tITO MeqeH~e x4C ~eopraHHueeR~e cOeRHHeH~ npH peaRo~ npHMeHeHHH OTJIHqaIOTCH oqeHb I~OpOTKHM nepHo~oM HoJIyBbIBe~eHHH Ha opraH~aMa. IIo~T~ Bee c0e~HHeHHH 1~(~ BLI~hlXalOTCH B BH~e 1 ~ 0 2 qepea HecHoJI/~K0 qaC0B. ~ } ~ HsyqeHHH BJ/HHHHH Ha cnepMaToreHe3Hc B o 3 p a c T a m ~ e r o B 6 H o c ~ e p e HOYlHqecTBa 14(], ~blJIH CHOHCTp y H p o B a n ~ 7~Be HaMep~,I C peryzmpyeMoi~ cpe~oi~. YIpI4MeHenne 9THX HaMep ;iaJio SOaM0h~-
HOCT~ y;~JmHHT~, aTOT nepno~ n ~epmaT~, M~mei~ ;~om,me B O~HOtt ~aMepe c aTMoceepoi~, co~ep~ameli uCO2, a SOHTpO~bHHX--B ~pyroiI, c ~on~po~,HOfl aTMoceep01L co~ep~a~el~ * This investigation was supported by U.S. Public Health Service Grant RH-243 with the New England Deaconess Hospital. 157
158
David L. Joftes TaKoe me HOnH'-IeCTBO1~(~02. 14C nocTynaeT B opraHnsM qepe3 yroae~Hcaoe pycao HpOBH, ocTasagc~, B ~THX yCaOBH.X R0CTaTOqHO K0aro R~IA CHHTeea HSBeCTHI,IMH (a MO~eT HeRoTOBIpMH eme HeHsBeCTHMMH)HyTHMH. OHIICMBaeTCHROHCTpyRI~HHH CHOTeMI,IperyaHpOBRH HaMep, a TaRme Heo6xo~nMI,ie MepM Hpo~OCTOpOH~HOCTH. Y[OBt~HIeHHecoRepmaHHH xaC B TI-~aHHXHcnt,ITyeMI,IX ~nHBOTHIxIXno cpaBHeHHm C coKep~aHneM ero B TRaHHX ROHTpOaBHhIX ;~ICHBOTHLIX onpeRea~eTca H'~H~I(OCTHI)IMC~HHTHJIaln~HOHHIaIM ctIeTtIHI~OM. BMeCTO ~a(~Oz MOH(HO Mcn0aI,3OBaTt, 7~pyrne pa3~oaRTnBHHe rae~,~ Man aap03oaH, ecan OHH MMeIOTCH B 6aRax no~ RaBneHneM, nnH nx MOP,nO renepHpoBaT5 BHyTpH KaMep n npMMeHHTB RJIH ~pyrnx nccaeKoBaHni~. E I N R I C H T U N G Z U R V E R A B R E I C H U N G V O N 14CO~ ALS GAS AN K L E I N T I E R E Es ist bekannt, dass laC-markicrtc unorganischc Vcrbindungcn bci kurzzeitiger Vcrabreichung cine schr kurze biologische Halbwcrtzeit haben. Fast das ganzc 14C wird als 14COz in wenigert Sturtden ausgeatmet. U m die m6gllchen Wirkungen einer gr6sseren Verffigbarkeit 14G in tier Biosphiire auf die Spermatogenese zu studieren, wurden zwei regelbare Gaskammern gebaut. Mit diesen Kammern wurde es m6glich die kurze Verweiheit zu umgehen und M~iuse auf liingere Zeit einer Atmosphiire mit einem Gehalt von riCO2 in einer Kammer und einer geregelten Atmosphiire mit demselben Gehah vo~ l'COn in der anderen Kammer auszusetzen. Das xaC erreicht deu Orgarfismus fiber den Blutkarbonatvorrat und verweilt unter diesen Bedingtmgen lartge genug, um zur Synthese durch bekannte (und vielleicht einige urtbekannt) Pfade verftigbax zu sein. Die Ausffihrung der Kammern und ihre Regelsysteme und auch die notwendigen Sicherheitsmassnahmen werden beschrieben. Ein Ansteigen des xaC Gehahes im Gewebe der bestrahltert Tiere gegenfiber dem Gewebe von Vergleichstieren wurde durch Fltissigkeits-Szintillationszahlung entdeckt. Andere radioaktive Gase ocler Aerosole kSnnten anstelle von x4COa verwendet werden, falls sic in Druckzylindern erhiiltlich sind oder in den Kammern zur Verwendung in anderen Uutersuchungea erzeugt werden k6rmten.
IN ORDER to s t u d y some o f t h e effects on future generations o f t h e increased a v a i l a b i l i t y o f r a d i o c a r b o n in t h e biosphere, (1-3) a p a i r o f controlled environment chambers have been b u i l t a n d a r e n o w in o p e r a t i o n . W h i l e t h e r e has b e e n m u c h s p e c u l a t i o n on t h e effects o f fallout r a d i a t i o n on future g e n e r a t i o n s b y C R O W (4), PAULING (5), LEIPUNSKY (6), a n d others, it is v e r y difficult to do definitive research in this area, a n d t h e r e is v e r y little definite inf o r m a t i o n . O n e a r e a in w h i c h i n f o r m a t i o n c a n b e a c c u m u l a t e d r e l a t i v e l y q u i c k l y is the cytogenetic effect o f 14C i n c o r p o r a t i o n into mouse testicular tissue. T h e mouse testis was selected b e c a u s e a g o o d d e a l o f cytogenetic c h a r a c t e r i z a t i o n has a l r e a d y been d o n e on it. T h e c h a m b e r s w h i c h h a v e b e e n c o n s t r u c t e d m a k e it possible to expose mice in a n e n v i r o n m e n t cont r o l l e d w i t h respect to t e m p e r a t u r e , h u m i d i t y . a n d gas c o n t e n t as well as r a d i o a c t i v i t y . O n e c h a m b e r is used to p r o v i d e t h e e x p e r i m e n t a l e n v i r o n m e n t , w h i c h consists o f a n a t m o s p h e r e to w h i c h a m e a s u r e d a m o u n t o f 14CO2 is a d d e d as a gas, a n d t h e a t m o s p h e r e o f the second c h a m b e r has a d d e d to it r i C O 2 in similar quantities, to serve for the c o n t r o l animals. I t is
well established t h a t air C O 2 (as distinguished from m e t a b o l i c CO2) c a n e n t e r t h e b l o o d b i c a r b o n a t e p o o l v i a t h e lungs despite t h e n e t outflow (7's) a n d b e fixed in t h e tissues b y m a n y b i o c h e m i c a l reactions. ( a - m A c c o r d i n g to BIANCO, GIUSTINA a n d LAZAVINI (12), C O 2 is a d i r e c t p r e c u r s o r o f D N A . I t was t h o u g h t t h a t it w o u l d b e m o r e c o n v e n i e n t to a d m i n i s t e r 14C as gaseous 14CO2, for c h r o n i c e x p e r i m e n t s w h i c h w o u l d s i m u l a t e n a t u r a l conditions m o r e closely than other methods of administration, and e l i m i n a t e the p r o b l e m o f a4CO2 b e i n g b r e a t h e d off r a p i d l y as occurs w i t h o t h e r m e t h o d s of administration. V e r y little w o r k has b e e n d o n e a d m i n i s t e r i n g 14CO~ v i a the lung, b u t m a n y studies h a v e b e e n p e r f o r m e d in w h i c h l a b e l e d i n o r g a n i c c a r b o n c o m p o u n d s were i n j e c t e d or fed. T h e results a r e p r e t t y m u c h t h e same b y either route. I n all cases v e r y r a p i d loss o f n e a r l y all 14CO~ v i a t h e lungs was observed. B U C H A N A N (7), w h o d i d the o n l y r e l a t i v e l y l o n g - t e r m studies i n v o l v i n g x4CO2 i n h a l a t i o n , c a l c u l a t e d t h a t o n l y 10-4-3 × 10 -5 o f tissue c a r b o n was fixed from air C O 2 o v e r a 3 4 - d a y p e r i o d . H e o b s e r v e d v a r i a t i o n s from
FIG. 1. View of experimental chamber showing the air lock on left end, glove ports and covers, sampling chamber and gas scrubbing towers. The tank of 14CO~ contaminated 12CO~, the control panel and clock, on the back of which panel are mounted the control and safety relays, and the freezer are all visible at the right. Pressure gauge is visible between the glove ports.
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FIG. 2. View of control c h a m b e r showing freezer a n d humidifier system to left. O n the floor are seen the air compressor a n d tank a n d the n a t u r a l C O 2 supply tanks a n d manifold. T h e heat exchanger is fastened u n d e r n e a t h the table above the compressor.
Apparatusfor administration of gaseous 14G0 2 t0 small animals
159
directs the air over a reservoir of water kept at a constant level by means of a float valve connected to the building cold water supply. The temperature of the water is maintained at approximately 35°C by DESIGN OF EXPOSURE APPARATUS means of an electric immersion heater under thermoSince long exposure intervals were anticipated, it static control. Each chamber has its own humidifier was thought that neither a closed system nor a gas recycling system would be suitable for the studies system. A lithium chloride gold grid humidity contemplated. It was known that animals would sensor [22] in each chamber operates a relay system need to be exposed to t4CO~ for periods of many [18] which actuates the control valve to direct the weeks to achieve significant gonadal radiation doses, air stream over the surface of the water when the and a closed or a recycling system which could work relative humidity of the chamber falls below a set for very long periods would be extremely complex. point, or bypasses the reservoir when the relative The continuous flow system decided upon most humidity exceeds another set point. Signals from the easily permits a fresh supply of oxygen, sweeps out humidity sensor cart be temporarily diverted manumetabolic COa as well as ammonia fumes, and allows ally from the relay system to an external meter by means of self-restoring switches so that visual checks addition of 14CO2 at a controlled known rate. The apparatus which has been constructed consists on the relative humidity can be made at will. The of two 320 1. rectilinear tanks made of 25 mm thick air, now conditioned so that it will maintain 45-52 Plexiglas, with sealable glove ports, an air lock sys- per cent relative humidity within each chamber, tem, and internal drinking water and electricity then passes through a check valve [6] intended to supplies (Figs. 1 and 2). The description will be to prevent a reversal of direction, and continues past more easily followed with the block diagram (Fig. the orifice of a branch line (this line will be described 3). * All plastic joints are stepped and have a bead later) through which a mixture of 12CO2 and x4CO2 or I2COz alone is added and into a mixing chamber which is triangular in cross-section cemented inside all corners, as well as 6 mm strips of Plexiglas [7] designed to mix the gases thoroughly by turbucemented over the raw edges, so that leaking gas lence. The turbulence is created by dividing the would have to traverse at ieast 6.3 cm of cemented mixing chamber into three compartments by two seam before escaping (see Item 34 of Fig. 3). I n plastic plates containing staggered 6 mm dia. holes. operation a heavy-duty air compressor [1] with a A needle valve [12] in the last compartment of the capacity of more than 100 1. per mln pumps air mixing chamber permits samples of gas to be drawn successively through a copper coil heat exchanger [2] off for testing. When the gas leaves the mixer, it (to dissipate the heat resulting from compression) and passes through a flowmeter [13] and then a main two stainless steel boxes baffled with stainless steel shut-off valve [14]. This valve opens into the end wool, which are contained in a freezer [3] at --25°C of a 3.1 cm i.d. Plexiglas cylinder which runs the where the moisture is frozen out of the air. An addi- length of the interior rear upper corner of the main tional pair of boxes are available to be substituted at chamber. This cylinder is sealed at its opposite end weekly intervals for those in use by quick-connect and has a narrow slit running its whole length, which fittings, while the ones previously in use are being slit is directed downwards and to the rear of the thawed, emptied, and dried. The quick-connect chamber [15], forcing the air (which at this point fittings are defrosted twice daily by heating tapes still has considerable velocity) against the rear wall which are wrapped around the fittings and controlled and floor of the chamber and diffusing it thoroughly. by automatic timers. During the summer when the The air leaves via an orifice placed in a front upper relative humidity is high, the air entering the com- corner of the chamber, through another shut-off pressor is drawn through an ordinary room air valve [25], and enters a sampling chamber [27] where dehumidifier. Upon leaving the freezer, the gas line another needle valve [28] makes it possible to sample divides into two essentially identical lines, one to the effluent gas mixture. From the sample chamber carry the control gas supply and the other the radio- the gas is directed through a second ftowmeter [26] active gas supply. A vacuum breaker installed just to measure outflow and assure that input and Output before the gas line division insures that should the are in the desired relationship. The gas leaving the compressor fail while the exhaust pump is working, flowmeter then passes through a two-way valve [29] which allows the gas to he passed to either of two no high vacuum will develop in the system. The air next enters a humidifying system [5] con- identical gas scrubbing tower systems [30, 30a]. Each sisting of a two-way control valve which bypasses or scrubbing tower system consists of three Plexiglas cylinders. Each cylinder can contain 1.5 1. of con• Numbers in square brackets refer to numbered centrated K O H solution. The effluent gas bubbles items in Fig. 3. through each cylinder in turn by means of four
tissue to tissue i n both the final fraction a n d the time at w h i c h e q u i l i b r i u m was achieved.
David L. Joftes
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CI202 Line
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r-
6
5
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17(1 16
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_L 31a
33 Fro. 3. Block diagram of 14CO s and control inhalation chambers ("a" indicates control chamber items) 1 Air compressor 19, 19a Thermostats 2 Heat exchanger 20, 20a Water supply with automatic valves 21 Air lock with purge valves 3 Freezer 22, 22a Lithium chloride gold grid humidity 4, 4a Heaters 5, 5a Humidifiers sensors 23, 23a Thermometers 6, 6a Check valves 7, 7a Mixing chambers 24, 24a Pressure gauges 25, 25a Main shut off valves, output 8, 8a Flow meters 26, 26a Flow meters showing bypass lines and 9, 9a Solenoid safety control valves valves 10, 10a Alarm relays controlling solenoids 11 14CO2 contaminated CO= supply 27, 27a Sampling chambers 28, 28a Valves for sampling output gas mixture 1l a Natural CO S supply 29 Three-way valve 12, 12a Valves for sampling input gas mixture 30 Triple gas scrubbing towers (double system) (from mixing chambers) 30a Triple gas scrubbing towers (single system) 13, 13a Flow meters 31, 31 a Vacuum line valves 14, 14a Main shut off valves, input 32 Vacuum pump 15 Chamber containing radioactive CO S 33 Vent to fume hood flue 16 Control chamber 34 Detail--cross-section through cemented plastic 17, 17a Glove port covers with purge valves joint showing how 6-5 cm leak path is achieved (gloves not indicated) in joining 2.5 cm stock. 18, 18a Humidistats
Apparatusfor administration of gaseous J-4C02 to small animals polyethylene filter candles attached to a stainless steel manifold inside the cylinder. All the cylinders are provided with individual stainless steel valves at the bottom for drawing off exhausted K O H solution and have stainless steel plugs at the top which can be removed to add fresh K O H solution. As noted above there are two systems so that one can be cleaned and recharged while the other is absorbing the CO 2 from the effluent gas mixture without shutting down. The gas leaving the scrubbing towers passes through a needle valve [31] used to control the rate of outflow and is exhausted through a vacuum pump [32] into a pipeline [33] carried up through a fume hood flue and out of the building, so that the minuscule amounts of 14CO2 which might evade K O H absorption will be safely diluted and diffused in the atmosphere. The exhaust line is wrapped in a thermostated heating cable so that condensed moisture does not freeze in it when the temperature drops to freezing. The room air and the effluent air are regularly monitored so that unsafe levels of x4CO2 will be detected should they occur. The 14CO2 branch llne, mentioned but not described above, leads from a tank of x~CO~ [11] which has been contaminated with the desired amount of a4CO2 (specific activity O.0485pc/mM) through the usual reduction valve to a normally closed solenoid valve [9] which is actuated to remain open unless a variety of unsafe conditions develop, in which case the current is cut offby means of a relay system [10], and the valve automatically closes to stop the flow of x4CO2. Under normal operating conditions, the gas flows from the solenoid valve through a flowmeter [ 13], which constantly monitors the rate of flow, and then it enters the main air stream just before the mixing chamber and continues as described above. An essentially identical branch line carries ordinary CO 2 from tanks [ 11a] to the control chamber air line. For ease in manipulating animals and supplies inside the chamber, glove ports are provided which are sealed by removable 2.5 cm Plexiglas covers [17] with neoprene " O " ring seals. Bolts fixed in the chamber walls and wing nuts every two inches around the perimeter of the covers insure a tight seal of the " O " rings which are let into the covers. Valves through the Plexiglas port covers make it possible to blow out and absorb any 14CO2 which passes through the neoprene gloves and collects inside them. This flushing is done routinely before unsealing the ports. An air lock [21] is used for passing animals and materials in and out of the chamber without permitting radioactive gas to escape into the room. The air lock is also provided with purge valves so that the radioactive gas may be blown out and absorbed before the outer door is opened. Both inner and outer air lock doors are sealed with neoprene
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" O " rings and are also fitted with bolts every 2 in. around their perimeters. All bolts are 1/4 in.-20 and have an individual neoprene " O " ring mounted under the bolt head where it bears against the plastic wall. Another chamber [16] identical with the one described, except that it has no air lock, and is provided with only a single set of gas scrubbing towers, is used as a control chamber. In this case the scrubbing towers function primarily to provide the same resistance to air flow as the experimental chamber scrubbing towers. It has been so designed that an air lock and additional scrubbing towers can be added should they prove to be necessary at a later date. The control chamber is supplied with ordinary CO 2 with only then aturally occurring amount of CO~ in it, and the rate of addition is the same as that of the radioactive gas, so that the total CO 2 content is as nearly the same as possible in both chambers. The chambers are warmed by external electric radiators placed close to the rear wall of each chamber. Each radiator is controlled by a thermostat inside its own chamber. Cooling is accomplished by maintaining the ambient room temperature a few degrees below the desired chamber temperature. In practice it has been found that the animals provide enough heat to maintain a higher temperature than the desired 22-23°C, so that cooling is necessary nearly all year round. Both chambers have a volume of 320 1., and the air flow through each is adjusted to 32 l./min so that a complete exchange of air occurs every I0 min. In order to overcome the reduction in flow rate due to constrictions in the lines, such as the small apertures of the flowmeters and the filter candies used as air bubblers, input air is pumped at a pressure of about 1½ to 2 p.s.i. The pressure inside the chambers is reduced by pumping air out at a slightly greater rate than that at which it is entering. Chamber pressure is kept between 1-4 mm Hg pressure below atmospheric so that if any leaks should occur, air is drawn into the chambers instead of escaping from them. Mercury manometers [24] installed through the front wall of each chamber monitor the internal pressure. SAFETY W h e n the construction o f these c h a m b e r s was first c o n t e m p l a t e d , the i d e a o f using Plexiglas was rejected since it is k n o w n t h a t this plastic p er m i t s C 0 2 to diffuse t h r o u g h it. H o w ever, o t h e r m at er i al s p r esen t ed m a n y o t h e r d r a w b a c k s w i t h o u t t h e t r a n s p a r e n c y a n d insulative v a l u e o f Plexiglas. F i n a l l y the a m o u n t w h i c h c o u l d diffuse t h r o u g h the plastic was c a l c u l a t e d (using a f o r m u l a supplied b y t h e
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David L. Joftes
manufacturer) for a chamber of the dimensions which it was proposed to build. I t turned out that for every 1 cm H g positive differential pressure, 9 mg per day of C O s could diffuse out of the chamber if C O 2 concentrations are the same inside and out. T h e chamber is intended to operate under fairly large increases of internal C O s concentration over that occurring naturally, and at a slight negative pressure. If, for argument's sake, a pressure of 1 cm H g and a five-fold higher internal concentration of C O s are assumed, a m a x i m u m of 50/~c 14C/day would be released into the room air along with the nonradioactive C O S. AEC regulations governing the permissible levels of 14C in this room (which has an 1100 cfrn air exchange) allow a release of 28 t~c/min. It was thought that while the 50 tic/day would certainly be acceptable, it would not be necessary to tolerate even this exposure if the chamber pressure were kept slightly negative, since the diffusion outward would be much decreased under negative pressure. I n practice only a few cpm/1, attributable to 14C were ever detected in the room air. It is necessary to provide a variety of safety devices to guard against several contingencies in order to assure the safety of personnel as well as the well-being of the animals in the chambers. T h e method selected to prevent excursion of gas from the chamber into the atmosphere depends upon control of a solenoid valve which is installed in each of the C O s branch lines immediately after the gas tank reduction valves. These solenoids are normally closed, and require electricity to remain open. A relay system operates to stop the flow of current to the solenoid, and thereby shut off the flow of COs: if there is failure of the room ventilation system; if there is an increase in pressure in the chamber to 1 cm of mercury; if there is failure of the x4CO2 tank reduction value; or if either the compressor or exhaust p u m p fails to function. Failure of the room ventilation is sensed by a flap in the exhaust flue of the room, which flap falls of its own weight when insufficient air is being drawn through the flue, and activates a microswitch. An increase in the chamber pressure is signalled by the rising mercury column of the manometer closing a circuit which consists of a pair of insulated platinum wires, the
uninsulated tips of which are positioned half a centimeter above the normal position of the mercury column on the up side of the m a n o m eter. Failure of the reduction valve on the 14CO 2 tank is sensed by a pressure switch which is set to activate the relay system if there is an increase in pressure in the line above 20 psi. Normal operating pressure is 7-9 psi in this line. I f the exhaust p u m p fails to operate, chamber pressure will increase and activate the relay system via the manometer. Failure of the compressor is monitored by a pressure switch which responds to the loss of air pressure. However, a vacuum breaker is installed in the main air line before it bifurcates, so that if the compressor fails but the exhaust p u m p continues to operate, air will be drawn into the system in suffÉcient quantity to prevent a v a c u u m of greater than 2 cm mercury from developing. U n d e r these conditions the animals would receive fresh air but the addition of C O 2 would be stopped. A general power failure would prevent operation of the relay system, but will also result in the closing of the solenoid valves, and so continued addition of C O s to the chambers will be prevented. T h e relay system is so arranged that if any one of the conditions mentioned above develops, both the 14CO2 and a~CO 2 solenoid valves will be closed and an electric clock will be stopped simultaneously. This is done so that control and experimental animals will be treated as m u c h alike as possible, even in the event of malfunction. T h e stoppage of the electric clock indicates how long the malfunction has continued. Should the malfunction correct itself, the clock starts again, but its discrepany with correct time will indicate the length of time of the malfunction. Diagnostic signal lights indicate proper function (amber) or malfunction (red) of various component circuits including the radiators, the freezer, and the humidifier systems. W h e n a malfunction occurs, a specific red signal lights and a buzzer sounds. These signals continue even if self-repair occurs, until a clear switch is operated manually. OPERATING CONDITIONS T h e experimental and control chambers have been in satisfactory operation since December, 1965. Each chamber holds 4 individual cages
Apparatusfor administration of gaseous a4C02 to small animals containing 4 or 5 mice each, or a total of 16 or 20 mice in each chamber. T h e r e have been some minor breakdowns which caused involuntary shutdowns of less than 2 hr each, and it is thought that in no case were these shutdowns significant to the results. During operation, rate of air input and output from the chambers and radioactive and normal C O 2 input are continually monitored by predictably flow meters. T h e degree of v a c u u m m a y be read at will from mercury manometers; relative humidity m a y be read at will as described above, and temperature m a y be read on dual thermometers installed in each chamber. All measurements are recorded twice daily on check sheets. Total C O s content of the input and effluent air are determined every other day by means of infrared absorption using a 1-m folded p a t h cell. At the same time 14C content is measured by absorption of C O S from samples of the input and effluent air into hyamine, aliquots of which are then submitted to scintillation counting. T h e room air before start-up was at background with respect to 14C content and contained approximately 0.04 per cent C O 2. T e n days after start-up it contained 7 dpm/1 above background; and 6 months later, after almost continuous operation, it had reached only 33 dpm/l. This level has never been exceeded. Rather, the level has dropped considerably to average 11 dpm/1. T h e m a x i m u m permissible level has been calculated to be 2,220 dpm/1 for the room in which the chambers are housed ~13). This room has a nominal volume of approximately 1300 ft 3 and an air exchange of approximately I I00 cfm. T h e chambers are operated at a temperature of 23-25°C. Pressure is maintained at approximately - - 4 m m Hg. T h e nominal chamber volume is 320 1. T h e input volume is 32.6 1./ min which allows for a total air exchange at least 6 times every hour in both the experimental and control chamber. O u t p u t volume is approximately 33.5 1/min. This removal at a greater rate than input allows us to maintain the negative pressure. In terms of the radioactivity input the experimental chamber receives 3,500 dpm/1 on the average during a normal run under present conditions. T h e control chamber gas is at
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background. T h e output from the experimental c h a m b e r is approximately the same, 3,500 dpm/l, indicating that the amount taken out by the animals is less than can be detected b y our method of air sampling. T h e control output is at background. These values have been obtained by scintillation counting and are within 10 per cent of the values calculated on a theoretical basis. Both the experimental and control chambers receive approximately 34 ml per min. of C O 2. This includes both the naturally occurring C O s in the air being p u m p e d in and the additional C O , containing the 1~C being added in the case of the experimental chamber or additional natural C O s being added in the case of the control chamber. I n this run the C O 2 input in both chambers averaged approximately 0.12 per cent and the output averaged 0.18 per cent from both chambers. T h e additional C O 2 in the output air is metabolic C O 2 breathed off by the animals. This means that the animals are living in an atmosphere where the C O , content is approximately 4½ times that of the free atmosphere in the Boston area. T h e 4½-fold increase in C O s concentration does not yield an appreciable increase in p C O 2. While this m a y seem surprising, H A L D ~ . and PRIESTLY(14) showed in 1905 with h u m a n subjects that an approximately 26-fold increase in inspired CO~ concentration from 0.03 to 0.79 per cent did not change the alveolar p C O a within their experimental error of 1 m g Hg, although there was a slight increase in tidal air and pulmonary ventilation without an increase in respiratory rate. Alveolar C O 2 concentration did not change. It seems reasonable that for this physiological response we m a y extrapolate from m a n to mouse under these conditions. BUCm~AN t~) has indicated and we have confirmed that only a negligible fraction of the isotope is removed by the animal from the throughput air. BUCHANANc7~ also indicated that total C O 2 concentration in the range under discussion had no effect on the amount of 14C retained. U n d e r these conditions an animal breathing a concentration of 0.04 per cent CO~ therefore will still have an alveolar concentration of 5-5 per cent which is an increase in concentration of the ambiant to alveolar air C O S of a factor of 137.5. O n the basis of HALDAN~ and
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David L. Joftes
PRIESTLY’S(~~) work, we can assume that the mice in the chambers are maintaining an alveolar CO, concentration of 5.5 per cent, or a factor of increase of approximately 30.5 over the CO, at O-18 per cent which they are breathing. With respect to the amount of 14C in the chambers, the stock supply of 14C0, contained very close to 0.002 &ml. The input per min of 2 1 ml CO, would, therefore, contain O-042 ,UC. The total 14C in the chamber at any instant after the first 10 min of gas addition would then be 0.42 ,UC. The specific activity in the 14C supply is O-0485 ,uc/mM. If effect is given to the dilution from the input air and the animal metabolic contribution, the chamber content has a specific activity of 0.03 pc/mM carbon. In the alveoli the CO, of the inspired air is further diluted by alveolar CO, to a specific activity of O*OOl ,uc/mM (computed). Based on the increase of CO, in the output gas it has been computed that the animals are putting out 3.04 ml (STP)/g/hr CO,. This compares favorably with BUCHANAN’S(~)figures of 3-3.3 ml (STP)/g/hr and thus adds confidence that our infrared measurements of CO, are accurate. ANIMAL
MAINTENANCE
Animals have been kept alive and in good health in the chambers for periods of 8-10 weeks. In the first test run when animals were kept for a period of 3 months without changing the cages in the chambers many of them developed a Pseudomonas infection of the urinary tract which resulted in urinary retention as evidenced by bladder distention. To prevent recurrence of this condition it is now routine to change the animals’ cages at two-week intervals or more frequently if indicated. The usual type of water bottle is used with a special stainless steel spout which contains 2 ball bearings, the lower of which fits closely against the in-curved opening of the spout. The upper bearing simply adds weight to insure a tight fit. This reduces dripping in response to the minor pressure changes which occur in the chambers. There is a cafeteria style water valve [20] installed in each of the chambers so that, using the gloves, the water bottles can be refilled whenever required. The animals are fed a commercial pelleted
dry food with no supplements of any kind. No gross evidence of vitamin deficiency has been observed even in the animals which remained in the chambers for 3 months. A large supply of the food is placed in a container in each of the chambers with the animals at the start of a run and more food is brought in through the airlock as needed. Food is added to the hoppers in the stainless steel cage tops as necessary, by means of the gloves. The cages used are of a solid bottom, clear plastic, nesting type. The bottom of one cage is sawed off and fitted with galvanized hardware cloth screen having approximately 4 wires per in. in both directions, in place of the original bottom. This modified cage is then placed in an intact cage which contains a commercially available absorbant bedding material which is treated to minimize urinary ammonia formation and reduce odor. The animals live on the screen and do not come in contact with the bedding. In practice either the top or bottom of the double cage unit is changed as required. The fresh and used cages enter and leave the experimental chamber via the air-lock so operation need not be suspended. The control chamber door is removed when necessary without stopping operations. REFERENCES 1. LIBBY W. F. Science, N.Y. 122, 57 (1958). 2. TOTTERJ. R., ZELLE M. R. and HOLLISTERH. Science, N. Y. 128, 1490 ( 1958). 3. LIBBY W. F. Statement delivered before the Federation of American Scientists, Washington, D.C. (1958). 4. CROW J. F. Hearings before the Special Subcommittee on Radiation of the Joint Committee on Atomic Energy, Congress of the U.S., 85th Congress, First Session on The Nature of Radioactive Fallout and Its Effects on Man, June 4, 1957, pp. lOG9-1028. 5. PAULINGL. Science, N.Y. 128, 1183 (1958). 6. LEIPUNSKY 0. I. Quoted in TOTTERet al. (Ref. 2). 7. BUCHANAN D. L. J. gen. Physiol. 34, 737 (1951). 8. BRUES A. M. and BUCHANAN D. L. Cold Spring Harb. Symp. pant. Biol. 13, 52 (1948). 9. KREBS H. A. Symposium, Sot. exptl Biol. 5, 1 (1951). 10. EVANSE. A. JR. Harvey Lectures 39, 273 (1944). 11. COHEN S. Exptl Cell Res. 29, 207 (1963). 12. BIANCOL., GIUSTINAG. and LAZAVINIE.Nature, Lond. 194 (4825), 289( 1962).
Apparatus for administration of gaseous 1~C0~ to small animals 13. Code of Fed. Reg, USAEC Title 10, Part 20. Standards for Protection Against Radiation. Appendix B, Table II. 14. I~LDANE J. S. and PRmSa'LEYJ. G. 2". Physiol. 32, 225 (1905).
ADDENDUM--PARTS LIST No attempt has been made in this description to specify or designate precisely the manufacturer and dimensions of the many parts used in building the
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chambers. However, all parts used were commercially available from electrical, plumbing, or air conditioning supply houses. The humidity controls were purchased from the Honeywell Corporation of Minneapolis, Minnesota. The dimensions of the air supply line and valves should be as large as possible consistent with economy. On the input side we used 1/4 in. (6 ram) plastic tubing of 2 in. (5 cm) i.d. The copper tubing coil used as a heat exchanger has a diameter of 3/4in. (18 ram) i.d. Valve apertures were at least 1/4 in. (6 ram). On the output side we used 3/4 in. soft copper tubing throughout.