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Respiration induced oxygen gradients in cultured mammalian cells Fengler, John Josef Paul 1988

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RESPIRATION INDUCED OXYGEN GRADIENTS IN CULTURED MAMMALIAN CELLS by John Josef Paul Fengler B.A.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Physics We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1988 © John Josef Paul Fengler, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date Osh&A \A. j968 DE-6 (2/88) ABSTRACT Oxygen i s known to s e n s i t i z e X - i r r a d i a t e d c e l l s to l e t h a l r a d i a t i o n damage. At low ambient oxygen tensions, however, the molecular mechanisms of the s e n s i t i z a t i o n process and the metabolic requirements of the c e l l may be forced to compete f o r the c e l l u l a r oxygen supply. The e f f e c t of c e l l r e s p i r a t i o n on the a v a i l a b i l i t y of i n t r a c e l l u l a r oxygen during i r r a d i a t i o n was consequently investigated by comparing the r a d i o s e n s i t i v i t i e s of r e s p i r i n g and non-respiring c e l l s . Cultured mammalian c e l l s were i r r a d i a t e d i n s i n g l e c e l l suspensions and t h i n f i l m monolayers at r e s p i r a t i o n i n h i b i t i n g (4°C) and at normal c e l l c u l t u r i n g (37°C) temperatures. Due to oxygen e q u i l i b r a t i o n and r a d i o l y t i c depletion problems, the r e s u l t s of the suspension culture experiments were inconclusive. By subsequently analyzing the d i f f u s i v e mass transfe r of oxygen i n the suspension medium, the s t i r r e r f l a s k was determined to be an inappropriate culture v e s s e l i n which to i r r a d i a t e c e l l s at constant low oxygen concentrations. A t h i n f i l m c e l l c ulture system i n which the oxygen concentrations to which the c e l l s were exposed during i r r a d i a t i o n could be more accurately c o n t r o l l e d was then developed. A comparison of the oxygen enhanced r a d i o s e n s i t i v i t i e s of the r e s p i r i n g and non-respiring c e l l s i n t h i n f i l m monolayers suggested that the metabolic depletion of oxygen at low oxygen tensions has a s i g n i f i c a n t e f f e c t on the l o c a l and i n t r a c e l l u l a r oxygen d i s t r i b u t i o n . These e f f e c t s are representative of those that would be produced i f r e s p i r a t i o n induced oxygen gradients ex i s t e d inside and immediately around r e s p i r i n g c e l l s . The magnitude of the d i f f e r e n t i a l r a d i o s e n s i t i v i t i e s was found to be dependent on c e l l shape and to have i i i values that agreed very well with t h e o r e t i c a l p r edictions based on the existence of such gradients. s i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES v i i LIST OF TABLES x ACKNOWLEDGEMENTS x i 1. INTRODUCTION 1 1.1 An Overview 1 1.2 The Role of Oxygen i n Cancer Therapy 3 1.2.1 Oxygen as a r a d i o s e n s i t i z e r 3 1.2.2 The oxygen e f f e c t mechanism 6 1.2.3 Oxygen and radioprotectors 9 1.2.4 Oxygen and chemotherapeutic drugs 11 1.3 Modelling C e l l Respiration 12 1.4 I n t r a c e l l u l a r Oxygen Gradients 16 1.5 Experimental Rationale and Thesis Objectives 17 2. CELL CULTURE 19 3. GENERAL EXPERIMENTAL METHODS 23 3.1 Clonogenic Su r v i v a l and Data Analysis 23 3.2 Respiratory I n h i b i t i o n 28 3.3 Choice of I r r a d i a t i o n Vessels 29 4. THE SINGLE CELL SUSPENSION 29 4.1 Materials and Methods 29 4.2 Experimental Protocol 32 4.3 Results 35 4.3.1 Radioresponse of r e s p i r i n g c e l l s 36 V 4.3.2 Radioresponse of the non-respiring c e l l s 39 4.3.3 Sources of error 44 4.4 Analysis of Suspension Culture Method 45 4.4.1 D i f f u s i o n rate constant 45 4.4.2 Oxygen consumption and r a d i o l y t i c depletion 50 5. THE THIN FILM MONOLAYER 57 5.1 Materials and Method 57 5.1.1 Choice of i r r a d i a t i o n vessel 60 5.1.2 Dosimetry considerations 61 5.1.3 Thin f i l m t o x i c i t y 63 5.1.4 Spot i n o c u l a t i o n 66 5.1.5 C e l l attachment time 68 5.1.6 C e l l shape i n t h i n f i l m monolayer 68 5.1.7 E q u i l i b r a t i o n time measurements 71 5.1.8 Low temperature t o x i c i t y 73 5.2 Experimental Protocol 76 5.2.1 Preparation of c e l l culture 76 5.2.2 I r r a d i a t i o n procedure 78 5.2.3 Assaying clonogenic s u r v i v a l 79 5.3 Results 81 5.3.1 F l a t c e l l monolayer 81 5.3.2 Round c e l l monolayer 87 5.4 Discussion and Analysis of Thin Film Method 91 5.4.1 Temperature mediated r a d i o s e n s i t i v i t y 91 5.4.2 Oxygen gradients i n t h i n f i l m culture 93 5.4.3 Evaluation of the t h i n f i l m technique 97 6. SUMMARY 98 v i 7 . BIBLIOGRAPHY 100 8. APPENDIX 106 8.1 The In t e r a c t i o n of Ionizing Radiation and C e l l s 106 8.2 Modelling Radiation Survival 109 v i i LIST OF FIGURES Page Figure 1 Alper and Howard-Flanders' model of r a d i o s e n s i t i z a t i o n by oxygen 5 Figure 2 The f i x a t i o n of DNA r a d i c a l s by oxygen 7 Figure 3 Radical scavenging by t h i o l s 10 Figure 4 Boag's model of r e s p i r a t i o n induced oxygen gradients inside and immediately surrounding si n g l e c e l l s i n suspension 14 Figure 5 Gradient l i m i t e d c e l l r e s p i r a t i o n at low oxygen tensions 15 Figure 6 Growth of V79 Chinese hamster c e l l s 22 Figure 7 V79 c e l l colony formation 24 Figure 8 T y p i c a l r a d i a t i o n s u r v i v a l data for V79 c e l l s 25 Figure 9 R e l a t i v e contributions of the two components of c e l l k i l l i n g described by the Linear Quadratic model of r a d i a t i o n s u r v i v a l 27 Figure 10 Experimental apparatus f o r the suspension culture i r r a d i a t i o n s 31 Figure 11 Protocol flowchart for the suspension culture experiments 33 Figure 12 Non-normalized suspension culture r a d i a t i o n s u r v i v a l data f o r r e s p i r i n g (37oC) c e l l s 37 Figure 13 Hypothetical biphasic r a d i a t i o n response of a heterogeously s e n s i t i v e c e l l population 38 Figure 14 Normalized and f i t t e d suspension culture r a d i a t i o n s u r v i v a l data for r e s p i r i n g (37oC) c e l l s 40 Figure 15 Normalized and f i t t e d suspension culture r a d i a t i o n s u r v i v a l data f o r non-respiring (4oC) c e l l s 41 Figure 16 V a r i a t i o n of the oxygen concentration i n a i r and i n a i r - e q u i l i b r a t e d water with temperature 43 v i i i Figure 17 Analysis of mass transf e r i n a suspension culture spinner f l a s k 46 Figure 18 Apparatus used to measure the d i f f u s i o n rate constant, k, of the spinner f l a s k 49 Figure 19 Experimental confirmation of d i f f u s i o n rate constant value 51 Figure 20 Respiration induced oxygen gradients between the gas and the i n t r a c e l l u l a r environment i n a s i n g l e c e l l suspension 53 Figure 21 Calculated cycle of r a d i o l y t i c depletion of oxygen i n an i r r a d i a t e d suspension culture 54 Figure 22 S t r i p chart recordings of r a d i o l y t i c depletion of oxygen i n a suspension culture 56 Figure 23 Possible solutions to oxygen e q u i l i b r a t i o n problems using the Whillans and Rauth equation 58 Figure 24 Experimental apparatus for t h i n f i l m c u l t u r e i r r a d i a t i o n s 62 Figure 25 B i o l o g i c a l dosimetry used to measure the a d d i t i o n a l dose to c e l l s attached to glass surfaces 64 Figure 26 T o x i c i t y of t h i n f i l m c u l t u r e 65 Figure 27 Spot i n o c u l a t i o n of glass P e t r i dishes 67 Figure 28 F l a t and round c e l l monolayers i n glass P e t r i dishes 69 Figure 29 C e l l attachment time to a glass surface 70 Figure 30 Time dependent change i n c e l l cross-s e c t i o n a l area a f t e r p l a t i n g 72 Figure 31 Upper l i m i t measurement of t h i n f i l m c ulture e q u i l i b r a t i o n time 74 Figure 32 T o x i c i t y of low temperatures 75 Figure 33 Protocol flowchart for t h i n f i l m c ulture experiments 77 Figure 34 S u r v i v a l data f o r f l a t t h i n f i l m c ultured c e l l s i r r a d i a t e d 82 i x Figure 35 Oxygen enhancement r a t i o f o r f l a t c e l l s i n t h i n f i l m culture 86 Figure 36 Su r v i v a l data f o r round t h i n f i l m c u l t u r e d c e l l s 88 Figure 37 Oxygen enhancement r a t i o f o r round c e l l s i n t h i n f i l m culture 90 Figure 38 C e l l cycle d i s t r i b u t i o n versus time on i c e p r i o r to i r r r a d i a t i o n 93 Figure 39 Gradients i n t h i n f i l m cultured c e l l s of d i f f e r e n t shapes 95 Figure 40 An approximate time scale of events a f t e r a radiation-DNA i n t e r a c t i o n 108 Figure 41 Single h i t , sin g l e target model of r a d i a t i o n s u r v i v a l I l l Figure 42 Proposed molecular basis f o r the Linear Quadratic model of r a d i a t i o n s u r v i v a l 112 X LIST OF TABLES Page Table I A summary of ea r l y studies of oxygen i n radiobiology 4 Table II Calculated oxygen concentrations i n suspension culture medium a f t e r a one hour e q u i l i b r a t i o n period 48 x i ACKNOWLEDGEMENTS I would l i k e to thank a l l of the members of the B r i t i s h Columbia Cancer Research Center who have a s s i s t e d me i n the completion of t h i s t hesis p r o j e c t . I am g r a t e f u l f o r both t h e i r t e c h n i c a l and moral support. I s p e c i f i c a l l y want to thank Ingrid Spadinger f o r her frequent and invaluable assistance that allowed me to be i n two places at the same time. Without such help several of the experiments i n t h i s thesis could not have been performed. F i n a l l y , I want to express my gratitude to my the s i s supervisor, Dr. Ralph Durand, to whom I owe a p a r t i c u l a r debt for the generosity with which he shared h i s advice and time, for h i s f i n a n c i a l support, and f o r h i s patience i n allowing me to f i n i s h t h i s work to my s a t i s f a c t i o n . 1 1. INTRODUCTION: 1.1 An Overview A cancer c e l l i s considered to be e f f e c t i v e l y dead i f i t i s unable to produce v i a b l e o f f s p r i n g . In the treatment of cancer, the induction of reproductive c e l l death i s t y p i c a l l y the r e s u l t of molecular i n t e r a c t i o n s i n v o l v i n g the c e l l ' s genetic material, the DNA, and therapeutic agents such as r a d i a t i o n , chemotoxins, or a combination thereof. Although i t i s d i f f i c u l t to optimize or even accurately evaluate the effectiveness of anti-tumor treatments i f the molecular mechanisms by which they function are not understood, i t i s often even more d i f f i c u l t to extract information about such mechanisms from the complex b i o l o g i c a l systems i n which they operate. Because the administration of a therapeutic agent to even the simplest of these systems w i l l r a r e l y produce an i s o l a t e d or e a s i l y interpreted experimental response, the i d e n t i f i c a t i o n of s u b c e l l u l a r cause-effect r e l a t i o n s h i p s and the accuracy with which they can be q u a n t i f i e d i s almost always b i o l o g i c a l l y l i m i t e d . I t i s , therefore, not s u r p r i s i n g that such l i m i t a t i o n s are also encountered i n attempts to determine the therapeutic r o l e of molecular oxygen. Although not a therapeutic agent per se, oxygen i s instrumental i n the f u n c t i o n of various cancer treatments. Oxygen i s , of course, also an i n t e g r a l and e s s e n t i a l component of c e l l u l a r energy metabolism. The importance of t h i s metabolic process i n normal c e l l function r e s t r i c t s the manner i n which c e l l u l a r oxygen can be experimentally manipulated. The nature of such in v e s t i g a t i o n s i s further r e s t r i c t e d by the t e c h n i c a l 2 d i f f i c u l t i e s associated with making non-disruptive measurements at the molecular l e v e l . An equally fundamental consequence of oxidative metabolism i s the interdependent r e l a t i o n s h i p that i s implied to e x i s t between the r e s p i r a t o r y and therapeutic demands on the c e l l u l a r oxygen supply. Oxygen consumed by r e s p i r a t i o n i s not a v a i l a b l e to act as a therapeutic co-factor and, given the already l i m i t e d oxygen supply to the poorly v a s c u l a r i z e d regions of tumor ti s s u e , the effectiveness of oxygen-enhanced cancer treatments may be a d d i t i o n a l l y a f f e c t e d by i n t r a c e l l u l a r and l o c a l i z e d metabolic depletion. The objective pursued i n t h i s thesis was to determine the consequences of t h i s concurrent demand on the c e l l u l a r oxygen supply. The means by which the r e l a t i o n s h i p between the r e s p i r a t o r y and therapeutic u t i l i z a t i o n of c e l l u l a r oxygen could be examined, however, were l i m i t e d by b i o l o g i c a l and te c h n i c a l r e s t r i c t i o n s s i m i l a r to those that would be encountered i n attempting to as c e r t a i n oxygen's r o l e as cancer therapy co-factor. An innovative approach was required to accommodate these r e s t r i c t i o n s i n two ways. F i r s t l y , the experimental r a t i o n a l e was formulated such that i t incorporated and used the c e l l u l a r metabolism of oxygen to i t s own advantage. Secondly, an experimental system was developed i n which the c e l l environment could be c a r e f u l l y and accurately c o n t r o l l e d . On t h i s basis, the e f f e c t of c e l l r e s p i r a t i o n on the a v a i l i b i l i t y and d i s t r i b u t i o n of i n t r a c e l l u l a r oxygen could be in v e s t i g a t e d from an informative and previously unexamined perspective. 3 1.2 The Role of Oxygen i n Cancer Therapy 1.2.1 Oxygen as a r a d i o s e n s i t i z e r Oxygen i s known to play a v a r i e t y of r o l e s i n d i f f e r e n t cancer treatment modalities. I t i s , however, predominantly known f o r i t s a b i l i t y to s e n s i t i z e c e l l s to the e f f e c t s of X-rays and other forms of i o n i z i n g r a d i a t i o n . A b r i e f h i s t o r i c a l outline h i g h l i g h t i n g some early research into the r a d i o b i o l o g i c a l r o l e of oxygen i s given i n Table I. Although the influence of oxygen on c e l l u l a r and tiss u e r a d i o s e n s i t i v i t y predates most current knowledge of radiobiology, i n t i t i a l theories regarding the cause of t h i s e f f e c t were l a r g e l y speculative. Oxygen's r o l e as r a d i o s e n s i t i z e r was not d e f i n i t i v e l y established u n t i l 1941, when Anderson and Turkowitz demonstrated that the absence of oxygen and not the biochemical state of anaerobiosis was responsible f o r a subsequently observed decrease i n the r a d i o s e n s i t i v i t y of yeast c e l l s (Alper, 1979). In the h i s t o r y of r a d i o b i o l o g i c a l research, two developments i n the mid 1950's dramatically increased awareness regarding the nature of r a d i o s e n s i t i z a t i o n by oxygen and i t s importance to radiotherapy. F i r s t , Thomlinson and Gray (1955) discovered that human lung cancer c e l l s were most r e s i s t a n t to radiotherapy i f they lay j u s t beyond the ca l c u l a t e d range of oxygenation by tumor blood flow. Shortly thereafter, Alper and Howard-Flanders (1956) developed an e m p i r i c a l l y based, mathematical model that established a quantitative r e l a t i o n s h i p between oxygen concentration and the r a d i o s e n s i t i v i t y of cultured b a c t e r i a . ( F i g . 1) These successive discoveries emphasized the l i n k between the research 4 Table I A Summary of Early Studies of Oxygen i n Radiobiology 1912 SWARTZ notes skin reaction to radium i s reduced by pressing a p p l i c a t o r t i g h t l y onto skin. 1921 HOLTHAUSEN shows Ascaris eggs are r e s i s t a n t to r a d i a t i o n i n absence of oxygen. 1923 PETRY finds c o r r e l a t i o n between presence of oxygen and r a d i o s e n s i t i v i t y of vegetable seeds. 1934 CRABTREE and CRAMER study r a d i a t i o n s u r v i v a l of human cancer c e l l s i n the presence and absence of oxygen. 1941 ANDERSON and TURKOWITZ use yeast c e l l s to show that decrease i n r a d i o s e n s i t i v i t y i s due to lack of oxygen not the state of anaerobiosis. 1952 READ measures growth i n h i b i t i o n of broad bean root by i r r a d i a t i o n as a function of oxygen concentration. 1955 THOMLINSON and GRAY discover r e l a t i o n s h i p between tumor blood flow and r a d i o s e n s i t i v i t y i n human lung cancers. 1956 ALPER and HOWARD-FLANDERS develop mathematical model of oxygen mediated r a d i o s e n s i t i z a t i o n as a function of concentration. (Source: Alper, 1979; H a l l , 1988) 5 Figure 1 Alper and Howard-Flanders' model of r a d i o s e n s i t i z a t i o n by oxygen. The r e l a t i v e r a d i o s e n s i t i v i t y of c e l l s i s p l o t t e d against a l o g a r i t h m i c a l l y scaled oxygen concentration and i s defined by the parameters m and K. 6 laboratory and the cancer therapy c l i n i c and p r e c i p i t a t e d an unprecedented i n t e r e s t i n oxygen-related r a d i o b i o l o g i c a l research. 1.2.2 The oxygen e f f e c t mechanism Despite several decades of research, much of what i s presently known about the mechanism by which oxygen-mediated r a d i o s e n s i t i z a t i o n occurs remains t h e o r e t i c a l ( Q u i n t i l i a n i , 1979; H a l l , 1988). The most widely accepted theory of the s e n s i t i z a t i o n mechanism postulates the following process (Fig. 2): A d i r e c t r a d i a t i o n - c e l l i n t e r a c t i o n or an encounter with a free r a d i c a l , such as those produced by the r a d i o l y s i s of water, causes a l e s i o n i n the c e l l target, the DNA. This l e s i o n may also be i n the form of a r a d i c a l which w i l l then react r e a d i l y with any nearby oxygen molecule to form a peroxy r a d i c a l . The peroxy r a d i c a l , i n turn, combines with hydrogen to form a stable hydroperoxide. The f i n a l step renders the l e s i o n v i r t u a l l y i r r e p a i r a b l e and, i n permanently damaging the DNA, may have an adverse a f f e c t on the reproductive a b i l i t y of the c e l l . This mechanism i s consistent with the f a c t that oxygen's s e n s i t i z i n g a b i l i t y decreases as the l i n e a r energy tr a n s f e r (LET) of the r a d i a t i o n increases. I f the amount of oxygen a v a i l a b l e to f i x r a d i o l y t i c a l l y induced lesions increases, c e l l u l a r r a d i o s e n s i t i v i t y i s observed to increase i n accordance with the Alper and Howard-Flanders model. The oxygen enhancement r a t i o , or OER, i s the f a c t o r by which the r a d i a t i o n dose that produces a p a r t i c u l a r b i o l o g i c a l endpoint under aerobic conditions must be m u l t i p l i e d to achieve the same endpoint i n anoxia. The value of the OER for c e l l k i l l i n g by X - i r r a d i a t i o n v a r i e s 7 Figure 2 The f i x a t i o n of DNA r a d i c a l s by oxygen. Lesions i n DNA caused d i r e c t l y by secondary electrons or by r a d i o l y t i c a l l y induced free r a d i c a l s may combine i r r e v e r s i b l y with molecular oxygen. 8 between 2 and 4 f o r a l l classes of c e l l s , but i s reduced to 1 f o r high LET r a d i a t i o n s such as a - p a r t i c l e s (Barendsen et a l . , 1966; Alper, 1979) . The s e v e r i t y of high LET r a d i a t i o n damage and the high number of l e s i o n s t h i s r a d i a t i o n induces are alone s u f f i c i e n t to k i l l the c e l l and make l e s i o n f i x a t i o n by oxygen redundant. The l e s i o n f i x a t i o n hypothesis i s also compatible with the r e s u l t s of time-resolved rapid-mixing and gas explosion experiments (Shenoy et a l . , 1975; Watts et a l . , 1978). These have confirmed that since the r a d i o l y t i c a l l y induced r a d i c a l s i n the DNA and i n s o l u t i o n have h a l f -l i v e s of the order of milliseconds and w i l l recombine with other molecules i n the absence of oxygen, the l e v e l of oxygen enhanced r a d i o s e n s i t i z a t i o n i s s o l e l y dependent upon the amount of oxygen present during the time of i r r a d i a t i o n . Suggested a l t e r n a t i v e or a d d i t i o n a l mechanisms, such as the e l e c t r o n a b s t r a c t i o n hypothesis (Adams, 1972), have also been put forward but t h e i r s i g n i f i c a n c e to the oxygen e f f e c t are l e s s c l e a r . M u l t i p l e oxygen s e n s i t i z a t i o n mechanisms and/or multiple s i t e s of r a d i a t i o n damage are occasionally invoked to explain unusual experimental r e s u l t s ( M i l l a r et a l . , 1979), but these have been d i f f i c u l t to repeat (Ling et a l . , 1981) and t h e i r proposed mechanisms remain unsubstantiated. I t i s generally accepted, therefore, that oxygen mediated r a d i o s e n s i t i v i t y i s quantitative rather than q u a l i t a t i v e , and that c e l l death i s caused by the same mechanism(s) at a l l oxygen tensions. Whether or not r a d i o s e n s i t i z a t i o n by oxygen i s s o l e l y due to l e s i o n f i x a t i o n as described above, however, remains to be confirmed. 9 1.2.3 Oxygen and radioprotectors The f u n c t i o n a l opposites of r a d i o s e n s i t i z e r s are radioprotectors. As t h e i r name indicates, radioprotectors protect c e l l s from r a d i a t i o n damage and can provide an a d d i t i o n a l method of increasing t i s s u e s e l e c t i v i t y i n the radiotherapeutic treatment of tumors. The most prominently studied radioprotective agents are t h i o l s (Biaglow et a l . 1983a). T h i o l s occur n a t u r a l l y i n the i n t r a c e l l u l a r environment i n the form of the non-protein sulphydryls glutathione, cysteine, cysteamine and coenzyme A. They can also be administered i n the form of drugs such as the phosphorothioate, WR-2721 (Yuhas and Storer, 1969; Purdie, 1979). A proposed explanation f o r the radioprotective properties of t h i o l s i s that they and other reducing species compete with oxygen for the free r a d i c a l s induced by i r r a d i a t i o n and thereby reduce the i n d i r e c t production of DNA lesio n s (Fig. 3). In addition, whereas the consequence of binding with oxygen i s postulated to be l e s i o n f i x a t i o n , t h i o l s bound to the r a d i c a l s i n damaged DNA are beli e v e d to promote r e p a i r by hydrogen donation and to restore the c e l l ' s reproductive capacity (Biaglow et a l . , 1983a; H a l l , 1988). Evidence obtained from experiments with WR-2721, however, supports a hypothesis which claims that these radioprotectors also act by oxygen depl e t i o n mechanisms (Durand, 1983a; Purdie et a l . , 1983; Durand, 1984). T h i o l s are known to oxidize spontaneously and to consume i n t r a c e l l u l a r oxygen i n the process (Gray, 1956). An oxygen d i f f e r e n t i a l could, consequently, be created between the i n t r a and e x t r a c e l l u l a r environments. Since target oxygen l e v e l s at the time of i r r a d i a t i o n determine the degree of oxygen enhanced r a d i o s e n s i t i v i t y , the 10 Figure 3 Radical scavenging by t h i o l s . T h i o l s are believed to protect c e l l s from r a d i a t i o n damage by combining with free r a d i c a l s before they are able to induce DNA l e s i o n s . 11 "radioprotection" a t t r i b u t e d to the presence of t h i o l s may, i n large part, a c t u a l l y be a decrease i n oxygen-mediated r a d i o s e n s i t i z a t i o n of the r a d i a t i o n target due to increased i n t r a c e l l u l a r hypoxia. This would also explain the observed c o r r e l a t i o n between increases i n the i n t r a c e l l u l a r t h i o l content and decreases i n the oxygen enhanced r a d i o s e n s i t i v i t y of c e l l s at low oxygen tensions (Cullen and Walker, 1980; C u l l e n et a l . , 1980;). 1.2.4 Oxygen and chemotherapeutic drugs Recently, i t has been discovered that oxygen also plays a c r i t i c a l r o l e i n the function of some chemotherapeutic drugs (Kennedy et a l . , 1980; Taylor and Rauth, 1982; Koch et a l . , 1984; Rauth et a l . , 1984). Chemotoxins with a high s p e c i f i c i t y f o r hypoxic c e l l s have been found to be strongly i n h i b i t e d from binding to t h e i r tumour target c e l l s by very low concentrations of oxygen. Because of t h e i r s e l e c t i v e t o x i c i t y to hypoxic c e l l s , both nitroheterocycles, such as misonidazole, and bioreductive a l k y l a t i n g agents, such as mitomycin C, were once considered promising candidates for mixed modality chemo-radiotherapy. The r a t i o n a l e f o r such therapies i s that hypoxic tumor c e l l s , normally r a d i o b i o l o g i c a l l y protected by the lack of oxygen, could be s p e c i f i c a l l y targeted by these chemotoxic compounds. However, since a large proportion of tumor c e l l s are neither continuously well oxygenated, nor c h r o n i c a l l y hypoxic, and the e f f e c t i v e t o x i c i t y of these drugs i s dramatically reduced by the presence of le s s than a micromolar of oxygen, treatments of t h i s type may be l e s s promising than i n i t a l l y a n t i c i p a t e d . 12 1.3 Modelling C e l l Respiration A d i r e c t consequence of oxygen's influence on c e l l u l a r radio- and chemo-sensitivity has been to focus research i n t e r e s t on fa c t o r s a f f e c t i n g the oxygen tension i n and around c e l l s , i n p a r t i c u l a r on c e l l r e s p i r a t i o n and on the transport of oxygen to c e l l s . C e l l s i n culture and i n ti s s u e e x i s t i n e s s e n t i a l l y aqueous environments, and the transport of oxygen from the gas overlying the culture medium, or from the blood supply to the ti s s u e , occurs p r i m a r i l y by d i f f u s i o n . The rate at which oxygen i s supplied to c e l l s i s , therefore, determined by the fac t o r s that govern the a b i l i t y of oxygen molecules to d i f f u s e through the medium or e x t r a c e l l u l a r f l u i d . The mechanics of the oxygen supply and c e l l r e s p i r a t i o n r e l a t i o n s h i p have been thoroughly analyzed by Boag (1969; 1970). Using the s i n g l e c e l l suspended i n a l i q u i d growth medium as an i d e a l i z e d model of the c e l l i n t i s s u e , Boag hypothesizes that c e l l s act as oxygen "sinks", depleting the oxygen i n t h e i r immediate environment as they r e s p i r e . As a r e s u l t a d i f f u s i o n gradient i s created around each sink, which i n turn provides the motivating force f o r the transport of more oxygen to the c e l l . A steady state of oxygen supply and consumption i s achieved when the d i f f u s i o n gradient becomes large enough to sustain a normal l e v e l of c e l l r e s p i r a t i o n or when the gradient s i z e i s l i m i t e d by the oxygen a v a i l a b l e from the source. In h i s model, Boag assumes a uniform d i s t r i b u t i o n of oxygen-consuming mitochondria throughout the c e l l . Cultured mammalian c e l l s are commonly used i n r a d i o b i o l o g i c a l research and have r e s p i r a t i o n rates of the order of 10" 1 7 moles c e l l " 1 s e c " 1 at 37°C (Boag, 1970, Biaglow, 13 1983) . Using t h i s value f o r the c e l l r e s p i r a t i o n rate and known values for c e l l s i z e and the d i f f u s i o n c o e f f i c i e n t of oxygen i n aqueous solu t i o n s , an evaluation of the e f f e c t s of c e l l r e s p i r a t i o n on the oxygen d i s t r i b u t i o n around the c e l l p r e dicts the c r e a t i o n of a steady state d i f f u s i o n gradient with a concentration d i f f e r e n t i a l of the order of 0.5 (M. According to Boag's model, approximately two t h i r d s of t h i s gradient would l i e outside of the c e l l ( F i g. 4). Since the actual d i s t r i b u t i o n of mitochondria i n r e s p i r i n g c e l l s i s often l o c a l i z e d around the c e l l n u c l e i , however, the i n t r a c e l l u l a r p o r t i o n of the gradient probably exceeds t h i s f i r s t approximation. C e l l u l a r r e s p i r a t i o n rates are known to remain independent of the ambient oxygen tension to r e l a t i v e l y low (5 to 20 pM) concentrations (Froese, 1962; Wilson et a l . , 1979; Marshall et a l . , 1986). Recent advances i n oxygen sensor technology have allowed accurate measurements of c e l l u l a r oxygen consumption below these concentrations and have revealed a commensurate decrease i n the r e s p i r a t i o n rate as surrounding oxygen tensions approached zero. Expanding on the Boag model, Marshall et a l . (1986) propose that, at very low oxygen tensions, the d i f f u s i o n gradient becomes too small to provide oxygen to a l l areas of the c e l l ( F i g . 5) and forces the c e l l ' s r e s p i r a t i o n rate to decrease. I f the oxygen tension i n the c e l l environment decreases even further, the e s s e n t i a l l y anoxic c e l l i s hypothesized to act s i m i l a r l y to a p o l a r i z e d electrode, i n s t a n t l y consuming any oxygen molecule that i t encounters. f 14 Figure 4 Boag's model of r e s p i r a t i o n induced oxygen gradients inside and immediately surrounding si n g l e c e l l s i n suspension. Approximately one t h i r d of the gradient i s predicted to l i e within the c e l l (r<a). ^ m i n f o r f u l l r e s p i r a t i o n a = c e l l r a d i u s K = 0 2 c o n s u m p t i o n r a t e p e r u n i t c e l l v o l u m e D = d i f f u s i o n c o n s t a n t f o r 0 2 i n w a t e r Figure 5 Gradient l i m i t e d c e l l r e s p i r a t i o n at low oxygen tensions. Reduced c e l l r e s p i r a t i o n rates are a t t r i b u t e d to d i f f u s i o n gradients that are too small to supply oxygen to a l l areas within the r e s p i r i n g c e l l . 16 1.4 I n t r a c e l l u l a r Oxygen Gradients The oxygen gradient mechanism i s a e s t h e t i c a l l y appealing because i t i s able to quantify the complex b i o p h y s i c a l process of c e l l u l a r r e s p i r a t i o n with conceptual and mathematical s i m p l i c i t y . From the perspective of cancer research, however, l o c a l i z e d and i n t r a c e l l u l a r oxygen gradients not only form the basis of an elegant model, but may also provide explanations f o r experimental observations i n v o l v i n g oxygen and the radio- and chemo-sensitivity of c e l l s . As noted previously, the radioprotective properties of t h i o l s may be l a r g e l y a t t r i b u t e d to the existence of such gradients. The consumption of i n t r a c e l l u l a r oxygen by t h i o l s at low oxygen tensions may r e s u l t i n a more hypoxic c e l l nucleus and "protect" the i r r a d i a t e d c e l l from the i r r e p a i r a b l e damage that might otherwise be caused by oxygen's l e s i o n f i x a t i o n properties. The existence of i n t r a c e l l u l a r oxygen gradients may also be c r i t i c a l i n evaluating the performance of, and a s c e r t a i n i n g the mechanisms by which oxygen-sensitive chemotherapeutic drugs function. Such gradients would c a l l into question the accuracy with which oxygen tensions at the c e l l target are being predicted and, consequently, the degree to which oxygen a c t u a l l y i n h i b i t s drug binding. I n t r a c e l l u l a r oxygen gradients may even disguise the nature and the mechanism of the oxygen e f f e c t i t s e l f . V a riations i n c e l l u l a r r a d i o s e n s i t i v i t y at low oxygen tensions, caused by factors a f f e c t i n g these gradients, may be mistakenly a t t r i b u t e d to a c e l l u l a r r e p a i r process or multiple r a d i a t i o n targets and oxygen s e n s i t i z a t i o n mechanisms. 17 I t i s notable that i n t r a c e l l u l a r oxygen d i f f u s i o n gradients are not purely t h e o r e t i c a l ; evidence supporting t h e i r existence has been documented. A comparison of the oxygen-dependent mitochondrial a c t i v i t y of i n t a c t c e l l s with that of i s o l a t e d mitochondria suggests that oxygen gradients e x i s t i n both i s o l a t e d r a t cardiac c e l l s and r a t hepatocytes (Jones and Mason, 1978; Wittenberg and Wittenberg, 1981; Gayeski et a l . , 1986). Fluorescent probes have been used to demonstrate the existence of s i m i l a r gradients i n mouse l i v e r c e l l s (Benson et a l . , 1980; Podgorski et a l . , 1981). I n d i r e c t l y , the r e s p i r a t i o n measurements made by Marshall et al.(1986) also imply the existence of i n t r a c e l l u l a r gradients. Cancer therapies frequently involve one or more oxygen-dependent mechanisms. I f r e s p i r a t i o n induced i n t r a c e l l u l a r oxygen gradients, such as those p r e d i c t e d by Boag and supported by the preliminary empirical evidence e x i s t , they could play a s i g n i f i c a n t r o l e i n the function of these mechanisms. An experimental analysis which would e i t h e r disprove or confirm the existence and nature of these gradients would also provide a c l e a r e r understanding of the r e l a t i o n s h i p between the therapeutic and r e s p i r a t o r y u t i l i z a t i o n of c e l l u l a r oxygen. 1.5 Experimental Rationale and Thesis Objectives The r a d i o s e n s i t i z i n g properties of oxygen are well established even i f they are not f u l l y understood. The oxygen enhancement r a t i o f or the X-ray induced k i l l i n g of cultured mammalian c e l l s requires approximately three times the dose received under aerobic conditions to be d e l i v e r e d under hypoxic conditions i f the same f r a c t i o n of c e l l s are 18 to be k i l l e d . Furthermore, oxygen mediated r a d i o s e n s i t i z a t i o n , as modelled by the Alper and Howard-Flanders equation, i s very s e n s i t i v e to changes i n oxygen concentration at low oxygen tensions. This r a i s e d the p o s s i b i l i t y of using r a d i o s e n s i t i v i t y as a gauge of the oxygenation status of the c e l l target. The existence of an oxygen-depleted zone around the c e l l nucleus could be confirmed by exposing both r e s p i r i n g and non-respiring c e l l s to the same low e x t r a c e l l u l a r oxygen tension during i r r a d i a t i o n . According to Boag's theory (1970), the r e s p i r i n g c e l l s should then deplete t h e i r i n t r a c e l l u l a r oxygen, create an oxygen gradient and, consequently, display l e s s r a d i o s e n s i t i v i t y than non-r e s p i r i n g c e l l s , f o r which the i n t r a c e l l u l a r and d i s t a n t e x t r a c e l l u l a r oxygen tensions remain the same. The magnitude of these r e s p i r a t i o n induced gradients could then be approximated from the observed r a d i o s e n s i t i v i t y d i f f e r e n t i a l and compared with t h e o r e t i c a l p r e d i c t i o n s . C e l l shape was l i k e l y to be an important f a c t o r i n the expression of these gradients, the average distance that oxygen molecules must d i f f u s e through the cytoplasm to reach uniformly d i s t r i b u t e d mitochondria being larger f o r s p h e r i c a l c e l l s than f or c e l l s growing i n a f l a t monolayer. I f the oxygen concentrations at the surface of both f l a t and s p h e r i c a l c e l l s are the same, the rounder c e l l s should have la r g e r i n t r a c e l l u l a r oxygen gradients and, hence, allow correspondingly l e s s oxygen to penetrate to the c e l l center. The challenge and the s p e c i f i c goal addressed i n t h i s t h e s i s , therefore, was to develop an experimental protocol that was capable of detecting these r e s p i r a t i o n induced i n t r a c e l l u l a r gradients. I t was hoped that during the course of the experimental i n v e s t i g a t i o n s , the following questions would be answered: 1 9 1. Is r a d i o s e n s i t i v i t y a s u f f i c i e n t l y s p e c i f i c and resolvable i n d i c a t o r of c e l l oxygenation? 2. Can r e s p i r a t i o n induced i n t r a c e l l u l a r oxygen gradients of the magnitude predicted by theory be shown to exist? 3. What i s the r o l e of c e l l shape i n determining the nature and magnitude of such gradients? 2. CELL CULTURE The c o n t r o l l e d manipulation of c e l l r e s p i r a t i o n and c e l l oxygenation required i n the proposed r a d i a t i o n s u r v i v a l experiments could best be c a r r i e d out i n a simple cultured c e l l system. Although i n v i t r o research i s subject to the c r i t i c i s m that i t includes the i m p l i c i t , and at times, questionable assumption that information obtained from cultured c e l l s reasonably r e f l e c t s the behavior or properties of c e l l s i n v i v o , for the purposes of t h i s t h e s i s , the use of a cu l t u r e d c e l l system was considered j u s t i f i e d by the i n t r i n s i c nature of the c e l l p roperties being investigated and the b e l i e f that the importance of these properties to normal c e l l function was l i k e l y to produce the same response regardless of the growth environment. A l l experimental r e s u l t s i n t h i s thesis were, subsequently, obtained with cultured V79-171 Chinese hamster lung f i b r o b l a s t s . These c e l l s are a sub-line of non-malignant f i b r o b l a s t s that were f i r s t i s o l a t e d and cultured by Ford and Yerganian (1958). A number of sub-20 l i n e s have since been cloned and are being used extensively i n r a d i o b i o l o g i c a l research. A major reason for the p o p u l a r i t y of these c e l l s among f a d i o b i o l o g i s t s i s t h e i r a b i l i t y to serve as i n v i t r o tumor models (Sutherland et a l . , 1970; Durand, 1972; Sutherland and Durand, 1976). F i b r o b l a s t s have also been dubbed "the weeds of the tissue c u l t u r i s t ' s garden" (Freshney, 1987), however, and t h e i r primary appeal i s probably due to t h i s more basic property. Cultured f i b r o b l a s t s are r e l a t i v e l y e a s i l y maintained and p r o l i f e r a t e r a p i d l y with minimal n u t r i t i o n a l and environmental requirements. The V79 c e l l s used i n the following experiments were grown i n Eagle's minimal e s s e n t i a l medium, MEM, supplemented with 10% f e t a l c a l f serum (Gibco). A 100 mm x 20 mm p l a s t i c P e t r i dish (Falcon) containing 10 ml of growth medium provided s u f f i c i e n t nutrients and space f o r the development of a confluent monolayer of the order of 10^ c e l l s . A bicarbonate/carbon dioxide b u f f e r maintained a pH of 7.2-7.4 i n the medium when the c e l l s were incubated i n 5% CO2. A 37°C incubation temperature was used to mimic the optimal i n v i v o conditions f o r mammalian c e l l growth. Unfortunately, these temperature and pH conditions also favor the growth of b a c t e r i a and fungi. To prevent contamination, therefore, the c e l l s , medium and the c u l t u r i n g equipment with which they came into contact were always handled under s t r i c t l y s t e r i l e conditions. The c e l l l i n e was propagated by means of routine bi-weekly sub-c u l t u r i n g . Since monolayer f i b r o b l a s t s anchor themselves f i r m l y to the bottom of the culture dish, a proteinase enzyme was required f o r the t r a n s f e r procedure. A f t e r a s p i r a t i n g the medium from the dish, a l l traces of serum p r o t e i n were removed by r i n s i n g the exposed c e l l s with 2 21 ml of a c i t r a t e b u f f e r s o l u t i o n containing 0.1% t r y p s i n (Gibco). Following an 8 minute incubation with a second 2 ml volume of enzyme so l u t i o n , the t r y p s i n was i n a c t i v a t e d and the loosened c e l l s suspended by adding 10 ml of medium containing 10% FCS. Any remnants of the i n t a c t monolayer were disaggregated to s i n g l e c e l l s by gently p i p e t t i n g the suspension. P e t r i dishes containing fresh medium were inoculated with a l i q u o t s of approximately 10^ c e l l s and were then incubated under the appropriate temperature and pH conditions. A t y p i c a l V79 growth curve i s seen i n F i g . 6a. A f t e r being transfered to a P e t r i dish containing fresh medium, the c e l l s pass through a short l a g period before beginning to divide at an exponential rate. The average c e l l c ycle time during exponential growth i s approximately 11 hours (Fig. 6b). I f s u f f i c i e n t nutrients and favorable growth conditions are supplied, the c e l l s w i l l continue to divide u n t i l they form a confluent monolayer that covers the base of the P e t r i dish. At t h i s point, population growth i s i n h i b i t e d and the c e l l s move out of c y c l e . I f renewed growth conditions are not provided, the depletion of nutrients and accumulation of c e l l waste w i l l r e s u l t i n the eventual death of the c e l l s . Figure 6 Growth of V79 Chinese hamster c e l l s i n 100 mm x 20 mm p l a s t i c P e t r i dishes. Panel (a) p l o t s the growth of the c e l l population per P e t r i d i s h with time and panel (b) shows the c a l c u l a t e d average c e l l cycle time during the exponential growth phase. Time (hours) 23 3. GENERAL EXPERIMENTAL METHODS 3.1 Clonogenic Su r v i v a l and Data Analysis The experiments i n t h i s thesis were based on the premise that the existence of i n t r a c e l l u l a r oxygen gradients at low oxygen tensions w i l l cause r e s p i r i n g c e l l s to be measurably less r a d i o s e n s i t i v e than non-r e s p i r i n g c e l l s . The foremost requirement i n the development of an experimental protocol was, therefore, to choose an appropriate c e l l u l a r r a d i o s e n s i t i v i t y assay. Because reproductive c e l l death i s an e a s i l y determined experimental endpoint, the r a d i o s e n s i t i v i t y of the c e l l s was most s u i t a b l y and e f f i c i e n t l y measured using the clonogenic s u r v i v a l assay (Puck and Marcus, 1956). The assay derives i t s name from the a b i l i t y of v i a b l e s i n g l e c e l l s i n culture to divide and produce "colonies" or aggregates of genetic clones (Fig. 7). With t h i s assay, the e f f e c t of a given treatment on the p r o l i f e r a t i v e a b i l i t y of c e l l s i s measured by the r a t i o of the number of c e l l colonies produced to the number of treated c e l l s i n i t i a l l y cultured. According to the Puck c r i t e r i o n , a treated c e l l i s considered a survivor i f i t i s capable of producing a colony containing 50 or more c e l l s (Puck and Marcus, 1956). The measured r a t i o i s , hence, termed the su r v i v i n g f r a c t i o n . The data obtained from the s u r v i v a l assay i s us u a l l y displayed i n a semi-logarithmic p l o t of the su r v i v i n g f r a c t i o n of a treated c e l l population versus the treatment dose. T y p i c a l r a d i a t i o n s u r v i v a l data f o r an X - i r r a d i a t e d mammalian c e l l population are shown i n F i g . 8a. In p r a c t i c e , the c a l c u l a t i o n of a surviving f r a c t i o n also includes a 24 Figure 7 V79 c e l l colony formation. These colonies were formed from s i n g l e c e l l s a f t e r one week of incubation. The colonies formed by the i r r a d i a t e d c e l l s ( l e f t ) are notably smaller and more poorly defined than those of the unirradiated population ( r i g h t ) . Figure 8 Ty p i c a l r a d i a t i o n s u r v i v a l data f o r V79 c e l l s i r r a d i a t e d under anoxic conditions. Panel (a) shows the raw data obtained with the clonogenic s u r v i v a l assay and panel (b) shows the same data normalized and f i t t e d with the Linear Quadratic s u r v i v a l model. i 1 1 1 1 1 1 1 1 | 1 1 1 1 1 1 1 r i i • i i i i i I I i 1 1 1 1 1 1 1— 0 20 40 0 20 40 Dose (Gray) 26 " p l a t i n g e f f i c i e n c y " c o r r e c t i o n to account for the c e l l s that f a i l to form colonies f o r reasons other than exposure to the s p e c i f i e d treatment. An experimental c o n t r o l or zero dose data point w i l l provide an approximate value f o r t h i s c o r r e c t i o n f a c t o r , but i n order to minimize the uncertainty associated with a si n g l e data point, i t i s u s u a l l y preferable to f i t a curve to the s u r v i v a l data and then to normalize the data with respect to the curve's zero int e r c e p t ( Fig. 8b). F i t t i n g a curve to the s u r v i v a l data requires a mathematical expression that can r e l a t e c e l l s u r v i v a l to the treatment dose. Several models describing the e f f e c t of r a d i a t i o n on c e l l v i a b i l i t y have been developed and, w i t h i n the bounds of experimental and b i o l o g i c a l uncertainty, f i t the r a d i a t i o n s u r v i v a l data equally w e l l . The Linear Quadratic model used to f i t the data i n t h i s thesis ( F i g . 9) postulates that there are two components of c e l l k i l l i n g , one that i s proportional to the r a d i a t i o n dose and dominates i n the i n i t i a l or shoulder region of the s u r v i v a l curve, and one that i s proportional to the square of the dose and becomes s i g n i f i c a n t i n the l a t t e r p o r t i o n of the curve ( S i n c l a i r , 1966; K e l l e r e r and Rossi, 1972; Chadwick and Leenhouts, 1973; 1981). Like other multiparameter s u r v i v a l models, the LQ model generally f i t s r a d i a t i o n s u r v i v a l data well, p a r t i c u l a r l y i n the f i r s t few decades of the s u r v i v a l curve, but also has the a d d i t i o n a l advantage of r e q u i r i n g the optimization of only two v a r i a b l e parameters. A more d e t a i l e d d i s c u s s i o n of the LQ model and other s u r v i v a l models can be found i n the appendix (Sec. 8.2). Figure 9 Relative contributions of the two components of c e l l k i l l i n g de by the Linear Quadratic model of r a d i a t i o n s u r v i v a l . T 1 I 1 1 1 r I l 1 1 1 I I i I 0 20 40 D o s e , D ( G r a y ) 28 3.2 Respiratory I n h i b i t i o n To obtain an accurate measurement of the e f f e c t of a treatment on c e l l v i a b i l i t y using the clonogenic s u r v i v a l assay, a s t a t i s t i c a l l y s i g n i f i c a n t f r a c t i o n of treated c e l l s must remain v i a b l e , and a l l f a c t o r s a f f e c t i n g the colony forming a b i l i t y of the c e l l s must be qu a n t i f i a b l e . The use of clonogenic s u r v i v a l as a measure of the r a d i o s e n s i t i v i t y of r e s p i r a t i o n i n h i b i t e d c e l l s , therefore, requires the t o x i c i t y of the r e s p i r a t o r y i n h i b i t i o n to be s u f f i c i e n t l y small not to obscure or i n t e r f e r e with the r a d i a t i o n s u r v i v a l c h a r a c t e r i s t i c s of the i n h i b i t e d c e l l s . Biochemical i n h i b i t o r s , such as rotenone and cyanide, have been used to induce varying degrees of r e s p i r a t o r y i n h i b i t i o n i n cultu r e d c e l l s . These are toxic at the dose l e v e l s required to completely i n h i b i t c e l l r e s p i r a t i o n (Durand and Biaglow, 1974; Durand and Biaglow, 1976), however, and may introduce a d d i t i o n a l complications to the r a d i o s e n s i t i v i t y assay. Under c e r t a i n conditions, r e s p i r a t o r y i n h i b i t o r s have even been found to protect c e l l s against r a d i a t i o n damage (Biaglow et a l . , 1983b). An a l t e r n a t i v e to using these r e s p i r a t o r y toxins was to cool the c e l l s and slow t h e i r metabolism n a t u r a l l y . The exposure of c e l l s to low temperatures i s an e f f e c t i v e and e a s i l y r e v e r s i b l e method of i n h i b i t i n g c e l l r e s p i r a t i o n and i s r e l a t i v e l y non-toxic i f used f o r short periods (see Sec. 5.1.8). The temperature induced i n h i b i t i o n of r e s p i r a t i o n was, consequently, adopted as the basis upon which r a d i o s e n s i t i v i t y comparisons were made. 29 3.3 Choice of I r r a d i a t i o n Vessels Because the most appropriate manner i n which to i r r a d i a t e the c e l l s was not i n i t i a l l y obvious, two d i f f e r e n t methods were employed and evaluated. The f i r s t s e r i e s of experiments was performed with c e l l s i n a s t i r r e d s i n g l e c e l l suspension culture. This was followed by a more extensive s e r i e s of experiments using a modified t h i n f i l m culture technique. In the i n t e r e s t s of c l a r i t y and the l o g i c a l development of the respective methods, a d e s c r i p t i o n of the r a t i o n a l e , the experimental protocol, and a discussion and analysis of the r e s u l t s w i l l be presented seperately f o r each of these techniques. 4. THE SINGLE CELL SUSPENSION 4.1 Materials and Methods In designing an experimental protocol, an attempt was made to optimize factors that would create the maximum l i k l i h o o d f o r a p o s i t i v e r e s u l t . C e l l shape was hypothesized to be such a f a c t o r . According to Boag's (1969) r e s p i r i n g c e l l model, c e l l shape can determine the magnitude of the r e s p i r a t i o n induced i n t r a c e l l u l a r oxygen gradient by increasing or decreasing the distance that oxygen molecules must penetrate to reach the mitochondria. From the perspective of maintaining a geometrical s i m p l i c i t y i n modelling the d i f f u s i o n process, and also to create the maximum p o t e n t i a l f o r "radioprotection" by oxygen depletion, i t was, therefore, desirable to use c e l l s that were sp h e r i c a l i n shape. The f i r s t s e r i e s of r a d i a t i o n s u r v i v a l experiments were 30 consequently performed with c e l l s i n a s t i r r e d suspension s i m i l a r to that used by Boag (1970). A mi t i g a t i n g f a c t o r i n the choice of t h i s experimental apparatus was the r e l a t i v e l y easy access to the necessary suspension culture equipment, eliminating the need f o r expensive purchases or time-consuming custom construction. A modified B e l l c o spinner f l a s k was used as the suspension culture and i r r a d i a t i o n v e s s e l ( Fig. 10). The f l a s k was water-jacketed, which allowed the temperature of the c e l l suspension to be c o n t r o l l e d by means of a r e c i r c u l a t i n g water bath (Haake). The oxygen content of the gas ove r l y i n g the s t i r r e d suspension was regulated by flowmeters and a mixing chamber (Matheson), which combined oxygen and nitrogen from compressed gas cyl i n d e r s (Canadian L i q u i d A i r ) i n the desired proportions and metered the mixture at a flow rate of 10 1 hr~^. To prevent excessive condensation or evaporation within the spinner f l a s k , the mixed gas was humidified by bubbling i t through a temperature c o n t r o l l e d gas washing b o t t l e before i t passed into the f l a s k . With the exception of two s i l i c o n stoppers and a t e f l o n coated s t i r bar, the e n t i r e gassing pathway was constructed of e i t h e r glass or s t a i n l e s s s t e e l . This was designed to minimize the unregulated d i f f u s i o n of oxygen into the system. As a f i n a l measure of the system's atmospheric i n t e g r i t y , the e f f l u e n t gas from the f l a s k was monitored by an oxygen analyzer (Applied Electrochemistry) and a record of the oxygen tension i n the f l a s k produced with the use a s t r i p chart recorder. To maintain the f l a s k as an i s o l a t e d system throughout the experiment, a sampling technique employing a s t a i n l e s s s t e e l cannula and valve was devised. The cannula t i p was h e l d below the surface of the s t i r r e d medium by a s i l i c o n stopper secured i n one of the ports at the 31 Figure 10 Experimental apparatus for the suspension culture i r r a d i a t i o n s . mixing tube and flowmeter stopcock water-}acketed f l a s k ( \ / humidif i e r §1 • 2 gas ana l y z e r X - r a y m a c h i n e s t i r r e r X c h a r t r e c o r d e r top of the f l a s k . This allowed samples to be drawn up into a disposable syringe without compromising the oxygen content of the gas overlying the suspension. During the sampling procedure, two samples were taken, the f i r s t of which was discarded to remove any c e l l s that might have been insi d e the cannula during the i r r a d i a t i o n . The c e l l suspension was i r r a d i a t e d through the side of the f l a s k by a Picker 270 kVp X-ray machine. To ensure that subsequent c e l l suspensions would be exposed to the same 4.0 Gy min" 1 dose rate, a p l e x i g l a s j i g was used to hold the spinner f l a s k i n place. (The dose rate was measured i n a mock experimental i r r a d i a t i o n using Victoreen 500 electrometer with 0.6 cc ion chamber probe.) A magnetic s t i r r e r beneath the j i g kept the c e l l s i n suspension at a non-vortexing 180 rpm. This s t i r r i n g speed caused no measurable damage to the c e l l s and allowed the gas exchange process to be mathematically modelled. 4.2 Experimental Protocol The experimental protocol f o r a t y p i c a l suspension culture i r r a d i a t i o n can be summarized as follows (Fig. 11): Seed plates were prepared approximately 2.5 days p r i o r to the experiment. Twenty-four p l a s t i c P e t r i dishes containing 10 ml medium (MEM + 10% FCS) were inoculated with 10 4 V79 Chinese hamster lung f i b r o b l a s t c e l l s and incubated at 37°C i n 5% CO2. Since v a r i a t i o n s i n the r a d i o s e n s i t i v i t y of the d i f f e r e n t c e l l populations can be caused by dif f e r e n c e s i n t h e i r c e l l cycle d i s t r i b u t i o n s ( S i n c l a i r and Morton, 1963), attempts were made to minimize such e f f e c t s by choosing the inoculate number so that the seed c e l l population would always be i n Figure 11 Protocol flowchart f o r the suspension culture experiments 2.5 days p r i o r to experiment: prepare 24 seed pl a t e s : 2x10^ V79 c e l l s i n 10 ml MEM + 10% FCS day of experiment: (repeat three times) 10 6 c e l l s p l a t e " 1 and t r y p s i n i z e 6 plates suspend i n MEM + 10% FCS @ 10 J c e l l s ml" 4 place 50 ml of suspension i n modified B e l l c o spinner f l a s k 4-e q u i l i b r a t e f or 1 hour @ given oxygen concentration and 37°C or 4°C gassing rate: 10 1 hr s t i r speed: 180 rpm 4 take sample and place i n previously prepared -» d i l u t i o n tube containing <-MEM + 10% FCS @ 20°C -1 i r r a d i a t e f l a s k @ 4.0 Gray min" 1 d i l u t e samples i f required and p l a t e i n 3 P e t r i dishes to produce 300 - 500 colonies 4 incubate f o r 6-7 days before s t a i n i n g and counting colonies count remainder o sample w/ Coulter counter 34 an exponential growth phase on the day of the experiment. The experimental procedure was s t a r t e d by r o u t i n e l y t r y p s i n i z i n g s i x of the dishes, each of which contained monolayers of approximately 10^ c e l l s , and suspending the c e l l s i n MEM + 10% FCS. The suspended 5 1 c e l l s were then pooled at a density of 10 c e l l s ml . A 50 ml a l i q u o t of t h i s suspension was pipetted into the modified Be l l c o spinner f l a s k , which was then placed into the p l e x i g l a s j i g attached to the X-ray machine and e q u i l i b r a t e d at the desired oxygen concentration and at e i t h e r 4°C or 37°C for one hour p r i o r to i r r a d i a t i o n . The gas flow rate was set at 10 1 hr""'" and the s t i r speed of the magnetic s t i r r e r was 180 rpm. Before the i r r a d i a t i o n was started, a c o n t r o l sample was taken and placed i n a previously prepared d i l u t i o n tube containing s u f f i c i e n t medium to make up a t o t a l volume of 10 ml. The c e l l suspension was i r r a d i a t e d i n cumulative 2.0 - 6.0 Gy increments with samples being taken a f t e r each increment. Sample sizes were minimized so that the volume of the suspension cul t u r e , and, hence, the c r o s s - s e c t i o n a l area being i r r a d i a t e d changed by le s s than 7% throughout the experiment. The sample al i q u o t s v a r i e d from 0.25 - 5.0 ml and were held i n t h e i r respective d i l u t i o n tubes at room temperature u n t i l the i r r a d i a t i o n procedure was completed. I f required according to predicted s u r v i v a l rates, each sample of c e l l s was further d i l u t e d with medium before a 0.5 ml volume ( s u f f i c i e n t to produce 300 - 500 colonies) was pipetted into each of three P e t r i dishes containing 10 ml of growth medium. In order to obtain an accurate estimate of the number of c e l l s pipetted into the dishes, the c e l l d e n s i t i e s of the d i l u t e d samples from which the c e l l s were plated 35 were measured using a Coulter c e l l counter. The culture dishes were then incubated at 37°C i n 5% CO^ and the i r r a d i a t i o n procedure was repeated f o r the next s i x seed plates at a d i f f e r e n t oxygen tension. When completed, t h i s protocol produced r a d i a t i o n s u r v i v a l data at four oxygen tensions. A f t e r a one week incubation period, the medium was removed from a l l of the dishes and the c e l l colonies were stained with a malachite green s t a i n i n g s o l u t i o n . A f t e r r i n s i n g and drip drying the dishes, colonies containing 50 or more c e l l s were counted and recorded. 4.3 Results The r a d i a t i o n s u r v i v a l r e s u l t s for the suspension culture experiments are displayed i n sets of four, corresponding to the four f l a s k s used f o r each pooled c e l l population. For three of the f l a s k s i n each set, the gas above the c e l l suspension contained a low l e v e l of oxygen. The fourth f l a s k was i r r a d i a t e d under aerobic conditions (95% a i r 5% CO2) and was used to characterize the general r a d i a t i o n s u r v i v a l properties of the seed c e l l population. A l l c a l c u l a t i o n s of experimental uncertainty were made i n accordance with the method proposed by Boag (1975). Using Poisson sampling s t a t i s t i c s , the variance i n c e l l s u r v i v a l was based on the t o t a l number of colonies counted at a given dose rather than on the v a r i a t i o n i n the number of colonies on each dish. 36 4.3.1 Radio-response of r e s p i r i n g c e l l s The raw c e l l s u r v i v a l data f o r i r r a d i a t i o n s performed at 37°C are p l o t t e d i n F i g . 12. The data are displayed i n t h i s manner because t h e i r b iphasic nature prevents the usual c u r v e - f i t t i n g and normalization procedure. Biphasic c h a r a c t e r i s t i s t i c s are p a r t i c u l a r l y noticable i n the data from f l a s k s i n which the gas contained 1% oxygen or l e s s . These p l o t s t y p i c a l l y have a steep slope over the f i r s t few dose points i n the shoulder region before breaking and decreasing more gently at higher doses. Multiphasic s u r v i v a l data i s u s u a l l y i n d i c a t i v e of heterogeneously s e n s i t i v e c e l l populations. The r i g i d c onfiguration of c e l l s i n tumors or tumor models, f o r example, l i m i t s the a b i l i t y of oxygen to d i f f u s e to c e l l s that are d i s t a n t from the oxygen supply (Durand, 1983b). S p a t i a l l y s t r a t i f i e d c e l l subpopulations with varying degrees of oxygen enhanced r a d i o s e n s i t i v i t y w i l l develop under such conditions. I f a representative c r o s s e c t i o n of tumor or tumor model c e l l s produces a biphasic r a d i a t i o n s u r v i v a l curve, the approximate proportions of hypoxic and oxic c e l l s can be deduced from the zero dose intercepts of the two phases (Fig. 13). A biphasic response i n a s t i r r e d s i n g l e c e l l suspension must be inte r p r e t e d d i f f e r e n t l y , however. Although degrees of s p a t i a l l y conferred oxygen-mediated r a d i o s e n s i t i v i t y may also e x i s t i n s t i r r e d suspensions (Whillans and Rauth, 1980), the method by which samples at the various dose points were obtained makes i t very u n l i k e l y that these r e s u l t s represent the existence of s p a t i a l l y s t r a t i f i e d subpopulations of c e l l s . A more probable explanation i s that the amount of dissolved Figure 12 Non-normalized suspension culture r a d i a t i o n s u r v i v a l data for r e s p i r i n g (37°C) c e l l s . A biphasic r a d i a t i o n response prevented use of conventional curve f i t t i n g and normalization procedures. Each panel shows the r e s u l t s of a four f l a s k experiment. C o •rH u ro 1 .0 O 2000 ppm ~z • 5000 ppm : A 10000 ppm O a i r 0.1: cn c •r-\ • H O . 0 1 > c D L0 \ A Os ^ A. (a) 0 \ A O 10 —1— 1 1 O i < i 200 ppm 1 > • 500 ppm : i<>g A O 1000 ppm ; a i r o -: O " (b) > \ 20 0 10 Dose (Gray) 20 30 Figure 13 Hypothetical biphasic r a d i a t i o n response of a heterogeously s e n s i t i v e c e l l population i n which 30% of the c e l l s are hypoxic. S p a t i a l v a r i a t i o n s i n r a d i o s e n s i t i v i t y producing t h i s type of response are commonly seen i n tumor models such as spheroids. i 1 I I | 1 I 1 1 1 1 r i i i i I i i i i L i L 0 10 20 D o s e ( G r a y ) 39 oxygen i n the suspension medium and, hence, the r a d i o s e n s i t i v i t y of the e n t i r e c e l l population changed over the course of the experiments (Koch et a l . ; 1973). In other words, the biphasic nature of the response i s due to a temporal, rather than s p a t i a l v a r i a t i o n i n c e l l oxygenation. The shape of the s u r v i v a l data implies that the oxygen concentration of the suspension medium was not i n equilibrium with that of the gas above i t at the end of the allowed one hour e q u i l i b r a t i o n period. I f an eq u i l i b r i u m c o n d i t i o n was achieved, i t was only a f t e r r a d i o l y t i c a l l y produced free r a d i c a l s depleted the oxygen surplus during the i n i t i a l stages of the i r r a d i a t i o n procedure. This i s supported by the steeper than expected dose response obtained over the f i r s t few dose points i n a l l but the highest oxygen content f l a s k s . Since r a d i a t i o n s u r v i v a l models do not account f o r v a r i a t i o n s i n the conditions of i r r a d i a t i o n , the biphasic nature of the s u r v i v a l data pre-empted the use of the usual c u r v e - f i t t i n g methods f o r the c a l c u l a t i o n of p l a t i n g e f f i c i e n c y values. Therefore, because the suspension medium only attained the desired oxygen tension a f t e r a dose of several Gray, the f i r s t two or three data points i n each of the data sets were ignored and the i n i t i a l p o r t i o n of the desired s u r v i v a l curve approximated by extrapolating back from the higher dose data. The inte r c e p t s u r v i v a l values were then used to normalize the curves f i t t e d to the high dose data ( F i g 14). 4.3.2 Radioresponse of the non-respiring c e l l s With the exception of the f l a s k i n which the gas mixture contained only 200 ppm oxygen, the c e l l s i r r a d i a t e d at 4°C do not d i s p l a y the 40 Figure 14 Normalized and f i t t e d suspension culture r a d i a t i o n s u r v i v a l data for r e s p i r i n g (37°C) c e l l s . Data were f i t t e d with the LQ model of r a d i a t i o n s u r v i v a l by ignoring the steeply decreasing i n i t i a l s u r v i v a l points for each curve. Figure 15 Normalized and f i t t e d suspension culture r a d i a t i o n s u r v i v a l data f o r non-respiring (4°C) c e l l s . Steeply f a l l i n g s u r v i v a l curves imply a lack of d i f f u s i v e e quilibrium between the oxygen concentrations of gas and l i q u i d phases of the suspension culture. At very low oxygen tensions i n the gas, dif f e r e n c e s i n the radioresponse become d i f f i c u l t to d i s t i n g u i s h . 42 biphasic s u r v i v a l c h a r a c t e r i s t i c s of those i r r a d i a t e d at 37°C (Fig. 15). The suspension cultures i r r a d i a t e d at 4°C are c o n s i s t e n t l y and more hi g h l y r a d i o s e n s i t i v e than those i r r a d i a t e d at 37°C with the same oxygen concentrations i n the gas. The dose responses are also considerably steeper than would be expected i f a d i f f u s i o n equilibrium had a c t u a l l y e x i s t e d f o r the various oxygen tensions i n the gas. This i s not s u r p r i s i n g when i t i s considered that i f a d i f f u s i v e equilibrium was not achieved at temperatures at which c e l l s were able to r e s p i r e and, therefore, a i d i n the removal of excess oxygen from the suspension medium, that i t was also not attained when such r e s p i r a t i o n was i n h i b i t e d ! The r a d i o l y t i c depletion of oxygen was by i t s e l f i n s u f f i c i e n t to cause the s u r v i v a l curves to break. A f a c t o r that undoubtedly contributed to the greater r a d i o s e n s i t i v i t y of the c e l l s i r r a d i a t e d at 4°C i s the increased s o l u b i l i t y of oxygen i n aqueous solutions (Boag, 1970). Oxygen i s almost twice as soluble i n water at 4°C as i t i s at 37°C ( F i g . 16). The s o l u b i l i t y of oxygen i n the gas phase also increases over t h i s temperature range, but by a considerably smaller degree. Low temperatures w i l l , therefore, not only decrease the rate at which oxygen d i f f u s e s out of so l u t i o n , but w i l l also r e s u l t i n a suspension medium with a s i g n i f i c a n t l y higher equilibrium oxygen concentration. The information salvaged from the r e s u l t s of these r a d i a t i o n s u r v i v a l experiments was, hence, not d i r e c t l y u seful to the stated objectives of t h i s t h e s i s . Although the s u r v i v a l curves d i d show a q u a l i t a t i v e increase i n r a d i o s e n s i t i v i t y with increasing oxygen tension, the data was frequently quite scattered and the r e s o l u t i o n of diffe r e n c e s i n radioresponses at low oxygen tensions was poor. The need 43 Figure 16 V a r i a t i o n of the oxygen concentration i n a i r and i n a i r - e q u i l i b r a t e d water with temperature at standard pressure (1 atm). The f r a c t i o n a l change i n the oxygen concentration over the range of 4°C to 37°C i s notably smaller i n a i r than i n water. ZL 400 c_ ra 2 OJ o 300 200 -- 9000 •rH CO 8500-5 o - 8000 0 20 40 Temperature (°C) [ 0 2 ] i n a i r - e q u i l i b r a t e d w a t e r : { 4 3 9 - 1 2 . 2 T + 0 . 2 3 9 T 2 - 0 . 0 0 1 9 T 3 } ( B o a g , 1969) [0 2 3 i n a i r a t 1 a t m : {P+ ( a 2 / ( 1 / [ 0 2 ] +b) 2 ) } 0 . 2 0 9 5 / R T R = 0 . 0 8 2 0 6 a t m ° K _ 1 M - 1 a = 1 . 3 6 a t m M " 2 b = 0 . 0 3 1 8 3 M " 1 4 4 to process the r e s p i r i n g c e l l data i n the manner described above, also added uncertainty to the r e s u l t s and reduced the confidence with which diff e r e n c e s between low oxygen tension s u r v i v a l curves could be claimed. Unfortunately, although differences i n r a d i o s e n s i t i v i t y due to the hypothesized i n t r a c e l l u l a r oxygen gradients would be most evident at low oxygen tensions, the poor r e s o l u t i o n of the data d i d not allow any relevant conclusions to be drawn. 4.3.3 Sources of error The suspension culture experiments were subject to several sources of e r r o r that contributed to preventing the detection of r e s p i r a t i o n induced, l o c a l i z e d differences i n c e l l r a d i o s e n s i t i v i t y . The a l t e r n a t i n g i r r a d i a t i o n and sampling procedure required the repeated opening and c l o s i n g of the shutter on an unpredictable 30 year o l d X-ray machine. I t i s suspected that t h i s may have r e s u l t e d i n the accumulation of errors i n dose measurement. D i l u t i o n and colony counting errors incurred through the use of the clonogenic assay, were magnified by the inexperience of the researcher and contributed to the random s c a t t e r of the data points. Most s i g n i f i c a n t l y , however, the combined suspension culture r e s u l t s i n d i c a t e that any v a r i a t i o n i n c e l l u l a r r a d i o s e n s i t i v i t y that might have been present due to the existence of r e s p i r a t i o n induced i n t r a c e l l u l a r oxygen gradients was obscured by the much greater and uncontrolled v a r i a b i l i t y i n the oxygen content of the suspension medium. This was not believed to be due to oxygen contamination of the gassing system, but to inherent e q u i l i b r a t i o n problems of the suspension culture system. A more 45 d e t a i l e d a nalysis of the mass transf e r c h a r a c t e r i s t i c s of the s t i r r e r f l a s k and suspension culture was required to determine whether t h i s major source of experimental uncertainty could be eliminated. 4.4 Analysis of Suspension Culture Method The a l l - g l a s s water-jacketed s t i r r e r f l a s k provides a convenient means of manipulating b i o l o g i c a l systems i n an i s o l a t e d environment. In view of i t s general d e s i r a b i l i t y as a ve s s e l i n which to perform environmentally c o n t r o l l e d i n v i t r o experiments, the s t i r r e r f l a s k was re-examined to determine whether or not the oxygen e q u i l i b r a t i o n problems i t posed could be overcome. 4.4.1 D i f f u s i o n rate constant A comprehensive review of the d i f f u s i o n of oxygen i n s t i r r e d c e l l suspensions has been conducted by Whillans and Rauth (1980). They envisioned conditions inside a s t i r r e r f l a s k to e x i s t as depicted i n Fi g . 17. A gas of a given oxygen concentration l i e s above suspension medium having a c e r t a i n depth and surface area, and time-dependent oxygen concentration. The medium i s well s t i r r e d , minimizing any gradients i n the bulk of the l i q u i d , but leaving a t h i n non-turbulent l a y e r of medium at the g a s - l i q u i d i n t e r f a c e . The concentration gradient between the gas and l i q u i d phases i n the f l a s k i s postulated to e x i s t across, and completely within t h i s layer of l i q u i d . The gradient i s described by Fick's Law (the time rate of change i n the number of molecules d i f f u s i n g i n a given d i r e c t i o n i s d i r e c t l y p r oportional to the 46 Figure 17 Analysis of mass transf e r i n a suspension culture spinner f l a s k . The mathematical model shown below was derived by assuming that a gas of oxygen concentration C l i e s above medium of depth d, surface area A and time-dependent oxygen concentration C ( t ) . The depth of the non-turbulent layer of medium at the g a s - l i q u i d i n t e r f a c e i s x and the rate of oxygen consumption i n the medium i s R. f r o m F i c k ' s Law: dC (t) / d t = A D / x V ( C g - C (t) ) =k ( C g - C (t) ) w h e r e k = A D / x V = D / x d a c c o u n t f o r o x y g e n c o n s u m e d : dC (t) / d t - k ( C g - C (t) ) - R ODE w i t h i . e . C ( 0 ) = C Q s o l u t i o n : - k t w h e r e C s = C g - R / k i s t h e s t e a d y s t a t e 47 negative concentration gradient i n that d i r e c t i o n ; Setlow and Po l l a r d , 1962) and i s expressed mathematically i n the form of an e a s i l y solved ordinary d i f f e r e n t i a l equation. The s o l u t i o n to the ODE reveals that the rate constant governing the exponential approach to d i f f u s i v e e q u i l i b r i u m between the oxygen concentrations of the gas and l i q u i d phases i s determined by three parameters of the system, s p e c i f i c a l l y the c o e f f i c e n t of d i f f u s i o n f o r oxygen i n the medium, the medium depth and the thickness of the non-turbulent layer at the g a s - l i q u i d i n t e r f a c e . By adapting t h i s analysis to the conditions e x i s t i n g during the suspension c u l t u r e i r r a d i a t i o n s i n the previous section, an approximate value f o r the oxygen concentration i n the c e l l suspension at the end of the one hour e q u i l i b r a t i o n period can be calc u l a t e d . The layer of l i q u i d at the g a s - l i q u i d i n t e r f a c e has a thickness of approximately 50 m^ f o r aqueous media being s t i r r e d at 200 rpm at 37°C (Davies and Rideal, 1963). The rate constant f o r the Bell c o s t i r r i n g f l a s k containing 50 ml of suspension medium has a c a l c u l a t e d value of 0.09 min" 1 under these conditions. When t h i s rate constant value i s used to ca l c u l a t e the oxygen concentrations that would e x i s t i n the same suspension medium a f t e r a one hour e q u i l i b r a t i o n period, the r e s u l t s confirm that the d i f f u s i o n process i s s t i l l f a r from equilibrium (Table I I ) . • Because the t h e o r e t i c a l c a l c u l a t i o n s involve a s i g n i f i c a n t degree of approximation, however, the value of the rate constant was also experimentally determined. The experimental apparatus, assembled as shown i n F i g . 18, was i d e n t i c a l to that used i n the i r r a d i a t i o n experiments with the exception that an oxygen probe was inse r t e d into the s t i r r e r f l a s k i n place of the sampling cannula. The medium i n the Table II Calculated oxygen concentrations i n suspension culture medium a f t e r one hour e q u i l i b r a t i o n period. t for C s+5% C (60) 0.5% 20.95% 75 min 0.60% 0.05% 20.95% 100 min 0. 15% 49 Figure 18 Apparatus used to measure the d i f f u s i o n rate constant, k, of the spinner f l a s k . m i x i n g t u b e a n d f l o w m e t e r \ h u m i d i f i e r p r o b e a m p l i f i e r E o x y g e n p r o b e / T s t i r r e r 02 g a s a n a l y z e r c h a r t r e c o r d e r 50 f l a s k (MEM + 10% FCS) also d i d not contain any c e l l s . The probe (Controls Katharobic) was used to measure the change i n the oxygen concentration of the suspension medium with time while i t was being s t i r r e d at 200 rpm. Nitrogen gas was metered to flow through the f l a s k at 10 1 hr"^ and oxygen content of the e f f l u e n t was measured by an oxygen gas analyser (Applied Electrochemistry). The oxygen content of both the e f f l u e n t gas and the medium was recorded from the d i g i t a l d isplays on the respective measuring devices and with a s t r i p chart recorder. By rearranging the Whillans and Rauth equation, the data c o l l e c t e d i n t h i s manner can be used to produce a s t r a i g h t l i n e semi-logarithmic p l o t ( F i g . 19). The negative slope of t h i s p l o t corresponds to the value of the rate constant. As can be seen from the p l o t , the experimental value f o r the rate constant at 37°C i s i n good agreement with the t h e o r e t i c a l l y c a l c u l a t e d value (0.09 min"^, p.47). The experimental data also confirms that the value of the rate constant decreases with temperature. This might have been expected i n view of the slow deoxygenation of the suspension culture that was implied by the r a d i a t i o n s u r v i v a l data obtained at 4°C. 4.4.2 Oxygen consumption and r a d i o l y t i c depletion The time requirements to reach a s t a t i c equilibrium between the oxygen concentrations of the gas and l i q u i d phases i n a s t i r r e r f l a s k could conceivably be f u l f i l l e d by extending the p r e - i r r a d i a t i o n e q u i l i b r a t i o n period. To determine the value of the oxygen concentration to which the c e l l s i n a suspension are a c t u a l l y exposed, Figure 19 Experimental confirmation of d i f f u s i o n rate constant value. By rearranging the Whillans and Rauth expression, the d i f f u s i o n rate constant can be determined from the slope of a semi-log p l o t of the measured oxygen concentration r a t i o versus time. C s = C g when R=0 (C (t) - C s ) / C Q - C S ) = e _ k t l 1 1 1 r 0 . 0 1 0 10 20 30 4 T i m e (min .) 0 52 the consequences of oxygen consumption must also be considered, however. According to the Whillans and Rauth model, the r e s p i r a t o r y depletion of oxygen i n the suspension medium w i l l e s t a b l i s h a dynamic equilibrium i n which the oxygen concentration of the suspension medium i s lower than that of the gas. The perpetual gradient between the suspension medium and the gas maintains the transport of oxygen to the c e l l s across the g a s - l i q u i d i n t e r f a c e . I f a l l of the hypothesized oxygen d i f f u s i o n gradients f o r f u l l y r e s p i r i n g c e l l s i n a s t i r r e d suspension are added (F i g . 20), a s t i r r e r f l a s k containing a 50 ml si n g l e c e l l suspension with a c e l l density of 10^ c e l l s ml"''- at 37°C would require a minimum oxygen concentration of 2.3 /iM i n the gas above the suspension. This f a c t o r must be subtacted from the oxygen concentration of the gas, i f the oxygen concentration to which the i n t r a c e l l u l a r r a d i a t i o n targets are exposed to i s a c t u a l l y desired. A p o t e n t i a l l y more d i f f i c u l t problem to circumvent, however, i s that of the r a d i o l y t i c depletion of oxygen. An incremental exposure and sampling procedure such as that used i n the suspension culture experiments, r e s u l t s i n a c y c l i c depletion and reoxygenation of the suspension medium. The rate at which oxygen i s consumed i s l i n e a r l y dependent upon the dose rate and f o r culture medium at 37°C the p r o p o r t i o n a l i t y constant has been determined to be approximately 0.04 fiK Gy" 1 (whillans and Rauth, 1980; P a l c i c and Skarsgard, 1984). R e c a l l i n g that the dose rate used i n the experiments was 4 Gy min"''' and again making use of the mathematical model by Whillans and Rauth, the change i n the oxygen concentration of the suspension medium f o r an incremental one minute i r r a d i a t i o n can be charted as shown i n F i g . 21. Since the sampling time i s also only one or two minutes i n duration, at t h i s dose 53 Figure 20 Resp i r a t i o n induced oxygen gradients between the gas and the i n t r a c e l l u l a r environment i n a sin g l e c e l l suspension cul t u r e . The magnitude of the gradient at the g a s - l i q u i d i n t e r f a c e i s c e l l density dependent. g a s  u n s t i r r e d l a y e r r e s p i r i n g c e l l L_l C i C S a r o u n d c e l l : C s - C i = K a 2 / 2 D @ g a s - l i q u i d i n t e r f a c e : C g - C s = R / k f o r f u l l r e s p i r a t i o n . C g > K a 2 / 2 D + R / k 54 Figure 21 Calculated cycle of r a d i o l y t i c depletion of oxygen i n an i r r a d i a t e d suspension c u l t u r e . The p l o t below shows a 1 minute i r r a d i a t i o n followed by a 9 minute recovery period assuming a dose rate of 4 Gray min" 1 and a depletion constant of 0.04 /*M Gray" 1. Time (min) 55 rate i t i s obvious that even i f an equilibrium at some low oxygen tension has been achieved, i t would be impossible to maintain once the i r r a d i a t i o n process i s started. The t h e o r e t i c a l predictions were again tested with the polarographic oxygen probe and the apparatus used to determine the s t i r r e r f l a s k rate constant. The s t r i p chart recordings i n F i g . 22 show the approach to equilibrium for medium i n contact with a gas mixture containing 0.5% oxygen. The p l o t s confirm the p r e d i c t i o n s of the model and, a f t e r the i n i t i a l one hour e q u i l i b r a t i o n period, show the r a p i d decrease i n the d i s s o l v e d oxygen content when the X-ray machine i s turned on and the more gradual recovery a f t e r the exposure i s completed. (The v e r t i c a l jumps i n the p l o t s are due to an increased probe background current when the X-ray machine i s turned on.) I t i s i n t e r e s t i n g to note that, judging from the shape of the p l o t s , the oxygen concentration i n the suspension medium i s considerably further from equilibrium p r i o r to the s t a r t of the intermittent r a d i a t i o n exposures at the lower temperature. In view of the r e s u l t s of t h i s a n a l y s i s , i t was concluded that a suspension culture was inappropriate f o r i r r a d i a t i n g c e l l s at constant low oxygen concentrations. The suspension culture experiments demonstrated that a d i f f e r e n t c e l l culture technique was required i f the oxygen concentration to which c e l l s were exposed during i r r a d i a t i o n was to be accurately c o n t r o l l e d . The methods of analysis used to i n v e s t i g a t e the mass transf e r c h a r a c t e r i s t i c s of the s t i r r e r f l a s k were subsequently u t i l i z e d to develop such a technique. 56 Figure 22 S t r i p chart recordings of r a d i o l y t i c depletion of oxygen i n a suspension cu l t u r e . Traces f o r one minute i r r a d i a t i o n s followed by two minute recovery periods at dose rate of 4.0 Gray min"''' were obtained at both 4°C and 37°C following a one hour e q u i l i b r a t i o n period with gas containing 0.5% oxygen. The v e r t i c a l jumps i n the recording patterns are due to an increased background current i n the i r r a d i a t e d oxygen probe. O J o 37°C 4°C ^JLP M M M x l O T i m e 57 5. THE THIN FILM MONOLAYER 5.1 Materials and Method Possible solutions to the problems encountered i n e q u i l i b r a t i n g and maintaining low oxygen concentrations i n i r r a d i a t e d s i n g l e c e l l suspension cultures were examined with the a i d of the Whillans and Rauth equation (Fig. 23). One p o s s i b i l t y was to reduce the rate of r a d i o l y t i c oxygen depletion by reducing the i r r a d i a t i o n dose rate. The amount by which the dose rate could be reduced, however, was l i m i t e d by the time constraints w i t h i n which the i r r a d i a t i o n procedure had to be completed. This l i m i t a t i o n d i d not allow a large enough reduction to the solve the e q u i l i b r a t i o n problems caused by c y c l i c r a d i o l y t i c oxygen depletion. By t h i s process of elimination, i t was decided that a s o l u t i o n would most l i k e l y be found by increasing the e q u i l i b r a t i o n time constant of the i r r a d i a t i o n v e s s e l . This can be accomplished, to some degree, by increasing the s t i r r i n g speed and using a l e s s viscous medium to decrease the thickness of the layer across which the d i f f u s i o n gradient i s developed. A more e f f e c t i v e method of increasing the rate constant, however, i s to increase the surface area to volume r a t i o of the suspension medium (Whillans and Rauth, 1980). Bubbling gas of the desired oxygen concentration through the medium i s an e f f e c t i v e , although, impractical example of t h i s , since suspended mammalian c e l l s are e a s i l y damaged by the r e s u l t i n g turbulence. I f the increased area to volume s o l u t i o n i s extrapolated to the i d e a l case, however, the gas-l i q u i d e q u i l i b r a t i o n time can be minimized i f the suspension medium i s 58 Figure 23 Possible solutions to oxygen e q u i l i b r a t i o n problems using the Whillans and Rauth equation. from Whillans and Rauth (19B0) : C (t) =CS+ (C 0-C s) e " k t C s=C g-R/k k=AD/xV=D/xd p o s s i b l e s o l u t i o n s : - reduce R by reducing dose r a t e - increase k by decreasing x - increase k by decreasing d 59 removed e n t i r e l y and the c e l l s are i r r a d i a t e d while covered by only a t h i n layer of l i q u i d . This i s the basis of t h i n f i l m c e l l c u l t u r e . Several versions of the t h i n f i l m culture technique have already been used to study the r a d i o s e n s i t i z a t i o n of c e l l s by oxygen at low oxygen tensions. To avoid oxygen depletion at u l t r a h i g h dose rates, Michaels et a l . (1978) and Ling et a l . (1981) have s u c c e s s f u l l y made use of a method pioneered by Epp et a l . (1972) i n which c e l l s are i r r a d i a t e d on a c o v e r s l i p made of a s p e c i a l oxygen impermeable p l a s t i c . The c e l l s were pl a t e d onto the c o v e r s l i p and then held i n a medium f i l l e d culture dish. The radioresponse of the c e l l s at low oxygen concentrations was determined by completely a s p i r a t i n g the medium from the dish, removing the c o v e r s l i p and i r r a d i a t i n g the exposed c e l l s i n a g a s - f i l l e d chamber. Due to the extremely short ( f r a c t i o n s of a second) e q u i l i b r a t i o n time required f o r the d i f f u s i o n of oxygen between the c e l l s and the gas, the r a d i o l y t i c depletion of oxygen within the t h i n f i l m of medium covering the c e l l s during the i r r a d i a t i o n was determined to be i n s i g n i f i c a n t using t h i s technique, even at dose rates of 20 Gy min" 1 (Michaels et a l , 1978). In a modified v e r s i o n of the t h i n f i l m culture technique, c e l l s are allowed to attach to the surface of glass P e t r i dishes (Koch and Painter, 1975; Koch, 1984). With t h i s technique, the growth medium i s aspirated from the dish and the c e l l monolayers are i r r a d i a t e d at various concentrations of oxygen a f t e r a small volume of medium (1 ml) i s returned to the dish. This method has the advantage that i t enables c e l l s to be maintained i n t h i n f i l m culture f o r extended periods (hours to days) without i n c u r r i n g c e l l drying or t o x i c i t y problems. I t i s also simpler to implement i n that i t u t i l i z e s r e a d i l y a v a i l a b l e labware. 60 Because medium i s returned to the dish before i r r a d i a t i o n , however, the r a d i o l y t i c and metabolic depletion of oxygen must s t i l l be considered i n determining the oxygen concentration at the c e l l surface with t h i s method (Koch, 1984). A hybrid of these techniques seemed i d e a l l y s u i t e d to the purposes of t h i s t h e s i s . A t h i n f i l m culture system was consequently developed -using a P e t r i d i s h c e l l culture system s i m i l a r to that used by Koch, but i n which the problems of r a d i o l y t i c oxygen depletion are eliminated by completely a s p i r a t i n g the growth medium before i r r a d i a t i o n . 5.1.1 Choice of i r r a d i a t i o n v e s s e l A primary consideration i n the use of t h i n f i l m culture was the nature of the culture v e s s e l surface. The c e l l s i n t h i n f i l m culture must be f i r m l y attached to a culture v e s s e l surface so that they are not removed during the a s p i r a t i o n of the growth medium. Under c e r t a i n conditions, t h i s could compromise the required c o n t r o l over the various low oxygen concentrations to which the c e l l s were exposed during the i r r a d i a t i o n procedure. At s u f f i c i e n t l y low oxygen tensions, c e l l s attached to the surface of commonly used p l a s t i c disposable t i s s u e culture dishes and f l a s k s can thwart attempts at c o n t r o l l i n g the c e l l u l a r oxygen supply by absorbing the oxygen molecules that d i f f u s e through and out of the p l a s t i c (Chapman et a l . 1970). The surface to which the c e l l s were attached i n the following t h i n f i l m experiments, therefore, had to have a low oxygen d i f f u s i v i t y . Glass c u l t u r e dishes have the desired oxygen impermeability and were the most r e a d i l y accessible substitutes f o r the ubiquitous p l a s t i c . 61 A t h i n f i l m culture system was consequently developed based on the use of 50 mm x 10 mm glass P e t r i dishes s i m i l a r to those used by Koch. C e l l s attached to these dishes were i r r a d i a t e d i n the apparatus shown i n F i g . 24. A dis h containing c e l l s and growth medium was sealed using a t i g h t l y f i t t e d pure gum rubber stopper ( F i s h e r ) . S t ainless s t e e l tubing served as both gas i n l e t and o u t l e t and was connected, by means of two short pieces of t h i c k walled rubber tubing, to the same gas metering and oxygen monitoring system used i n the previous suspension culture experiments. Constant monitoring ensured that neither the tubing nor the stopper compromised the oxygen content of the gas above the plated c e l l s . P r i o r to i r r a d i a t i o n , a vacuum pump was used to c a r e f u l l y , and as completely as possible, aspirate the growth medium from the dish through a s t a i n l e s s s t e e l cannula that passed through the rubber stopper. The metabolic a c t i v i t y of the c e l l s was c o n t r o l l e d by means of a s p e c i a l l y adapted r e c i r c u l a t i n g water bath that maintained the dish temperature at 4oC or 37oC. The c e l l s were i r r a d i a t e d from below by a P h i l i p s 250 kVp X-ray machine f i t t e d with a 0.5 mm Cu f i l t e r . 5.1.2 Dosimetry considerations The d e c i s i o n to use glass culture dishes provided an oxygen c o n t r o l l e d environment, but also introduced dosimetry complications. Due to the high atomic density of the glass, X - i r r a d i a t i o n scatters low energy electrons that can t r a v e l beyond the surface of the dis h (Dutreix and Bernard, 1966; S i n c l a i r , 1969). These electrons s i g n i f i c a n t l y increase the dose to c e l l s attached to the glass. Complications a r i s e from the f a c t that the increased dose i s d i f f i c u l t to measure using 62 Figure 24 Experimental apparatus f o r t h i n f i l m culture i r r a d i a t i o n s . m i x i n g t u b e a n d f l o w m e t e r s t o p c o c k r u b b e r P e t r i d i s h \ s t o p p e r d w a t e r b a t h T-r X - r a y m a c h i n e \ h u m i d i f i e r 0 2 g a s a n a l y z e r c h a r t r e c o r d e r 63 conventional dosimetry techniques. A b i o l o g i c a l dosimetry measurement was, therefore, made by i r r a d i a t i n g aerobic c e l l s attached to both p l a s t i c and glass culture dishes. By comparing the two sets of r a d i a t i o n s u r v i v a l data, i t was determined that the e f f e c t i v e dose received by c e l l s attached to the glass surface was 1.5 times that of the dose to c e l l s attached to the p l a s t i c f or the same X-ray exposure ( F i g . 25). This value compares favorably with those found i n the l i t e r a t u r e (Blakely et a l . , 1979; E l k i n d and Whitmore, 1967; Chapman et a l . , 1970). An electrometer (Victoreen 500) with a 0.6 cc ion chamber probe had been used to measure the photon dose at the dish surface (5 Gy min"''") f o r the apparatus assembled as shown i n F i g . 24. The r a d i a t i o n dose a c t u a l l y received by c e l l s attached to the glass could then be c a l c u l a t e d by m u l t i p l y i n g t h i s measured X-ray dose by the b i o l o g i c a l l y determined f a c t o r . 5.1.3 Thin f i l m t o x i c i t y Another consideration i n using t h i n f i l m culture techniques concerned the t o x i c i t y of the gassing procedure and the l i k e l i h o o d of c e l l s drying out during extended t h i n f i l m exposure. In a t y p i c a l i r r a d i a t i o n procedure the rubber stopper was inserted into the P e t r i dish and the a i r above the growth medium was purged from the dish by i n c r e a s i n g the flow of the humidified gas mixture. When the gas analyzer i n d i c a t e d that oxygen content of the e f f l u e n t gas was s u f f i c i e n t l y low, the medium was completely aspirated from the dish through the cannula. This was followed by an immediate reduction i n the gas flow rate from 30 to 10 1 hr"^". This flow rate was maintained 64 Figure 25 B i o l o g i c a l dosimetry used to measure the a d d i t i o n a l dose to c e l l s attached to glass surfaces. Low energy electrons increased the dose to c e l l s i r r a d i a t e d i n the glass culture dishes by a f a c t o r of 1.5 over those i r r a d i a t e d i n the p l a s t i c dishes. 65 Figure 26 T o x i c i t y of t h i n f i l m culture f o r exposure of c e l l s to gas flowing at 10 1 hr ^. No measurable e f f e c t on the p l a t i n g e f f i c i e n c y was noticed f or exposure times of up to eight minutes. c o •rH - P L) CD L_ L L cn c • i—i > •rH > ZD tn 0 . 1 -T i m e (min) 66 during the remainder of the i r r a d i a t i o n procedure. Measurements of the p l a t i n g e f f i c i e n c i e s f o r mock-irradiated c e l l s exposed to increasing t h i n f i l m exposure times are displayed i n F i g . 26. These show no increase i n t o x i c i t y f o r exposure periods of up to eight minutes. Since the maximum expected i r r a d i a t i o n time i n the planned r a d i a t i o n s u r v i v a l experiments was four minutes i n length, c e l l drying or t o x i c i t y was not regarded as a f a c t o r i n c e l l s u r v i v a l . 5.1.4 Spot i n o c u l a t i o n In using P e t r i dishes as t h i n f i l m culture vessels, i t was noticed that the small amount of growth medium that remained i n the dish (< 0.1 ml) a f t e r the a s p i r a t i o n procedure tended to accumulate i n the outer rim of the bottom of the dish. This accumulation could s i g n i f i c a n t l y a f f e c t the oxygenation of the c e l l s attached i n t h i s area, the d i f f u s i o n e q u i l i b r a t i o n time constant for an u n s t i r r e d layer of medium being r e l a t e d to the square of the medium depth (Boag, 1969). Since a s i m i l a r e f f e c t occurs with larger volumes of medium (the meniscus created by the walls of dish leaving a thicker layer of medium above the outer perimeter of the dish surface) Koch (1984) avoided t h i s problem by r e s t r i c t i n g the attachment of c e l l s to the center of the dish. The P e t r i dishes were prepared using a "spot" i n o c u l a t i o n procedure i n which a small amount of c e l l suspension i s c a r e f u l l y micropipetted into the center of a dish already containing the required growth medium. The dish was then allowed to s i t undisturbed while the c e l l s attached themselves to the glass surface i n a c e n t r a l , two to three centimetre spot ( F i g . 27). By Koch's measurement, les s than 3% of the c e l l s were 6 7 Figure 27 Spot i n o c u l a t i o n of glass P e t r i dishes. C e l l s were only allowed to attach to a 1-2 cm area i n the center of the glass dish, thereby avoiding the v a r i a t i o n i n depth of the t h i n f i l m layer that occurs at the outer rim of the dish. The stained spot innoculated and normally plated c e l l s below demonstrate the difference i n monolayer configuration. 68 attached outside of a c l e a r l y defined c e n t r a l area using t h i s method. 5.1.5 C e l l attachment time In order to e s t a b l i s h a time frame for an experimental protocol, an i n v e s t i g a t i o n of the time required for c e l l s to attach to the glass surface was c a r r i e d out. Glass P e t r i dishes containing growth medium were spot inoculated with a known number of c e l l s and l e f t to s i t undisturbed at room temperature. At defined time i n t e r v a l s the medium from one of the dishes would be aspirated and the remaining c e l l s trypsinzed, suspended and counted. The r e s u l t s shown i n F i g . 28 i n d i c a t e that maximum c e l l recovery i s achieved a f t e r approximately f o r t y minutes. A minimum one hour attachment period was consequently adopted as the experimental standard. 5.1.6 C e l l shape i n t h i n f i l m monolayer The a n t i c i p a t e d importance of c e l l shape i n e s t a b l i s h i n g r a d i o b i o l o g i c a l ^ s i g n i f i c a n t i n t r a c e l l u l a r oxygen gradients was a key f a c t o r i n the d e c i s i o n to perform the i n i t i a l suspension culture experiments. Thin f i l m culture, however, presented an opportunity to determine d i r e c t l y whether or not c e l l shape plays a s i g n i f i c a n t r o l e i n the formation of such gradients. This was investigated by performing two seperate s e r i e s of experiments using the t h i n f i l m c u l t u r e technique. In the f i r s t s e r i e s , c e l l s were allowed to attach to the glass P e t r i dishes and were incubated for 24 hours p r i o r to i r r a d i a t i o n . This allowed c e l l s to spread out and form a f l a t monolayer i n the dish 69 Figure 28 F l a t and round c e l l monolayers i n glass P e t r i dishes. The c e l l s shown below were photographed (scale 65:1) a f t e r overnight incubation (upper) and a f t e r a 2 hour attachment period at room temperature (lower). Both were plated i n 100 pi a l i q u o t s at densities of 10 7 c e l l s ml""'". 70 Figure 29 Time required f o r c e l l s to attach to a glass surface was measured by the a b i l i t y to recover c e l l s at various times a f t e r p l a t i n g i n P e t r i dishes. Maximum c e l l recovery was achieved approximately one hour a f t e r p l a t i n g . 40 0 6 0 120 T i m e a f t e r P l a t i n g (min) 71 (F i g . 29a). The experiments then went on to determine the s u r v i v a l c h a r a c t e r i s t i c s of c e l l s exposed to gas mixtures containing various low l e v e l s of oxygen and i r r a d i a t e d while i n t h i s configuration. The change i n the area of a V79 c e l l as i t spreads out a f t e r attaching to a surface i s p l o t t e d i n F i g . 30. The change i n shape of attached c e l l s with time was determined with the a i d of an imaging microscope and the data shows that, at room temperature, a c e l l w i l l maintain a minimal crossection or rounded shape f o r several hours a f t e r p l a t i n g . A second seri e s of r a d i a t i o n s u r v i v a l experiments was subsequently performed, but d i f f e r e d from the f i r s t i n that the c e l l s were only allowed a one hour room temperature attachment period before being i r r a d i a t e d . These c e l l s were f i r m l y attached to the surface of the P e t r i dish, but also retained an e s s e n t i a l l y round shape throughout the remainder of the experiment (Fig. 29b). The oxygen mediated r a d i a t i o n s u r v i v a l of c e l l s with t h i s round shape was then also determined. 5.1.7 E q u i l i b r a t i o n time measurements Attempts were made to measure the g a s - l i q u i d e q u i l i b r a t i o n time f o r c e l l s i n both the round and f l a t configurations. For these measurements, the medium was removed from dishes and the c e l l s exposed to a 95% N£ / 5% CO2 atmosphere for increasing time i n t e r v a l s p r i o r to the onset of i r r a d i a t i o n . The c e l l s were i r r a d i a t e d with an X-ray dose that produced a known l e v e l of s u r v i v a l f o r hypoxic c e l l s . Oxygen enhanced c e l l k i l l i n g was expected i f a g a s - l i q u i d equilibrium had not been achieved p r i o r to the s t a r t of the i r r a d i a t i o n . A f t e r an 72 Figure 30 Time dependent change i n c e l l c r o s s - s e c t i o n a l area a f t e r p l a t i n g . Average change i n c e l l area with time f o r 40 V79 c e l l s was measured with the a i d of a computer c o n t r o l l e d microscopic imaging technique and shows that c e l l s remain r e l a t i v e l y round for 2-3 hours a f t e r p l a t i n g . These measurements were made at 37°C and represent the most r a p i d possible f l a t t e n i n g of the c e l l s . 1 i 1 1 1 1 " r 3 0 0 -O J CO CD < CD 2 0 0 100 V79 c e l l s a t 37° C \ l 1 l I i I • L 0 2 4 6 8 T i m e ( h o u r s ) 73 equivalent exposure to flowing a i r , a c o n t r o l was i r r a d i a t e d at a dose that would produce a comparable l e v e l of s u r v i v a l . Judging from the r e s u l t s of previous s i m i l a r t h i n f i l m experiments (Michaels et al.,1978; Ling et al.,1981), i t was expected that oxygen l e v e l s would be extremely r a p i d l y e q u i l i b r a t e d . Since the shortest possible p r e - i r r a d i a t i o n time i n t e r v a l was f i f t e e n seconds, these expectations were confirmed by the r e s u l t s i n F i g . 31 which show no enhanced r a d i o s e n s i t i v i t y f o r eit h e r c e l l c onfiguration. 5.1.8 Low temperature t o x i c i t y The spot inoculated P e t r i dishes were pre-cooled on i c e before the s t a r t of the i r r a d i a t i o n procedure to ensure the r e s p i r a t o r y i n h i b i t i o n of the c e l l s . Because the P e t r i dishes were i r r a d i a t e d i n d i v i d u a l l y and sequentially, the t o t a l time required from the beginning of the f i r s t i r r a d i a t i o n to the completion of the l a s t could extend f o r as long as one hour. An experimental i n v e s t i g a t i o n was, therefore, c a r r i e d out to determine the e f f e c t of extended exposure to low temperatures on c e l l s u r v i v a l . Glass P e t r i dishes containing c e l l s i n spot monolayers were placed on i c e and then i r r a d i a t e d i n f i f t e e n minute i n t e r v a l s at 4°C following the i r r a d i a t i o n protocol i n Sec. 5.2.2. The c e l l s were exposed to a low concentration of oxygen during the i r r a d i a t i o n , which produced a known l e v e l of r a d i a t i o n s u r v i v a l . The i r r a d i a t e d c e l l s and an u n i r r a d i a t e d c o n t r o l were immediately trypsinzed, p l a t e d and incubated f o r one week i n the manner also described i n the following protocol s e c t i o n . The r e s u l t i n g clonogenic s u r v i v a l data i s p l o t t e d i n F i g . 32 and shows no measurable decrease i n p l a t i n g e f f i c i e n c y and no 74 Figure 31 Upper l i m i t measurement of t h i n f i l m culture e q u i l i b r a t i o n time. C e l l s were i r r a d i a t e d i n nitrogen (21 Gray) at various times a f t e r a s p i r a t i o n of the medium and were expected to show an increased r a d i o s e n s i t i v i t y i f any oxygen remained i n the dish at the onset of i r r a d i a t i o n . No increase i n s e n s i t i v i t y was noted f o r the minimum possible time of f i f t e e n seconds. A i r c o n t r o l data ( i r r a d i a t e d with 7 Gray) revealed no t h i n f i l m t o x i c i t y . 1 . 0 c o o a i r • n itrogen - P u ra L L 0 . 1 cn c ; ,8-n- - Q - o - - - o - - -45--! > > . 0 1 4 7 0 60 120 T i m e ( s e c ) 75 Figure 32 T o x i c i t y of low temperatures. P l a t i n g e f f i c i e n c i e s of c o n t r o l and i r r a d i a t e d (17 Gray i n 200 ppm O2 at 4°C) c e l l s were measured a f t e r various times on i c e p r i o r to i r r a d i a t i o n . Toxic e f f e c t s were noted a f t e r approximately f o r t y - f i v e minutes i n both the i r r a d i a t e d and t h e c o n t r o l c e l l s . 1 1 r T i m e on I c e (min.) 76 time dependent synergism between the r a d i a t i o n and the low temperature fo r the f i r s t f o r t y - f i v e minutes of the c e l l s ' time on i c e . To ensure a consistent p l a t i n g e f f i c i e n c y f o r a l l of the c e l l s being i r r a d i a t e d at 4°C, therefore, the P e t r i dishes were pre-cooled f o r a maximum of t h i r t y minutes p r i o r to i r r a d i a t i o n . 5.2 Experimental Protocol 5.2.1 Preparation of c e l l culture The f l a t c e l l monolayer was used i n the f i r s t s e r i e s of t h i n f i l m c u l t u r e experiments and was prepared as follows ( Fig. 33): Seed plates were made up 3 days before an experiment by adding 10^ V79 c e l l s to the 10 ml of MEM & 10% FCS (Gibco) growth medium contained i n each of s i x 100 mm x 20 mm p l a s t i c P e t r i dishes (Falcon). The seed plates were then incubated at 37°C i n 5% C0 2. On the day p r i o r to the experiment, a l l s i x seed plates were t r y p s i n i z e d i n the usual manner and the c e l l s suspended i n 50 ml of growth medium. The c e l l suspension was t r a n s f e r r e d to a 50 ml c o n i c a l culture tube (Falcon) and spun down by c e n t r i f u g i n g at 300 rpm f o r 5 minutes. The medium supernatant was c a r e f u l l y aspirated and 2-3 ml of fr e s h medium was added to the c e l l p e l l e t . The p e l l e t was then disaggregated and the c e l l s suspended by gentle vortexing. The c e l l 7 -1 density of t h i s suspension was measured and d i l u t e d to 10 c e l l s ml Depending on the number of data points desired, 20-25 small 50 mm x 10 mm glass P e t r i dishes were prepared on an incubator tray. Five m i l l i l i t r e s of pre-warmed growth medium were added to each dish. With 77 Figure 33 Protocol flowchart f o r t h i n f i l m culture experiments: 3 days p r i o r to experiment: prepare 6 seed pla t e s : 10^ V79 c e l l s i n 10 ml MEM + 10% FCS day before experiment ( f l a t c e l l c o n f i guration): - t r y p s i n i z e seed plates and suspend c e l l s i n medium - centrifuge @ 300 rpm f o r 5 minutes - aspirate supernatant and resuspend c e l l s i n medium @ 10 7 c e l l s ml" 1 4 prepare 20-25 small glass P e t r i dishes on incubator tray and add 5 ml MEM + 10% FCS 4 pip e t t e 100 / i l a l i q u o t of c e l l suspension into each dish and allow c e l l s to attach @ 20°C f o r 2 hours day of experiment: 4°C i r r a d i a t i o n s : place 10-12 P e t r i dishes on ice 15-30 minutes p r i o r to i r r a d i a t i o n 37°C i r r a d i a t i o n s : i r r a d i a t e d immediately upon removal from incubator i r r a d i a t i o n procedure (repeat f o r each P e t r i d i s h ) : seal P e t r i dish with rubber stopper and place i n water bath 4-increase gas flow from 10 to 30 1 hr"^ 4-aspirate medium a f t e r a i r has been purged and reduce gas flow rate to 10 1 hr"^ 4 i r r a d i a t e c e l l s @ 5 Gray min"^ 4 remove P e t r i dish and add 5 ml of medium at 20°C 4 t r y p s i n i z e P e t r i dishes (without r i n s i n g ) 4 d i l u t e and plate c e l l s i n 3 dishes to produce 300-500 colonies per P e t r i dish 4 s t a i n and count colonies a f t e r one week incubation 78 the tray on a v i b r a t i o n free surface, a 100 /*1 a l i q u o t of the c e l l suspension was very c a r e f u l l y micropipetted into the center of each glass d i s h and the c e l l s allowed to s e t t l e and attach to the surface. A f t e r a 2 hour room temperature attachment period, the P e t r i dishes were incubated at 37°C i n 5% CO2 f o r an a d d i t i o n a l 24 hours. The procedure f or preparing the glass P e t r i dishes with round c e l l monolayers was the same with two exceptions. Since an overnight incubation of the glass dishes was not required, the seed plates were prepared only two days p r i o r to an experiment. Secondly, i n order to take advantage of the maximum roundness of the c e l l s , the i r r a d i a t i o n procedure was s t a r t e d immediately following the minimum one hour room temperature attachment period. 5.2.2 I r r a d i a t i o n procedure I r r a d i a t i o n s were c a r r i e d out sequentially, f i r s t at 4°C and then at 37°C. P r i o r to i r r a d i a t i o n , the c e l l s were e i t h e r pre-cooled on ice or, as required i n the case of the round c e l l s being held at room temperature, pre-warmed by incubating at 37°C. For the low temperature i r r a d i a t i o n s , the required number of P e t r i dishes were e i t h e r removed from the incubator i n the f l a t c e l l monolayer experiments, or from the room temperature tray i n the case of the round c e l l experiments, and placed on i c e approximately 15 minutes before the f i r s t i r r a d i a t i o n . For the 37°C i r r a d i a t i o n s , the remaining P e t r i dishes i n the round c e l l experiments were incubated a maximum of 30 minutes before i r r a d i a t i o n i n order to minimize any changes i n c e l l shape. The gassing system was assembled and allowed to e q u i l i b r a t e at the 79 desired oxygen concentration p r i o r to the s t a r t of the low temperature i r r a d i a t i o n s . (The 4°C i r r a d i a t i o n procedure was u s u a l l y c a r r i e d out f i r s t because r e f r i g e r a t i o n u n i t of the water bath required a r e l a t i v e l y long time to e q u i l i b r a t e . ) The i r r a d i a t i o n of the c e l l s then followed a repeated sequence of steps. With the apparatus prepared as shown i n F i g . 24, a P e t r i d i s h would be removed from the i c e , sealed with the rubber stopper and placed i n the 4°C water bath. The gas flow would then be increased to 30 1 h r " 1 and the e f f l u e n t monitored using the oxygen analyzer. When the oxygen l e v e l i n d i c a t e d that the a i r had been purged from the stoppered P e t r i dish, the growth medium was c a r e f u l l y a spirated through the cannula by t i l t i n g the dish. Due to surface tension e f f e c t s and the s l i g h t l y convex shape of the glass, almost a l l r e s i d u a l medium was drawn to the periphery of the dish. The gas flow was immediately reduced to 10 1 h r " 1 and the desired r a d i a t i o n dose was d e l i v e r e d to the c e l l s . A f t e r the i r r a d i a t i o n was completed, the stopper was removed from the dish and 5 ml of fresh room temperature growth medium added. The covered P e t r i dish was then held at room temperature u n t i l the remainder of the i r r a d i a t i o n s at that temperature had been completed. The same procedure was followed f or the i r r a d i a t i o n s at 37°C with the exception that the P e t r i dishes were removed from the incubator immediatly p r i o r to i r r a d i a t i o n . 5.2.3 Assaying clonogenic s u r v i v a l A set of i r r a d i a t e d spot monolayers were t r y p s i n i z e d by c a r e f u l l y a s p i r a t i n g the medium covering the c e l l s , adding 1 ml of pre-warmed 0.1% t r y p s i n i n c i t r a t e b u f f e r and incubating the dishes at 37°C f o r 5 80 minutes. The dishes were not r i n s e d with t r y p s i n a f t e r the a s p i r a t i o n of the medium because t h i s r e s u l t e d i n unacceptably high c e l l losses, p a r t i c u l a r l y when the c e l l s were i n the round configuration. The t r y p s i n i z e d monolayers were suspended and d i l u t e d i n previously prepared d i l u t i o n tubes containing the appropriate amounts of medium. Aliquots from the d i l u t e d c e l l suspensions were added to 100 mm x 20 mm p l a s t i c P e t r i dishes (Falcon) containing 10 ml of growth medium. Samples of the d i l u t e d c e l l suspensions obtained from each i r r a d i a t e d monolayer were pipett e d into three p l a s t i c dishes i n volumes s u f f i c i e n t to produce 300-500 c e l l colonies. A p o r t i o n of the d i l u t e d c e l l suspension was also centrifuged, the medium supernatant aspirated, and the c e l l s resuspended i n an ethidium bromide nuclear s t a i n . A f t e r vigorous vortexing to lyse the c e l l s and ensure the penetration of the s t a i n into the c e l l n u c l e i , fluorescent flow cytometry was used to characterize the c e l l c ycle d i s t r i b u t i o n of the experimental population (Vindelov, 1977). The c e l l density of the remainder of the d i l u t e d suspension was measured using a Coulter c e l l counter to determine the number of c e l l s p l ated i n each p l a s t i c P e t r i dish. A f t e r a one week incubation, the medium was removed from the p l a s t i c dishes and the c e l l colonies stained using a malachite green s o l u t i o n . The colonies were then scored manually and the number of colonies i n each dish was recorded. 81 5.3 Results 5.3.1 F l a t c e l l monolayer The s u r v i v a l data obtained with t h i n f i l m culture c e l l s i r r a d i a t e d i n the f l a t c o n f i g u r a t i o n are shown i n F i g . 34. The two s u r v i v a l curves i n each panel represent the r a d i a t i o n responses of r e s p i r i n g and non-r e s p i r i n g V79 c e l l s that were i r r a d i a t e d while being exposed to a gas of the stated oxygen tension. The r e s u l t s f o r a l l i r r a d i a t i o n s at oxygen tensions below 1000 ppm i n the gas above the monolayer were confirmed by one or more repeated experiments and the s u r v i v a l data were pooled to reduce random error. Each curve was produced by a computer c a l c u l a t e d l e a s t squares f i t of the Linear Quadratic c e l l s u r v i v a l model to the data obtained with the clonogenic assay. A l l variances i n the s u r v i v a l data were again c a l c u l a t e d according to the method proposed by Boag (1975). The p l o t t e d s u r v i v a l curves display the established c h a r a c t e r i s t i c s of oxygen mediated r a d i a t i o n s u r v i v a l , showing the expected increase i n c e l l u l a r r a d i o s e n s i t i v i t y with increasing oxygen tension. The data corroborate the findings reported by Ling et a l . (1981) by showing that oxygen mediated r a d i o s e n s i t i z a t i o n i s evident even at very low oxygen tensions (100 ppm i n gas for the non-respiring c e l l s ) . I r r a d i a t i n g the c e l l s at the two d i f f e r e n t temperatures d i d not always produce a n t i c i p a t e d r e s u l t s , however. Most s u r p r i s i n g perhaps, i s the marked di f f e r e n c e i n the hypoxic radioresponses of the c e l l s at 4°C and 37°C. These curves represent the data pooled from several 82 Figure 34(a-d) Su r v i v a l data f o r f l a t t h i n f i l m cultured c e l l s i r r a d i a t e d at 4°C (closed c i r c l e s ) and 37°C (open c i r c l e s ) under various low concentrations of oxygen. Figure continues on following page. 1 . 0 0 . 1 .So - M CJ (TJ C_ u_ c n > •rH > ZJ 0 01 1 0.01k Dose (Gray) 83 Figure 34(e-g) 84 experiments i n which the repeat experiments displayed i d e n t i c a l r e s u l t s . This r u l e d out the p o s s i b i l i t y that the observed d i f f e r e n c e i n r a d i o s e n s i t i v i t y i s the r e s u l t of random a r t e f a c t s . The r a d i o s e n s i t i v i t y d i f f e r e n t i a l i s also not due to the presence of oxygen. In a d d i t i o n to having c l o s e l y monitored the oxygen content of the gas to ensure that the c e l l s were t r u l y hypoxic during the i r r a d i a t i o n procedures at both temperatures, i t i s not l i k e l y that a r e s p i r i n g c e l l c u lture would r e t a i n a greater amount of r e s i d u a l oxygen than a non-r e s p i r i n g one. Possible explanations f o r t h i s r e s u l t are examined i n greater d e t a i l i n a discusion of the t h i n f i l m r e s u l t s . Manipulating c e l l r e s p i r a t i o n by means of temperature produced a more predictable e f f e c t at higher oxygen tensions. A notable feature of the data i s that as the p a r t i a l pressure of oxygen i n the gas increases from near zero to a few hundred parts per m i l l i o n , the r a d i o s e n s i t i v i t y of the non-respiring c e l l s increases more r a p i d l y than that of the r e s p i r i n g c e l l s . The d i f f e r e n c e i n the dose responses of the r e s p i r i n g and non-respiring c e l l s reaches a maximum and then becomes les s marked as the oxygen content of the gas increases even further. This pattern i s consistent with the cr e a t i o n and maintenance of small oxygen gradients i n r e s p i r i n g c e l l s that eventually become less r a d i o b i o l o g i c a l ^ s i g n i f i c a n t as the oxygen concentration i s increased. The d i f f e r e n c e i n radioresponses at 4°C and 37°C for a given oxygen tension i n the gas are not s o l e l y due to the existence of oxygen gradients, however, and the i m p l i c i t oxygen s o l u b i l i t y differences at these two temperatures must also be considered. (As the temperature increases from 4°C to 37°C, the proportional change i n s o l u b i l i t y of oxygen i s greater i n aqueous solutions than i t i s i n a i r ; F i g . 16) 85 Because the same p a r t i a l pressure of oxygen i n the gas was maintained throughout the i r r a d i a t i o n procedures at both temperatures, the oxygen concentration was commensurately higher i n the aqueous c e l l cytoplasm at 4°C. Although adjusting the oxygen content of the gas between i r r a d i a t i o n s at the two temperatures to compensate f o r such s o l u b i l i t y d i f f e r e n c e s was a t t r a c t i v e i n theory, i t was judged too d i f f i c u l t to implement accurately using the a v a i l a b l e oxygen monitoring and gas mixing equipment. The true radioprotective e f f e c t of r e s p i r a t i o n i n the f l a t c e l l c o n f i g u r a t i o n can be seen more c l e a r l y i n a semilogarithmic p l o t of the OER versus oxygen concentration (Fig. 35). The enhancement r a t i o values were c a l c u l a t e d at doses required to k i l l 99% of the c e l l s f o r each oxygen tension and p l o t t e d against the corresponding temperature corrected oxygen concentrations. The non-respiring c e l l data were f i t t e d a n a l y t i c a l l y (Alper, 1979) with the Alper and Howard-Flanders model (m = 3.1 and K = 3.0 fM) and display r e s u l t s t y p i c a l of the oxygen enhanced k i l l i n g of mammalian c e l l s . The small s h i f t to the r i g h t i n the 37°C OER data corresponds to a 0.15 /J.K d i f f e r e n t i a l between the oxygen concentrations of the r e s p i r i n g and non-respiring c e l l s . The r e s p i r i n g c e l l data were f i t t e d with a curve defined by the same parameters as the curve f i t t e d to the non-respiring c e l l s , but i n which the oxygen concentration was adjusted by t h i s f a c t o r . The presence of t h i s s h i f t i s p r e c i s e l y the r e s u l t that might be expected i f small, l o c a l i z e d , respiration-induced oxygen gradients e x i s t e d within and immediately around the r e s p i r i n g c e l l s . 86 Figure 35 Oxygen enhancement r a t i o f o r f l a t c e l l s i n t h i n f i l m c u l t u r e . Data f i t t e d a n a l y t i c a l l y with the Alper and Howard-Flanders equation were determined to be defined by the curve parameters m = 3.1 and K = 3.0 fiK. Enhancement of r e s p i r i n g c e l l s (open c i r c l e s ) was found to l a g that of non-respiring c e l l s by 0.15 /M. TT| 1—I II Mll| 1—I I II I ll| 1—I I II 111| 1—I I I I 1111 1—r n i i — i i i 11111 i — i i 11 n i l i m i » i 0 . 0 1 0 . 1 1.0 10 100 I I I 1 1 1 1—I—I—I I I 0 .1 1.0 [0 2 ] (UM) 87 5.3.2 Round c e l l monolayer The r a d i a t i o n response for the c e l l s i n the round configuration was s i m i l a r to that of the f l a t c e l l s (Fig.36). The r e s u l t s f o r a l l i r r a d i a t i o n s at oxygen tensions below 1000 ppm i n the gas were again confirmed by one or more repeated experiments and the s u r v i v a l data pooled. In a s i m i l a r gradient invoking pattern, the r a d i o s e n s i t i v i t y of the non-respiring c e l l s again increased more r a p i d l y with increasing oxygen tensions than that of the r e s p i r i n g c e l l s . However, although quan t i t a t i v e comparisions at any low p a r t i a l pressures of oxygen are again d i f f i c u l t to make given the i m p l i c i t s o l u b i l i t y differences at 4°C and 37°C, the d i f f e r e n c e s i n c e l l u l a r r a d i o s e n s i t i v i t y at the two temperatures are q u a l i t a t i v e l y more dramatic f o r the round c e l l s than f o r f l a t c e l l s i r r a d i a t e d under i d e n t i c a l conditions. The quantitative e f f e c t of c e l l r e s p i r a t i o n on the oxygen mediated r a d i o s e n s i t i v i t y of round c e l l s i n t h i n f i l m culture i s most c l e a r l y displayed i n a K curve p l o t (Fig. 37) The OER values were again c a l c u l a t e d f o r doses that give a 1% c e l l s u r v i v a l and p l o t t e d against the corrected oxygen concentrations. C e l l shape d i d not a f f e c t the oxygen enhancement of the non-respiring c e l l s and the a n a l y t i c a l l y f i t t e d curve was defined by i d e n t i c a l curve parameters (m = 3.1 and K = 3.0 uM). The s i m i l a r i t y of the oxygen enhanced r a d i o s e n s i t i v i t y of f l a t and round non-respiring c e l l s implies that the r a d i o l y t i c depletion of i n t r a c e l l u l a r oxygen i s i n s i g n i f i c a n t i n the c r e a t i o n of oxygen gradients w i t h i n and around the t h i n f i l m cultured c e l l s . Conversely, the oxygen enhanced r a d i o s e n s i t i v i t y of r e s p i r i n g round c e l l s i s s h i f t e d considerably further to the r i g h t than that f o r the f l a t c e l l s , implying 88 Figure 36(a-d) Su r v i v a l data f o r round t h i n f i l m cultured c e l l s i r r a d i a t e d at 4°C (closed c i r c l e s ) and 37°C (open c i r c l e s ) under various low concentrations of oxygen. Figure continues on following page. 40 0 20 Dose (Gray) 89 Figure 36(e-f) 90 Figure 37 Oxygen enhancement r a t i o f o r round c e l l s i n t h i n f i l m c u l t u r e . Data f i t t e d a n a l y t i c a l l y with the Alper and Howard-Flanders equation were again defined by the curve parameters m = 3.1 and K = 3.0 fM. Enhancement of r e s p i r i n g c e l l s (open c i r c l e s ) was found to l a g that of non-respiring c e l l s by 0.35 M^. TT| 1—1 i—i i i i 11*1 1—I M 11 T 11 1—I I 11111| 1—r~ nl i i ' » ' i " » i i i i i ' II ni i i i i i n i l i L 0 . 0 1 0 .1 1.0 10 100 9 1 the existence of larger r e s p i r a t i o n induced i n t r a c e l l u l a r gradients i n c e l l s i n t h i s t h i n f i l m configuration. The 37°C data were also f i t t e d with the same K curve, but at oxygen concentrations adjusted to lag those of the non-respiring c e l l s by 0.35 /xM. Since the d i f f e r e n t i a l c l e a r l y demonstrates the dependence of gradient s i z e on c e l l shape and i s i n e x c e l l e n t agreement with the c e l l r e s p i r a t i o n p r e d i c t i o n s made by Boag (1969; 1970), these r e s u l t s are very compatible with theories that propose oxygen gradients not only e x i s t i n the medium immediately surrounding r e s p i r i n g c e l l s , but extend into the c e l l s themselves. 5.4 Discussion and Analysis of Thin Film Method 5.4.1 Temperature mediated r a d i o s e n s i t i v i t y In order to demonstrate the existence of i n t r a c e l l u l a r oxygen gradients, low temperatures were employed to i n h i b i t c e l l r e s p i r a t i o n during the t h i n f i l m i r r a d i a t i o n s . I t was a n t i c i p a t e d that i r r a d i a t e d c e l l s would d i s p l a y a temperature dependent r a d i o s e n s i t i v i t y due to the influence of c e l l r e s p i r a t i o n on the l o c a l and i n t r a c e l l u l a r oxygen d i s t r i b u t i o n . Further i n v e s t i g a t i o n was prompted, however, by the discovery of an unexpected temperature mediated increase i n the r a d i a t i o n s u r v i v a l of hypoxic c e l l s i r r a d i a t e d at 4°C. As previously noted, the p o s s i b i l i t y that the low temperature increase i n c e l l s u r v i v a l was a random a r t e f a c t was e a s i l y eliminated by repeating the experiments several times. The hypoxic r a d i a t i o n s u r v i v a l data f o r c e l l s i n the round and f l a t configurations, f o r example, were 92 pooled from f i v e and seven experiments, r e s p e c t i v e l y , a l l of which displayed s i m i l a r responses. Since p o s i t i o n i n the c e l l cycle i s known to a f f e c t c e l l u l a r r a d i o s e n s i t i v i t y ( S i n c l a i r and Morton, 1963) , a second p o s s i b i l i t y was that t h e i r exposure to low temperatures selected a subpopulation of the 4°C c e l l s or simply arrested the c e l l s i n a less s e n s i t i v e p o r t i o n of the c e l l c y c l e . Investigating the e f f e c t s of low temperatures on c e l l s u r v i v a l , as described i n the Methods section 5.1.8, however, showed no s i g n i f i c a n t decrease i n c e l l v i a b i l i t y f o r c e l l s that had been placed on ice f o r up to f o r t y f i v e minutes (Fig. 32). Because the maximum " i c e time" f o r the t h i n f i l m i r r a d i a t i o n s was t h i r t y minutes, the 4°C r a d i a t i o n s u r v i v a l data does not represent the response of a selected, r a d i o r e s i s t a n t subpopulation of c e l l s . A d d i t i o n a l l y , samples of c e l l s that were being removed from the ice and t r y p s i n i z e d i n f i f t e e n minute i n t e r v a l s , during the same low temperature experiment, were stained with an ethidium bromide nuclear s t a i n and analyzed using fluorescent flow cytometry techniques (Vindelov, 1977). A p l o t of the c e l l cycle d i s t r i b u t i o n displays no detectable changes i n the population samples over the e n t i r e two hours that the c e l l s were held at or below 4°C (Fig. 38). No di f f e r e n c e s were seen i n s i m i l a r p l o t s obtained from c e l l s i r r a d i a t e d at 4°C and 37°C during the seri e s of t h i n f i l m experiments. The lower s e n s i t i v i t y of the non-metabolising c e l l s i s , therefore, also not a t t r i b u t a b l e to temperature induced s h i f t s i n the c e l l cycle d i s t r i b u t i o n . A review of the l i t e r a t u r e reveals that a s i m i l a r low temperature r a d i o p r o t e c t i v e e f f e c t was noted by B e l l i and Bonte (1963) i n experiments with cultured Hela c e l l s . Although no evidence f o r a Figure 38 C e l l cycle d i s t r i b u t i o n versus time on ice p r i o r to i r r r a d i a t i o n Ethidium-bromide labeled DNA p r o f i l e s of c e l l populations from P e t r i dishes exposed to low temperatures f o r increasing periods of time p r i o r to i r r a d i a t i o n reveal no changes i n c e l l cycle d i s t r i b u t i o n over the observed 2.5 hour period. 0 Gray 17 Gray 94 p a r t i c u l a r molecular mechanism was found, i t was suggested that exposing c e l l s to low temperatures during i r r a d i a t i o n may r e s u l t i n slowed rate processes and the molecular recombination of r a d i c a l s before deleterious reactions can occur. Ben-Hur et a l . (1974) have, however, documented r e s u l t s that seem to contradict these, observing that low temperatures potentiated l e t h a l r a d i a t i o n damage i n a V79-753B-3M subline when the c e l l s were i r r a d i a t e d i n a b u f f e r s o l u t i o n at 0°C. A t h i r d r e s u l t i s claimed by Koch and Burki (1977) who found no measurable diffe r e n c e i n the r a d i a t i o n s u r v i v a l c h a r a c t e r i s t i c s of yet another V79 subline when these c e l l s were i r r a d i a t e d at 4°C and 37°C. Although i t i s possible that the v a r i a b i l i t y of these r e s u l t s r e f l e c t s r e a l d ifferences between c e l l l i n e s and sublines, they are most l i k e l y due to differences i n experimental protocol (the length of time for which the c e l l s were held at low temperatures and the nature of the i r r a d i a t i o n and post-i r r a d i a t i o n c o n d i t i o n s ) . 5.4.2 Oxygen gradients i n t h i n f i l m culture The r o l e of c e l l shape i n determining the c e l l u l a r r a d i o s e n s i t i v i t y i n t h i n f i l m culture can be envisioned as depicted i n F i g . 39. Glass P e t r i dishes containing t y p i c a l spot inoculated c e l l monolayers were c a r e f u l l y weighed before and a f t e r removing the culture medium. I t was determined that l e s s than 0.1 ml of l i q u i d remained once the medium was aspirated. Hypothetically, t h i s residium would wet the dish surface with a t h i n f i l m of medium an average of a few tens of microns i n depth. In p r a c t i c e , however, surface tension e f f e c t s w i l l reduce the depth of the f i l m above the c e l l s considerably, perhaps to a Figure 39 Gradients i n t h i n f i l m cultured c e l l s of d i f f e r e n t shapes. A proposed mechanism by which the v a r i a t i o n i n oxygen enhanced r a d i o s e n s i t i v i t y of f l a t and round t h i n f i l m cultured c e l l s can be explained. Approximate values f o r differences i n d i f f u s i o n distances and i n t r a c e l l u l a r oxygen concentrations are given f or the f l a t and round c e l l s . 96 thickness the order of a s p h e r i c a l c e l l radius (<10 fim). According to Ling et a l . (1981), the f i l m i s t h i n enough to allow an immediate exchange of d i f f u s i n g molecules between the gas and the surface of the c e l l . Oxygen d i f f u s e s through the f i l m and into the c e l l s down a concentration gradient created by c e l l r e s p i r a t i o n . In t h i n f i l m c u l t u r e , round c e l l s s i g n i f i c a n t l y increase the d i f f u s i o n distance from the gas above the f i l m of medium to the oxygen consuming mitochondria w i t h i n the c e l l and, hence, also to the c r i t i c a l r a d i a t i o n target, the c e l l nucleus. The magnitude of the oxygen gradient must increase commensurately to maintain f u l l c e l l r e s p i r a t i o n . Consequently, i f the oxygen concentration immediately above the t h i n f i l m i s the same for c e l l s of both shapes, the n u c l e i of round c e l l s are l i k e l y to be exposed to a lower oxygen concentration than those of f l a t c e l l s . This r e s u l t s i n the observed l a g i n oxygen-enhanced c e l l k i l l i n g of the round c e l l s . Furthermore, when the differ e n c e i n the magnitude of the oxygen gradients f o r the two c e l l shapes i s of the same order as the oxygen concentration of the gas (a few hundred parts per m i l l i o n at 37°C) the diffe r e n c e s i n the radioresponses are most acute. As the oxygen concentration of the gas increases, the differ e n c e i n oxygen concentration at the c e l l nucleus f o r the two c e l l shapes becomes les s r a d i o b i o l o g i c a l ^ s i g n i f i c a n t and the oxygen enhancement r a t i o s become in c r e a s i n g l y s i m i l a r . 97 5.4.3 Evaluation of the t h i n f i l m technique The t h i n f i l m c e l l c ulture technique has been shown to be i d e a l l y s u i t e d f o r experiments i n which c o n t r o l of the oxygen concentration to which c e l l s are exposed at low oxygen tensions i s required. The stated objectives were met s u c c e s s f u l l y using t h i s technique and the questions posed i n the statement of the hypothesis have been answered d e c i s i v e l y . Differences i n the r a d i a t i o n responses of t h i n f i l m cultured c e l l s could be accurately resolved at very low oxygen tensions and the technique d i d not d i s p l a y the oxygen e q u i l i b r a t i o n or depletion problems associated with the suspension culture method. Random experimental error was r e l a t i v e l y low f o r these experiments and the greatest v a r i a b i l i t y i n the r e s u l t s seemed to be b i o l o g i c a l i n o r i g i n . The r e s u l t s of the experiments were e a s i l y repeated within v a r i a t i o n s that can be a t t r i b u t e d to s l i g h t l y d i f f e r i n g c e l l cycle d i s t r i b u t i o n s i n the experimental populations. The i r r a d i a t i o n of c e l l s i n t h i n f i l m culture, as i t i s presented i n t h i s t h e s i s , i s somewhat longer and more cumbersome than others (Michaels et a l . , 1978; Ling et a l . , 1981) i n that i t requires two t r y p s i n i z a t i o n procedures, once p r i o r to and once a f t e r i r r a d i a t i o n . This d i d not seem to adversely a f f e c t the q u a l i t y of the experimental r e s u l t s , however, and allowed the use of s t a t i s t i c a l l y superior s u r v i v a l assays. No t o x i c i t y or contamination problems were encountered despite the complete removal of the medium covering the c e l l s p r i o r to i r r a d i a t i o n and the various other c e l l handling procedures. F i n a l l y , although i t was not a f a c t o r i n these experiments, the dosimetry problems associated with c e l l s attached to glass surfaces would present d i f f i c u l t i e s i f absolute dose responses are desired. For such a p p l i c a t i o n s , a p l a s t i c culture surface with a low oxygen permeability such as that used by Michaels et al.(1978) may be a more su i t a b l e a l t e r n a t i v e . 6. SUMMARY The contents of t h i s thesis can be c l e a r l y summarized by a b r i e f review of i t s most s a l i e n t points. These are as follows: 1. The need to understand the e f f e c t of c e l l r e s p i r a t i o n on the a v a i l a b i l i t y of i n t r a c e l l u l a r oxygen at low ambient oxygen concentrations was reviewed by c i t i n g established examples of the r o l e of oxygen i n the r a d i o s e n s i t i z a t i o n , radioprotection and chemosensitization of c e l l s . 2. A hypothesis was formulated based on the use of r a d i o s e n s i t i v i t y as an i n d i c a t o r of i n t r a c e l l u l a r oxygen concentration and, hence, as a method of determining the existence of t h e o r e t i c a l l y predicted, r e s p i r a t i o n induced i n t r a c e l l u l a r oxygen gradients. 3. The e q u i l i b r a t i o n and the r a d i o l y t i c and metabolic depletion of oxygen i n s t i r r e d suspension cultures were examined and showed that t h i s c u l t u r i n g method i s inappropriate f o r a p p l i c a t i o n s i n which accurately c o n t r o l l e d low, but non-zero c e l l u l a r oxygen concentrations are desired. A very r a p i d l y e q u i l i b r a t i n g , non-toxic t h i n f i l m c u l t u r i n g technique was developed to solve the problems incurred during the suspension culture experiments. The d i f f e r e n t i a l r a d i o s e n s i t i v i t y of r e s p i r i n g and and non-r e s p i r i n g t h i n f i l m cultured c e l l s was established and interpreted to be a consequence of r e s p i r a t i o n induced i n t r a c e l l u l a r oxygen gradients. The magnitude of the implied d i f f e r e n c e i n c e l l oxygenation was shown to be dependent upon c e l l shape and was i n good agreement with t h e o r e t i c a l predictions regarding such gradients. 100 7. B I B L I O G R A P H Y ADAMS, G. E. (1972) Radiation chemical mechanisms i n r a d i a t i o n biology. Adv. Radiat. Biol., 3, 125-208 ALPER, T. (1979) Cellular Radiobiology. Cambridge: Cambridge U n i v e r s i t y Press ALPER, T. and P. HOWARD - FLANDERS (1956) The r o l e of oxygen i n modifying the r a d i o s e n s i t i v i t y of E. coli B. Nature, 178, 978-979 BARENDSEN , G. W. , C. J . ROOT , G. R. VAN KERSEN , D. K. BEWLEY , S . B. FIELD and C. J . 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Biol.: Oxygen Transport to Tissue IV (H. I. Bicher and D. F. Bruley, eds.) New York: Plenum Press, 159, 419-434 DU R A N D , R. E. (1972) Growth and Radiation Survival Characteristics of Cells Grown as an in Vitro Tumor Model. Doctoral d i s s e r t a t i o n , U n i v e r s i t y of Western Ontario. DU R A N D , R. E. and J . E. B I A G L O W (1977) R a d i o s e n s i t i z a t i o n of hypoxic c e l l s of an in vitro tumor model by r e s p i r a t o r y i n h i b i t o r s . Radiat. Res., 69, 359-366 DU R A N D , R. E. and J . E. B I A G L O W (1974) M o d i f i c a t i o n of the r a d i a t i o n response of an in vitro tumour model by c o n t r o l of c e l l u l a r r e s p i r a t i o n . Int. J. Radiat. Biol., 26, 597-601 102 DUTREIX, J . and M. BERNARD (1966) Dosimetry at inter f a c e s f o r high energy X and gamma rays. B r i t . J. Radiol. 39, 205-210 ELKIND, M. M. and G. F . WHITMORE (1967) The Radiobiology of Cultured Mammalian Cells. Gordon and Breach: New York EPP, E. R. , H. WEISS, B. DJORDJEVIC and A. SANTOMASSO (1972) The r a d i o s e n s i t i v i t y of cultured mammalian c e l l s exposed to single high i n t e n s i t y pulses of electrons i n various concentrations of oxygen. Radiat. Res., 5 2 , 324-332 FORD, D. K. and G. YERGANIAN (1958) Observations on the chromosomes of Chinese hamster c e l l s i n tiss u e c u l t u r e . J. Nat. Cancer Inst., 21, 393-425 FRANKO, A. J . and C. J . KOCH (1983) The r a d i a t i o n response of hypoxic c e l l s i n EMT6 spheroids i n suspension culture does model data from EMT6 tumors. Radiat. Res., 96 497-504 FRESHNEY, R. I., (1987) Culture of Animal Cells: A Manual of Basic Technique, 2 ed., New York: Alan R. L i s s , Inc. FROESE, G. (1962) The r e s p i r a t i o n of as c i t e s tumor c e l l s at low oxygen concentrations. Biochim. Biophys. Acta, 57, 509-519 GAYESKI, T. E. J . and C. R. HONIG (1986) Shallow i n t r a c e l l u l a r 0 2 gradients and absence of perimitochondrial 0 2 "wells" i n h e a v i l y working red muscles. Adv. Expt. Med. Biol.: Oxygen Transport to Tissue VIII ( I . S . Longmuir, ed.) New York: Plenum Press, 200, 487-494 GRAY, L. H. (1956) A method of oxygen assay applied to a study of the removal of dissolved oxygen by cysteine and cysteamine. Progress in Radiobiology ( J . S . M i t c h e l l , B. E. Holmes and C. L. Smith, eds.) Edinburgh: O l i v e r and Boyd, 267-274 HALL, E. J . (1988) Radiobiology for the Radiologist, 3 rd ed., P h i l a d e l p h i a : J . B. Lippencott Co. JONES, D. and H. S . MASON (1978) Gradients of 0 2 concentration i n hepatocytes. J. Biol. Chem., 253, 4874-4880 KELLERER, A. M. , and H. H. Rossi (1972) The theory of dual r a d i a t i o n action. Curr. Top. Radiat. Res., 8, 85-158 KENNEDY, K . A., S . ROCKWELL and A. C. SARTORELLI (1980) P r e f e r e n t i a l a c t i v a t i o n of mitomycin C to cytotoxic metabolites by hypoxic tumor c e l l s . Cancer Res., 40, 2356-2360 KOCH, C. J . (1984) A t h i n - f i l m c u l t u r i n g technique allowing r a p i d gas-l i q u i d e q u i l i b r a t i o n (6 sec) with no t o x i c i t y to mammalian c e l l s . Radiat. Res. 97, 434-442 103 K O C H , C. J . and H. J . B U R K I (1977) Enhancement of X-ray induced p o t e n t i a l l y l e t h a l damage by low temperature storage of mammalian c e l l s . Br. J. Radiol., 50, 290-293 K O C H , C. J . , J . K R U U V and H. E. F R E Y (1973) V a r i a t i o n i n r a d i a t i o n response of mammalian c e l l s as a function of oxygen tension. Radiat. Res., 5 3 , 33-42 K O C H , C. J . and R. B . P A I N T E R (1975) The e f f e c t of extreme hypoxia on the re p a i r of DNA single-strand breaks i n mammalian c e l l s . Radiat. Res. 64, 256-259 K O C H , C. J . . C. C. S T O B B E and K. A. B A E R (1984) Metabolism induced binding of C-misonidazole to hypoxic c e l l s : K i n e t i c dependence on oxygen concentration and misonidazole concentration. Int. J. Radiat. Oncol. Biol. Phys., 10, 1327-1331 L E G R Y S , G . A. and E. J . H A L L (1969) The oxygen e f f e c t and X-ray s e n s i t i v i t y i n synchronously d i v i d i n g cultures of Chinese hamster c e l l s . Radiat. Res., 37, 161-172 L I N G , C. C , H. B . M I C H A E L S , L . E. G E R W E C K , E. R. E P P and E. C. P E T E R S O N (1981) Oxygen s e n s i t i z a t i o n of mammalian c e l l s under d i f f e r e n t i r r a d i a t i o n conditions. Radiat. Res., 86, 325-340 M A R S H A L L , R. S . , C. J . K O C H and A. M. R A U T H (1986) Measurement of low l e v e l s of oxygen and t h e i r e f f e c t on the r e s p i r a t i o n i n c e l l suspensions maintained i n an open system. Radiat. Res., 108, 91-101 M I C H A E L S , H. B . , E. R. E P P , C. C. L I N G and E. C. P E T E R S O N (1978) Oxygen s e n s i t i z a t i o n of CHO c e l l s at u l t r a h i g h dose rates: Prelude to oxygen d i f f u s i o n studies. Radiat. Res., 76, 510-521 M I L L A R , B . C , E. M. F I E L D E N and J. J . S T E E L E (1979) A biphasic r a d i a t i o n s u r v i v a l response of mammalian c e l l s to molecular oxygen. Int. J. Radiat. Biol., 36, 117-120 M U R T H Y , M. S . S . and A. R. L A K S H M A N A N (1976) Dose enhancement due to backscattered secondary electrons at the int e r f a c e of two media. Radiat. Res., 67, 215-223 P A L C I C , B . and L . D. S K A R S G A R D (1984) Reduced oxygen enhancement r a t i o at low doses. Radiat. Res., 100, 328- 339 P O D G O R S K I , G . T. , I. S . L O N G M U I R , J . A. K N O P P and D. M. B E N S O N (1981) Use of an encapsulated fluorescent probe to measure i n t r a c e l l u l a r P Q 2 • Cell. Phys., 107, 329-334 P U C K , T. T. and P . I. M A R C U S (1956) Action of x-rays on mammalian c e l l s . J. Exp. Med., 103, 653-666 P U R D I E , J . W., (1979) A comparative study of the radioprotective e f f e c t s of cysteamine, WR-2721 and WR-1065 i n cultured human c e l l s . Radiat. Res., 77, 303-311 104 P U R D I E , J . W. , E . R. I N H A B E R , H . S C H N E I D E R , J . L. L A B E L L E (1983) Interaction of cul t u r e d mammalian c e l l s with WR-2721 and i t s t h i o l , WR-1065: implications f o r mechanisms of radioprotection. Int. J. Radiat. Biol., 43, 517-527 Q U I N T I L I A N I , M. , (1979) M o d i f i c a t i o n of r a d i a t i o n s e n s i t i v i t y : The oxygen e f f e c t . Int. J. Radiat. One. Biol. Phys., 5, 1069-1076 R A U T H , A. M. , R. A. M C C L E L L A N D , H . B . M I C H A E L S and R. B A T T I S T E L L A (1984) The oxygen dependence of the reduction of nitroimidazoles i n a r a d i o l y t i c model system. Int. J. Radiat. Oncol. Biol. Phys., 10, 1323-1326 S E T L O W , R. B . and E . C. P O L L A R D (1962) Molecular Biophysics Reading, Massachusetts: Addison-Wesley Publishing Company, Inc. S H E N O Y , M. A., J . C. A S Q U I T H , G . E . A D A M S , B . D . M I C H A E L and M. E . W A T T S (1975) Time resolved oxygen e f f e c t s i n i r r a d i a t e d b a c t e r i a and mammalian c e l l s : A rapid-mix study. Radiat. Res., 62, 498-512 S I N C L A I R , W. K . , (1969) Radiation Dosimetry, Vol. I l l , 2nd ed.(Attix, F . H . , W. C. Roesch and E . T o c h i l i n ) New York: Academic Press S I N C L A I R , W. K . , (1966) The shape of r a d i a t i o n s u r v i v a l curves of mammalian c e l l s cultured in vitro. Biophysical Aspects of Radiation Quality, Technical Report Series No. 58, 21-43. S I N C L A I R , W. K . and R. A. M O R T O N (1963) Var i a t i o n s i n x-ray response during the d i v i s i o n cycle of p a r t i a l l y synchronized Chinese hamster c e l l s i n c u l t u r e . Nature, 199, 1158-1160 S U T H E R L A N D , R. M. and R. E . D U R A N D (1976) Radiation response of m u l t i c e l l spheroids: An i n v i t r o tumor model. Curr. Top. Radiat. Res., 11, 87-139 S U T H E R L A N D , R. M. , W. R. I N C H , J . A. M C C R E D I E and J . K R U U V (1970) A multicomponent r a d i a t i o n s u r v i v a l curve using an in vitro tumor model. Int. J. Radiat. Biol., 18, 491-485 T A Y L O R , Y. C. and A. M. R A U T H (1982) Oxygen tension, c e l l u l a r r e s p i r a t i o n , and redox state as the var i a b l e s i n f l u e n c i n g the c y t o t o x i c i t y of the r a d i o s e n s i t i z e r misonidazole. Radiat. Res., 91, 104-123 T H O M L I N S O N , R. H . and L. H . G R A Y (1955) The h i s t o l o g i c a l structure of some human lung cancers and the possible implications f o r radiotherapy. Br. J. Cancer, 9, 539-549 V A R N E S , M. E . , J . E . B I A G L O W and S. W. T U T T L E (1986) Role of c e l l u l a r non-p r o t e i n t h i o l s i n oxygen consumption and peroxide reduction. Adv. Expt. Med. Biol.: Oxygen Transport to Tissue VIII ( I . S. Longmuir, ed.) New York: Plenum Press, 200, 565-571 105 V I N D E L O V , L . L . , (1977) Flow microfluorometric analyses of nuclear DNA i n c e l l s from s o l i d tumors and c e l l suspensions. Virchows Arch. B. Cells Pathol., 24, 227 W A L K E R , H. C. and B . M . C U L L E N (1987) The importance of NPSH on the r a d i o s e n s i t i z i n g e f f e c t of oxygen i n Chinese hamster V-79 c e l l s . Int. J. Radiat. Biol., 51, 19-27 W A T T S , M . E. , R . L . M A U G H A N and B . D. M I C H A E L (1978) Fast k i n e t i c s of the oxygen e f f e c t i n i r r a d i a t e d mammalian c e l l s . Int. J. Radiat. Biol., 33, 195-199 W H I L L A N S , D. W . and A. M . R A U T H (1980) An experimental and a n a l y t i c a l study of oxygen depletion i n s t i r r e d c e l l suspensions. Radiat. Res., 84, 97-114 W I L S O N , D. F. , M . E R E C I N S K A , C. DROWN and I. A. S I L V E R (1979) The oxygen dependence of c e l l u l a r energy metabolism. Arch. Biochem. Biophys., 195, 485-493 W I T T E N B E R G , J . B . and B . A. W I T T E N B E R G (1981) F a c i l i t a t e d oxygen d i f f u s i o n by oxygen c a r r i e r s . Oxygen and Living Processes: An Interdisciplinary Approach (D. L . G i l b e r t , Ed.), New York: Springer-Verlag, 186-199 Y U H A S , J . M . and J . B . S T O R E R (1969) D i f f e r e n t i a l chemoprotection of normal and malignant t i s s u e s . J. Nat. Cancer Inst., 42, 331-335 106 8. APPENDIX 8.1 The In t e r a c t i o n of Ionizing Radiation with C e l l s I o n i z i n g r a d i a t i o n i s defined as a r a d i a t i o n having s u f f i c i e n t energy to remove an o r b i t a l e l e c t r o n from an atom or molecule with which i t i n t e r a c t s . The i o n i z a t i o n of an atom can occur as a r e s u l t of two modes of r a d i a t i o n action. Charged p a r t i c l e radiations (electrons, protons, a - p a r t i c l e s , etc.) act by d i r e c t l y removing o r b i t a l electrons from the atoms through which they pass. Electromagnetic radiations (X and 7-rays) act i n d i r e c t l y , f i r s t producing f a s t moving secondary electrons, which then proceed to ionize other atoms and molecules. Neutrons are also a form of i n d i r e c t l y i o n i z i n g r a d i a t i o n , i n t e r a c t i n g with atomic n u c l e i to produce charged p a r t i c l e s ( f a s t moving protons, a-p a r t i c l e s and other nuclear fragments) through which i o n i z a t i o n processes occur. An i o n i z a t i o n event releases approximately 33 eV and i s s u f f i c i e n t to break strong chemical bonds. (A C=C bond has an associated energy of 4.9 eV.) The density with which these i o n i z i n g events occur i n i r r a d i a t e d matter i s dependent upon the l i n e a r energy t r a n s f e r (LET) of the r a d i a t i o n . P a r t i c u l a t e r a d i a t i o n has a much higher LET than electromagnetic r a d i a t i o n and the nature of p a r t i c u l a t e radiation-matter i n t e r a c t i o n s i s also much more complex. The in t e r a c t i o n s of X and 7-rays with matter have, by comparison, been well defined. For electromagnetic r a d i a t i o n with energies below 10 MeV, the production of the secondary electrons i s known to occur p r i m a r i l y as a r e s u l t of e l a s t i c c o l l i s i o n s between photons and free electrons (Compton e f f e c t ) 107 and the release of bound o r b i t a l electrons by photon a n n i h i l a t i o n ( p h o t o e l e c t r i c e f f e c t ) . The most c r i t i c a l target of r a d i a t i o n a c t i o n i n c e l l s i s the DNA. The i n t e r a c t i o n of a DNA strand with a f a s t moving e l e c t r o n or photon can cause a rupturing of the phosphate sugar back bone or the formation of a re a c t i v e DNA free r a d i c a l (atom with unpaired e l e c t r o n i n i t s outer o r b i t a l s h e l l ) . Both of these types of DNA damage could adversely a f f e c t the reproductive a b i l i t y of the c e l l . In ad d i t i o n to d i r e c t radiation-DNA i n t e r a c t i o n s , damage may also be a caused by free r a d i c a l s that are r a d i o l y t i c a l l y induced i n l i q u i d environment close to the DNA. The r a d i o l y t i c products of water ( c e l l s are composed of 80% water) includes the l^O* ion r a d i c a l which r a p i d l y combines with another water molecule to produce hig h l y r e a c t i v e hydroxyl free r a d i c a l . Approximately two th i r d s of r a d i a t i o n damage incurred by c e l l s during the X - i r r a d i a t i o n i s due to in t e r a c t i o n s with such free r a d i c a l s . As discussed i n the main body of the thesi s , r a d i a t i o n damage to c e l l s can be modified by both r e p a i r and f i x a t i o n . The radioprotective e f f e c t of t h i o l s i n c e l l s has been p a r t i a l l y a t t r i b u t e d to the chemical r e p a i r of r a d i c a l s by hydrogen donation. Conversely, r a d i o l y t i c a l l y induced l e s i o n s i n DNA can also be made permanent by combining with oxygen. An approximate time scale of events a f t e r a radiation-DNA i n t e r a c t i o n i s depicted i n F i g . 40. The ph y s i c a l stage, during which the charged p a r t i c l e passes through the atom or molecule and i o n i z a t i o n occurs, i s completed i n les s than 1 0 " ^ seconds. Ion r a d i c a l s continue to e x i s t f o r approximately 1 0 " ^ seconds and free r a d i c a l s f o r 1 0 8 Figure 40 An approximate time scale of events a f t e r a radiation-DNA i n t e r a c t i o n . - 10 - 1 6 i o n i z a t i o n o f a toms by r a d i a t i o n 10 - 1 1 f o r m a t i o n of f r e e r a d i c a l s d i r e c t a c t i o n c o m p l e t e CJ OJ in 1 0 ~ 5 l i f e t i m e of s e c o n d a r y e l e c t r o n s OJ E • r H - 10" - 10* r e a c t i o n s w i t h f r e e r a d i c a l s c o m p l e t e a l l r a d i o c h e m i c a l r e a c t i o n s c o m p l e t e 10- c e l l u l a r damage a p p a r e n t 1 0 5 l e t h a l e f f e c t s o b s e r v e d i n a n i m a l s 1 0 9 approximately 10"^ seconds. A l l radiochemical events are completed one second a f t e r i r r a d i a t i o n and the various b i o l o g i c a l expressions of the damage become evident hours, days or months a f t e r the i n i t i a l i n t e r a c t i o n . 8 . 2 Modelling Radiation Survival Radiation s u r v i v a l curves are a commonly u t i l i z e d as tools of qua n t i t a t i v e analysis i n i n vitro radiobiology. However, i n ad d i t i o n to simply measuring the e f f e c t of r a d i a t i o n on c e l l s u r v i v a l , the shape of these curves has given r i s e to speculation about the r e l a t i o n s h i p between the mechanism and the b i o l o g i c a l expression of r a d i a t i o n action. A common feature of s u r v i v a l curves f o r cultured mammalian c e l l s i r r a d i a t e d with low LET radiations i s the exisitence of a "shoulder" i n the i n i t i a l p o r t i o n of the curve. This feature has been the source of several theories regarding the response of c e l l s to r a d i a t i o n damage. Some of these theories i n t e r p r e t the shoulder to be a consequence of a l e s i o n r e p a i r mechanism. The re p a i r mechanism i s hypothesized to ameliorate r a d i a t i o n induced lesions by biochemically r e s t o r i n g the damaged DNA. The steeper decline i n s u r v i v a l at higher doses i s a t t r i b u t e d to the eventual saturation or i n a c t i v a t i o n of the mechanism. An a l t e r n a t i v e theory proposes that the existence of the shoulder i s a r e f l e c t i o n of the innate a b i l i t y of most i r r a d i a t e d c e l l s to withstand a c e r t a i n amount of r a d i a t i o n damage before succumbing to more l e t h a l doses. I t has been proposed that the mathematical models used to f i t curves to r a d i a t i o n s u r v i v a l data also reveal clues that connect the 110 molecular mechanisms of r a d i a t i o n a c t i o n with t h e i r b i o l o g i c a l consequences. The exponential nature of r a d i a t i o n s u r v i v a l has l e d to the development of theories which attempt to explain the induction of l e t h a l r a d i a t i o n damage i n terms of Poisson d i s t r i b u t e d " h i t s " on s p e c i f i c c e l l u l a r targets. The simplest such theory i s the "single h i t , s i n g l e target" theory which postulates that a s i n g l e c r i t i c a l event at a r a d i a t i o n target i s s u f f i c i e n t to i n a c t i v a t e the c e l l . The most basic mathematical representation of t h i s hypothesis i s a simple negative exponential curve (Fig. 41) which has been found to c l o s e l y model the i n a c t i v a t i o n of i r r a d i a t e d molecules and some b a c t e r i a l c e l l s . A more elaborate r a d i a t i o n s u r v i v a l model, the Linear Quadratic model, was used to f i t the mammalian c e l l s u r v i v a l data i n t h i s thesis and i s based upon the theory that the primary mode of r a d i a t i o n a c t i o n on c e l l s i s the breaking of DNA strands. The l e t h a l r a d i a t i o n event i s postulated to be an unrepaiared DNA double strand break. In an i r r a d i a t e d c e l l , a double strand break, or " h i t " , can occur as a r e s u l t of a s i n g l e i o n i z a t i o n event that breaks both strands, or by two separate i o n i z a t i o n events, each breaking a s i n g l e strand. Using the s i n g l e h i t , s i n g l e target hypothesis, the p r o b a b i l i t y that e i t h e r such l e t h a l event w i l l occur i s proportional to the dose and to the to the square of the dose, r e s p e c t i v e l y . The net p r o b a b i l i t y of r a d i a t i o n s u r v i v a l i s the product of these two i n d i v i d u a l p r o b a b i l i t i e s (Fig. 42). Although i t s continuous curving does not match in vitro data as c l o s e l y at lower l e v e l s of s u r v i v a l , the LQ model f i t s data i n the f i r s t two decades of s u r v i v a l extremely well. Other more complex models of r a d i a t i o n s u r v i v a l possess a multitude of adjustable parameters (as opposed to two f o r the LQ model) and generally o f f e r no s i g n i f i c a n t Figure 41 Single h i t , s i n g l e target model of r a d i a t i o n s u r v i v a l produces s t r a i g h t exponential s u r v i v a l curve. Dose 112 Figure 42 Proposed molecular basis f o r the Linear Quadratic model of r a d i a t i o n s u r v i v a l . The model can be derived as the product of the i n d i v i d u a l s u r v i v a l p r o b a b i l i t i e s r e s u l t i n g from two modes of DNA double strand break production. T o t a l s u r v i v a l : s = e - ( a D + b D 2 ) w h e r e a=k ^ a n d b = k 2 113 improvement i n the accuracy with which a curve i s f i t t e d to r a d i a t i o n s u r v i v a l data over t h i s range of c e l l s u r v i v a l . 

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