Tobacco Documents Online

v THE EFFECTS OF TOBACCO SMOKE ON LUNG TISSUE AS MEASURED BY ELECTRON SPIN RESONANCE John R. Rowlands, David G. Cad'ena, Jr. and Arthur L. Gross* Southwest Research Institute . .. 8500 Culebra Road San Antonio, Texas This work was supported by the National Cancer Institute, National In:,titutes'of Health under Contract No. PI-I43-65-100. *Present Address Pico- Laboratories, Incorporated 8222 Broadway San Antonio, Texas 0

I. INTRODUCTION The presence of free radicals in tobacco smoke has been previously reported. During the course of a program concerned with free radicals ; and alkylating agents in tobacco smoke it was observed that the concen- tration oi free radicals in tobacco smoke condensate varied considerably as a function of methods used for collecting and subsequent treatment with solvents. In order to determine the biological effects of free radicals it became apparent that a method was needed whereby smoke could be directly applied to lunb tissue without first colltcting and processing the condensate. The method reported herein enabled lung tissue to be directly exposed to cigarettc smoke in a manner similar to normal smoking and resulted in the observation of distinct changes in the lung tissue as measured by electron spin resonance. •

II. EXPERIMEN'TAL Large adult rabbits of unkown strain and obtained locally were used. Each animal was sacrificed by cervical dislocation and the lungs and the trachea were excised intact as rapidly as possible. This was accomplished within ten minutes after the animals were sacrificed. The trachea was then attached to the apparatus shown in Figure 1 using thin twine to hold it in place. The smoking apparatus (Figure 1) consists of a bell jar, valving, and a Phipps and Bird smoking machine that was modified to partially exhaust the bell jar, hold it in the exhausted state, and then admit atmospheric air into the jar. The timing of the cycle is controlled by the cam and motor arrangement contained in the smoking machine. The smoking parameters used were: puff frequency ---------------------30 seconds puff duration----------------------- 2 seconds inhalation time------------------- - 4 seconds butt length-- ---- ----------------- 15 mm The puff volume varied with the capacity of the lungs during each experiment. The volume was adjusted so that the lungs would fully inflate without rupturing. During the initial phases of this work several lungs did rupture thereby allowing smoke to enter the bell jar. This was later prevented by - first using the mininial puff volume that would inflate the lungs and step-wise increasing the voiur.-±e until the lungs would inflate with a turgid appearance. King size, non-filtered cigarettes, manu actured in the United States were 10Q3546928

~ ~ CIGARET,T:E: ;~ b h1ORMAL-~ ::: I I ::) NIO~-M?.LLY ~ LY i CLOSE.. ~ ..._.__~t=V E CLOSED c i VALUE _11 ~ i I L , .~~ _ FIGURE 1. SMOKING APPA tiATUS

used. The cigarettes were not preconditioned prior to use.. They were inserted into a holder composed of rubber tubing and were ignited by means of a lit cigarette of the same brand. A total of six cigarettes were smoked into the lungs for each experiment. As soon as the smoking was completed the lungs were removed from the apparatus and homogenized at room temperature for thirty seconds in an ©mnimixer. A cylindrical sample of the homogenate was then prepared and placed into a Varian liquid nitrogen dewar. Electron spin resonance spectra . of the sample were then recorded at 77 0 K using a Varian V 4500 X band spectrometer using 100 mc/sec modulation. A typical spectrum is shown in Figure 2 together with the spectrum obtained from an unsmoked lung using the same instrument parameters, and sample size. The observed spectrum was reasonably reproducible although small changes in the relative magnitudes of the three line pattern superimposed~ on the broad resonance P line were noted from sample to sample indicating that the observed pattern is composite. A considerable amount of additional data is required before • we can hope to arrive at any definite conclusions about the species giving rise to the signal. However, the similar pattern that Swartzob-erved in irradiated blood suggests that the observed spectrum may be produced by the interaction of the free radicals in tobacco smoke with the red blood Q . O cells. This is being further investigated on further lung experiments and ~ on a series of model systerns including metal porphyrin complexes. In ~ Fi;ure 3 we have included the spectrum obtained from freeze dried smoke ~ exposed lung tissue. Figure 3A represents the spectrum obtained at 77 °i{,

B FIGURE 2. (A) ESR'OF LUNG WHICH HAS SMO1'11D SIX CIGARETTES (B) EPR OF CONTROL

and Figure 3B the same sample run at room temperature. The room - temperature signal consists of a single narrow line which is typical of the electron spin resonance signal obtained from tobacco sinoke condensate. On refreezinb to T?°K the electron spin resonance signal observed reverts measurement at low temperature is consistent with it being due to a paramagnetic metal complex resonance, which becomes saturated and to that shown in Figure 3A. The reappearance of the broad signal upon hence not detected at room temperature. Conclusions ~ It is. apparent from this work that the smoking system that is reported has proven to be a reliable method for exposing intact lung tissue to fresh cigarette smoke comparab2e to the manner of' human exposure. The results are not biased by changes in the smoke that occur as a result of trapping and processing. We cannot at this time be certain of the origin of our observed electron spin resonance signal. However, due to the similarity of it to that ob ! served by Swartz upon irradiation of red blood cells we anticipate that both his and our observations may be explained by free radical attack of the haernoglobin molecule at the sixth coordination position leading to a covalent hexacoordinated complex. N ~ ~ W L1"1 ~ ~ • ~ ~ _:.~ ,.:.....: .._ _.~.::.::.,.,4.:..,

FIGURE 3A. EPR OF VACUIUVM L~TF %~-UO_~=JL L"u NG AT 770X . . .. .~.a=.....j . .. . . ..:.. _ . _. . . .. f.S:! .~ .._ . ~ _

[j~Viy~ .,:\ o FIGURF, 3B. EPR OF VACUUM L`RTED LUZG AT ROOM ' Ai'URE .

APPENDIX A. 0