Tobacco Documents Online

~_ `_

C C Accessiion No. 71-162 Copy N'o . Issued To CHARGE N0, & TITLE: 1901 - Biochemical Modification of Tobacco PHILIP M 0RRI1 S U, ,S, R E S E A R C H C E N T E R TY'P E R E P 0 R'T ; DATE: OANNUAL QSEMIANNU'AL QCOMP'LETION OSPECIAL December, 1971 PERIOD COVERED: June 1 - September 30, 1971 REPORT TDTLE','THE PHYSIOLOGICAL AND BIOCHE:iICAL RESPONSES'TO C'IGARETTE,SM0KE AND~ CIGARETTE SMOKE COMPONENTS II. The Insignificance of Cell Death in the Depressed Growth of Yeast Cells Exposed to Whole Smoke. ~ ~ af z ~/ WRITTEN BY 'L ~ `/ ia 4' D. T. Walsh ana W. K. Neal, II. APPROVED1B'Y R. M. Ilkeda DiI STR IBUTDON ; 3 n, j /Dr. H. Wakeham ~,="-- Mr. .L . F. Meyer ~yDr. J1. F. DeBarde leben " Dr. 3Mr. T'. F. S. Osdene E. Resnik N Mr. W. Mu t t e r()1=~~ ~~ Mr. J. S. Osmalov ~'-- r1r . R. W. Jenkins Dr, N. B. Rainer M r. R. N. Thomson k-;3 Dr. F. Will, III. c-,'-? Mr . L. Weissbecker 5 Mr. F'. L. Daylor'~'-`'`' ~/Dr. R. Fagan,l--° -A 3Dr. R. F. Dawson ~'Dr. P. A. Eichorn ~~Mr. J. D. Hinc~~'N-' ,-~~'Dr. C. B. Hoelz (;4' Mr. B. J. Kosakowski_,~,',~Dr. T. S~. La.szlo .~;~Dr. R. M. Ikeda ' 0 ~ Dr. Mr. D. H. A. B. Lowitz Merritt `~ /7Mr. ifDr. R. D. D. Carpenter -~cDr. W. K. Neal, M. Teng, --77Mr. D. T. Walsh 1000350190 II. KEYWQRDS, cigarette smoke optical density viability growth methylene blue glucose medium ETSS' medium ~ ~ '7 yeast ~..,

INTRODUCTION: Death is one possible biological response of cells to cigarette smoke (1,3,5,6). There are two pressing reasons for wanting to know th~e deathiresponse to smoke. First, we want to know if there are any lethal effects of smoke. Second, we need to~noxmalize our biochemical and physi;ologicali experiments for viable cell numbers, so that we might properly evaluate our data. The first confrontation with the cell death problem came when Neal demonstrated non-logarithmic growth and a drop ini02 conisumption~ and C02 output for yeast cells exposed to whole smoke (3)'. To explain this data he proposed~ the "kill curve Hypothesis",which states that the growth curve of the control is described by the equation~N = No e kt + D. C The constant "'D" represen~ts the dead cells inithe culture. Neal proposed that growth is inhibited temporarily, and then resumes non!- logarithmi~~.cally. As of the writing of hiis paper, plating techniques or methylene blue absorptionistudies had not showed whether cell death was involved. This report describes the development of a methylene blue staining procedure for detecting,dead yeast cells. A study of growth and 02 conisumption by yeast exposed to whole smoke was continued in conjunction with this cell death stud,y. The data show that cell death is not an, important factor. The depressed growth curve of yeast exposed to whole = smoke is caused by temporary growth inhibition. Concomitant with this growth inhibition is a temporary respiratory arrest. Whether the 1000350191 respiratory arrest is related to the growth inhid~bition cannot be ascertained by the available data. J

2 Materials and Method's : Organism: The yeast employed' was Saccharomyes cereviisiae, F1!eischman!s dry strain. Growth conditions and'culituring techniques used for maintain- ing the yeast were as previously described (1). Experimental Procedure Two 100-m1 cultures were used in the early experiment, where only viability counts were taken. In later experiments where many samples had to be taken from the cultures, 200 ml of culture were prepared. Two types of yeast culture were used in this experiment~ yeast grown in 2'0 ethanol (2o ETSS) as the malincarbon source (high respiratory), and'yeast grown with 10o glucose (10% SS) as the main carbon source (low respiratory). A water bath shaker (25°'C) provided the cultures with maximum aerationiand temperature control. Tygon capillary tubing was placed in each culture to remove sterile samples withou~t stopping the shaker. The cultures were treated withia constant smoke dose of 6.66 cc/OD•mil when they reached a cell density of 0.15 OD (2'). The smoke was bubbled into the culture by a glass syringe equipped with a Pasteur pipette. The cigarette used in these experiments was P. 'Mi. 5X7kE, which wasstored in the cold (36°'F). Prior to: use, thie cigarettes were equilibrated for two days in a control!led environmental room ('5°F and 60~ R. H.). A worm gear-driven smoking machine (P. M. 4S4'6, p 31) gave a standard puff of 35 cc/2 sec duration. Only the first puff after lighting was used (4 mm mark). This assures us of approximately constant smoke l00o35o1s2 ~

-3- composition. The flasks were kept under slow agitation during treatment, C with 500 RPM maintained 3ah!ile preparing the next smoke treatment. The smoke was allowed to interact for 20 minutes,and then cell density (OD1660), 012 consumption and viability counts were taken. An 02 poliarograph (model YS1-51) measured the 02 consumption. Specific respiratory rates were calculated a!s u moles 02 min 1.0D660.ML 1. For the viability counts, a 5-mi sample of the yeast was exposed tolmethylene blue and then live and dead cells were counted on the hiemac7tometer. Dead yeast cells are stained by methylene blue while living cells are not. The procedure for staining the cells is given in fig. 1, modified from Townsend and Lindegren (4). In the staining procedure the media was removed! to: 1) give a constant time for smoke-cell interaction and 2), prevent any interaction of smoke components with the dye which might cause continued death of the cells. If this was not done the % of dead cells increased during the counting time. Aliso,deionized HZ0 can not be used tolwash the cells because this causes clumping of the cells (5'sQ3/33)1. Cell density and respiratory rate were measured'before the smoking and periodically following the 2'0 minute:effect period. Viability counts were taken before s;:ol:inig, 20 min. after smoking, and during plateaus of the growth curves. Results: Cell Death: titi'hen l'og phaise yeast are exposed~ to 6.6 cc/0D•ml wholl'e cigarette smoke, there is no significant cell death at any time. The data in table I shows there is no essential difference between the control and~ experimental 1000350193 ~

C -4- cultures of 2°s ETSS yeast exposed to whole smoke for 2&minutes. Next we monitored cell death three times d'uring the course of an experiment. Viability counts were taken~before smoking, twenty minutes after smoking, and approximately two hours after smoking (table II). Cell death was not evident at any time. At two hours after smoking, the growth curve was on a plateau (fig. 2), but the viability count shows this is not due to cell death. In experiment 5393/41,double th~e standard dose of srnoke was given and again thiere was no significant kill (table III). The smoking of yeast whose main carbon source is glucose (10%) also: gives no significant kill (table IC') . Growth Inhibition: With cell death not a factor, growth inhibition possibly expl'ains the depressed! growth curve of smoked yeast. Smoked cultures at the time of plateau consisted mostly of single cell~s,indicating cessationlof cell division. When the growth curve for smoked 2%ETSS yealst was recorded for several hours, the curves plateaued after smoking, rose for sor.:e time and then plateaued again to be followed'by a rise (fig. 3). This rise and plateau is similar to cultures in synchrony. Respiratorv Inhibition: Bymonli~toring~ the respira~tor}~~rate before and after smoking, the yeast, we confirm that there is a sharp decline in 02 consumption immediately after smoki~ng the yeast (fi~gs. 3, 4) (11 . It is inter- es,tingto note that the declining respiratoryrateof glucose-grol~,n ~ yeast is not substantially altered in smoked yeas~t (fig. 4). O 01 ~ CW G? O ~ ~_

-5- The length of respiratory inhibition and growth inhibition ~ appears dependent on the.dose. If twice the standard dose is administered to yeast, the temporary respiratory inhibition period and growth inhibition are longer, but still there is recovery. (fig. Discussion: The data presented here indicate that in our experiments cell death is not an important factor as a biological response to smoke. Our data on~ growth, respirati=and metabolic C&2 output can now be normalized withirespect to cell numbers without fear of the artifact of a large number of dead cells. This means the kill curve hypothesis is not valid and we are looking directly at growth and respiratory inh~ibition. Our hypothesis to explain the temporary growth and respiratory inhibition is that there are non-lethal compounds in cigarette smoke which interfere with the basic cell machinery. The reduced 012 consumption folloitiing smoking of yea.st shows interference with the respiratory system. The microscopic observaltion of mostly single cells in smoked cultures shows interference with normal budding and growth. Non-logarithmic growth curves indicate growth interference. Other possible targets for smoke components would be glycolysis and the citric acid cycle, the main pathways for the breakdown of glucose in the cell. Neal C1)~ monitored the COZ output of yeast exposed'~ to smoke and found a temporary decrease in COZ output. Since C02 is produced in both of these metabolic' pathways, this could point to either pathway. So, diverse:cell mechanisms are affected indicatingthalt more than one component of smioke,s istaking a biologically active role. 1000350195 ~

It is important to~note that all of these effects are temporary ~ and th~e cu~lture finally recovers its normal growth and respiratory rates. This recovery can be accelerated if the smoked yeast are subcultured into fresh media. We interpret this to meanithe ceIl'must be able to either metabolize, neutralize, or store the inhibitory compounds and/or the compounds escape from the medium~. C L

REFERENCES: (1) Kennedy, J. R'. and A. M. Elliott. 1970. Cigarette Smoke: The effect of residue on mitochondria:l structure. Science 168: 1097-1098. PM 50418/13. Neal, W. K., II. 1971. The physiological and'biochemical responses to cigarette smoke and smoke components. 1. The response of yeast cultures to whole smoke. PN1 Report Acc. No. 71-022. (4) Townsend, F. G. and C. C. Lindegren. 1953. Structures in the yeast cell revealed in wet mounts. Cytologia 18:183-201. (5) Weiss, W. and W. ti4eiss. 1967. The gas phase toxicity l of cigarette smoke for paramecium. Arch. Environ. Health 14:682-686. (6) Weissbecker, L. 1971. Biological effects of smoke. PM Report Acc. No. 71-0140. N 0 0 0 w 0 ~ ~ ~

TABLE I V'IAB'ILITY COUNT OF 2% ETSS YEAST EXPOSED T0'WHOLE SMOKE FOR 20 MIN, CELL COUNT (X 105 FOR CELLS/ML) EXPERIMENT CONTROL EXPERIMENTAL LIVE DEAD % DEAD' LIVE DEAD~ ZDEAD 5393/34 137 1 01.73 120 1 0.83 144 2 1.38' 126 0 0.00 119 2 1.68' 122 3 2.46 120 2 1.66 119 01 0.00 5393/361 135 111 0 0 0.00 0.00 13 41 128 1 0 0.75 01.00 120 0 0.00 118 2 1.69 ~ 539'3/38 114 1 0.&8 112 0 0.00. 105 01 0.00 121 1 0.83 132' 0 0.00 114 2 1.75 5393/40 114 112 3' 2 2.63 1.77 135~ 104 2 1 1.48' 0.96: 10~5 0 01.00 115 0 0.00, mean '0 0.82 mean ~ 0.82 "T" test sho,;s no significant difference N O C O W C1i. _ COCL"