BEGCEEEECAL EFFECTS $17 EHRABiATEB 33163133 FIN HUMAN CELLS {R CUL’E’UEE That‘s {‘M i’ko aw“ 05 Mi. D. WCHEGRR STATE UNWERSETY Noemi Diaz =- Samtiago 7:970 rHEsv-z 0-169 111111111111 IMIWIWIHWD W 31293 01085 0729 This is to certify that the thesis entitled BIOCHEMICAL EFFECTS OF IRRADIATED SUGARS ON HUMAN CELLS IN CULTURE presented by Noe-1 Din-Santiago has been accepted towards fulfillment of the requirements for M degree in 504 Safe me 1. ’ f p 7-:/’/ y . \ V '(2CZW7 '/7[£w/€&/CM Major professor Date ///213’//970 -'=i§47- a w. 3.3) 1'- _ - ‘ we“ ‘ ”Awl? U 3 my?» : ABSTRACT BIOCHEMICAL EFFECTS OF IRRADIATED SUGARS ON HUMAN CELLS IN CULTURE By Noemi Diaz -Santiago In the present study two criteria were used to test the toxicity of gamma -irradiated sugar solutions on human amnion AV3 cells in culture: a) the ability of the cells to grow and multiply after having been incubated with irradiated sucrose for various periods, and b) the effect of irradiated glucose, fructose and sucrose on the synthesis of DNA, RNA, and protein in the same cells. Minimum essential medium (MEM) containing 5. 8 x io'ZM of 3 Mrad irradiated sucrose'was incubated with AV3 cells for 6, 12 and 24 hours at 37° C. After these periods the medium containing irradiated sucrose was replaced by medium containing non —irradiated sucrose and cell counts were made at 2, 4 and 6 days. The duration of the incubation of the cells with the irradiated sugar-was critical for their growth and replication. The percentage of growth inhibition Noemi Diaz —Santiago for the incubation periods of 6, 12 and 24 hours were: 40, 60, and 90 respectively. The synthesis of DNA, RNA and protein in human amnion cells was assessed by following the incorporation of tritiated pre - cursors added to MEM in the presence or absence of 5. 8 X IO-ZM sucrose, glucose or fructose solutions which had been irradiated at l, 2 and 3 Mrad. For a period of 6 hours of incubation with the 3 Mrad. irradiated sugars the following percentages of inhibition of DNA, RNA and protein synthesis (in that order) were: 49, 26 and 38 for sucrose; 20, 14 and 24 for glucose; and 51, 26 and 34 forfruc- tose. Other factors suchas the effect of pH, the activity of DNA polymerase, the presence of catalase in the medium containing 3 Mrad irradiated sucrose andthe time elapsed between irradiationof the sugar solution and testingfor its effects on the DNA synthesis in AV3 cells were also investigated. A considerable reduction in the toxicity of irradiated glucose was observed by maintaining. the pH of the MEM containing irradiated glucose medium at 7. 2 during the incubation period with the cells. The activity of DNA polymerase from an AV3 cells extract was assessed following polymer formation measured by the incor- poration of a nucleotide labeled with 3H into acid -insoluble material Noemi Diaz -Santiago (presumably DNA). The enzyme was not affected by the presence of irradiated sucrose in the substrate. It was also observed that a concentration of 40 ,LLg / ml (66 units/ ml) of catalase only partly eliminated the deleterious effects of 3 Mrad irradiated sucrose to human amnion cells. Sucrose solutions irradiated at 3 and 4 Mrad and stored for seven months at room temperature did not show a reduction on the inhibitory effects on the synthesis of DNA in AV cells. 3 Evidence was presented to show that the damage caused to human amnion cells by the radiolysis products of sucrose and fructose cannot be repaired. The duration of the incubation of the cells with the irradiated sugar, the radiation dose applied, the pH of the medium and the stage of development of the cells are critical for their sur- vival. It seemed that only the fraction of the cell population specifically involved in DNA synthesis (the S phase of development) was severely affected by the radiolysis products of the sugars. This effect is time dependent: the longerthe incubation period the higher the number of cells rendered unable to grow and multiply. BIOCHEMICAL EFFECTS OF IRRADIATED SUGARS ON HUMAN CELLS IN CULTURE By Noemi/Di/az-Santiago A THE SIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1970 667274 TO MY PARENTS ii ACKNOWLEDGMENTS The author wishes to express her sincere appreciation to Professor’Dr. Pericles Markakis for his guidance and encourage- ment during this study and the preparation of the manuscript. She is also grateful to Dr. C. L. Bedford and Dr. W. M. Urbain for their suggestions and help in the preparation of this manuscript. The author is indebted to Dr. J. E. Trosko for his valuable advice and discussions during the course of this project, for pro- viding the cell cultures, reading the manuscript, and for the opportunity to work in his laboratory. The author also wishes to thank Mrs. Miriam Isoun and Mrs. Virginia Mansour for their technical assistance. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES IN TRODUC TION REVIEW OF THE LITERATURE . MATERIALS AND METHODS Culture Irradiation Labeled Compounds . . DNA, RNA, and Protein Synthesis Cell Viability Study. . . Activated Carbon . . . DNA Polymerase Activity. Catalase Activity. Irradiated Sucrose in Storage RESULTS AND DISCUSSION . A. Cell Viability B. Synthesis of DNA 1. N «10501th Effects of irradiated water and dose rate Effect of irradiated sucrose on DNA synthesis - - preliminary experiment The effect of activated carbon . Effect of pH . Irradiated glucose vs. DNA synthesis Irradiated fructose vs. DNA synthesis Irradiated sucrose-vs. DNA synthesis iv Page vi . viii 16 16 18 18 20 20 23 29 29 32 C. Synthesis of RNA. . . . 1. Irradiated glucose vs. RNA synthesis 2,. Irradiated fructose vs. RNA synthesis 3. Irradiated sucrose vs. RNA synthesis D. Protein Synthesis . . . 1. Irradiated glucose vs. protein synthesis 2. Irradiated fructose vs. protein synthesis 3. Irradiated sucrose vs. protein synthesis E. The Effect of Ageing of Irradiated Sucrose Solution on the DNA Synthesis F. DNA Polymerase Activity. G. The Influence of Catalase on the Medium Containing Irradiated Sucrose H. Reducing Sugar Content of Irradiated Sucrose Discussion SUMMARY AND CONCLUSIONS Summary Conclusions LIST OF REFERENCES APPENDIX Page 32 32 35 35 35 35 38 38 42 42 48 52 53 58 58 59 61 66 Table LIST OF TABLES Percentage of DNA synthesis inhibition in AV cells observed using irradiated sucrose stored from O to 7 months at room tempera- ture DNA polymerase activity in extracts of AV cells incubated with 3 Mrad irradiated sucrose for 1 hour at 37° C 3 The effect of catalase on 3 Mrad irradiated sucrose, measured by the incorporation (in counts / min) of tritiated thymidine into the DNA of AV3 cells Percent of total reducing sugars from 1, 2 and 3 Mrad irradiated sucrose solution . Average percent inhibition of DNA, RNA and protein synthesis for AV3 cells in contact with 3 Mrad irradiated sugars . Effect of duration of incubation on the growth of AV 3 cells incubated with 3 Mrad _2 irradiated sucrose solution (5. 8 X 10 M) Percent inhibition of DNA synthesis in AV3 cells incubated with 5. 8 X 10'2M sucrose, glucose and fructose solutions irradiated at 1, 2 and 3 Mrad (average; individual in parentheses) . vi Page 42 49 51 52 53 66 67 Table Pag e Percent inhibition of RNA synthesis in AV cells incubated with 5.8 X 10’ M sucrose, glucose and fructose solutions irradiated at 1, 2 and 3 Mrad (average; individual in parentheses)................... 68 Percent inhibition of protein s nthesis in AV3 cells incubated with 5. 8 X 10‘ M sucrose, glucose and fructose solutions irradiated at 1, 2 and 3 Mrad (average; individual in parentheses)................... 69 vii Figure LIST OF FIGURES Effect of duration of incubation on the growth of AV3 cells exposed to 3 Mrad irradiated sucrose solution 5. 8 X IO‘ZM in MEM Effect of water irradiated at different dose rates on the 3H -thymidine uptake in DNA synthesis by AV3 cells . . . 3H- thymidine uptake ,in DNA synthesis by Av3 cells incubated with 5. 8 x 10’2M irradiated sucrose solutions (preliminary experiment) 3H ~thymidine uptake in DNA synthesis by AV3 cells incubated with 2% irradiated sucrose solutions treated with activated carbon post -irradiation 3H -thymidine uptake in DNA synthesis by Av cells incubated with 5. 8 x 10'2M glucose and fructose solutions irradiated at 3 Mrad 3H -thymidine uptake inDNA synthesis by Av3 cells incubated with 5. 8 x 10’2M glucose and fructose solutions irradiated at 4 Mrad 3H -thymidine uptake in DNA synthesis by Av3 cells incubated with 5. 8 x 10-2M sucrose solution irradiated at 3 Mrad and unirradiated sucrose solutions at pH 6. 40 in MEM . ' viii Page 17 19 21 22 24 25 27 Figure 10. 11. 12. 13. 14. 15. 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 Mrad irradiated sucrose, glucose and fructose solutions (5.8 x 10-2M in MEM at pH 7.2). 3H ~thymidine uptake in DNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad . . . -2 lrradlated glucose solutlons (5.8 X 10 M in MEM at pH 7.2) 3H -thymidine uptake in DNA synthesis by AV cells incubated with 1, 2 and 3 Mrad irradiated fructose solutions (5.8 X 10‘2M in MEM at pH 7.2) 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated sucrose solutions (5.8 X 10‘ M in MEM at pH 7.2) 3H -uridine uptake in RNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated glucose solutions (5.8 X 10'2M in MEM at pH 7. 2) 3H -uridine uptake in RNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated fructose solutions (5.8 X 10‘2M in MEM at pH 7. 2) 3H -uridine uptake in RNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated sucrose solutions (5.8 X 10‘2M .inMEM at pH 7.2) 3H -leucine uptake in protein synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated glucose solutions (5.8 X 10'2M in MEM at pH 7. 2) Page 28 30 31 33 34 36 37 39 Figure 16. 17. 18. 19. 20. 21. 22. 3H -leucine uptake in protein synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated fructose solutions (5.8 X 10‘2M in MEM at pH 7. 2) 3H -leucine uptake in protein synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated sucrose solutions (5. 8 X 10'2M in MEM at pH 7.2) 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 and 4 Mrad irradiated sucrose solutions (5.8 X 10’2M) immediately after irradiation 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 and 4 Mrad irradiated sucrose stored for 2 months at room temperature. Sucrose solutions (5.8 x 10'2M) 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 Mrad irradiated sucrose solution stored for 2 months at room temperature. Sucrose concentration was 5. 8 x IO‘ZM in MEM at pH 7.15. 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 and 4 Mrad irradiated sucrose stored for 4 months at room temperature. Sucrose concentration was 5. 8 X IO’ZM in MEM at various pH' s 3H -thymidine uptake in DNA synthesis by ' Av3 cells incubated with 3 and 4 Mrad irradiated sucrose stored for 7 months at room temperature. Sucrose concentration was 5. 8 x IO'ZM in MEM at various pH' s Page 40 41 43 44 45 46 47 INTRODUC TION Studies on the feasibility of preserving foods by ionizing radiations were undertaken shortly after World War II. Early work in the United States demonstrated a number of possible industrial applications on a theoretical and laboratory basis (Brasch and Huber, 1947). An extensive program on the radiation sterilization of foods was started in 1953 bythe U. S. Army Quartermaster Corps, which was attracted by the advantages of preserving food for long periods of time without the use of heat or refrigeration. The fundamental requirement for obtaining Governmental approval of the application of irradiation to a given food is the pro- duction of evidence that such food is wholesome for human consump~ tion. Although the recognized method of producing such evidence is by animal feeding trials, the results of studies using a less compli- cated "model system" should not be overlooked. It is well established that ionizing radiation exerts biological effects not only by direct action on the organism, but also by changing the environment in‘which organisms live. Some of these indirect effects are toxic. While there are considerable data on the cytotoxic and radiomimetic effects of irradiated culture media and sugar solutions, their precise biochemical nature is yet to be clarified. In the present study two criteria were used to test the toxicity of gamma irradiated sugar solutions on human amnion AV3 cells in culture: a) the ability of the cells to multiply after having been in contact with sucrose for various periods and, b) the synthesis of DNA, RNA, and protein in the same cells. Other factors such as the pH of the irradiated solutions, the time elapsed between irradiation and testing, the duration of the exposure of the cells to irradiated sugar, and the effect of catalase on the irradiated media were also studied. REVIEW OF THE LITERATURE A number of studies have been conducted in which organisms were grown in the presence of irradiated substances. Such organisms included bacteria, yeasts, higher plants, the fruit fly, and mammalian cells. Thevradiomimetic effects of irradiated media have been known for many years. As early as 1887, while investigating the killing of the spores of the anthrax bacillus by sunlight, Roux came to the conclusion that germination of the spores could be inhibited by materials which were formed as a result of the oxidation of the carbohydrate fraction of the medium (Blank and Arnold, 1935). Coblentz and Fulton (1924) and Woodrow, Bailey and Fulmer (1927) showed that the ability of a medium containing carbohydrates to support the growth of micro -organisms was diminshed if the medium was irradiated with ultraviolet energy previous to inocula - tion. Blank and Kersten (1935) showed that modified extract agar Could be so altered by the action of soft X -rays that it would no no longer support the growth of Bacillus subtilis. They concluded that inhibition results from the formation of a toxic material in the agar, the major carbohydrate of the medium. This discovery led to the investigation of the action of radiation on other carbohydrates. Blank and Arnold (1935) observed that Bacillus subtilis could be similarly inhibited by the addition to the culture medium of an irradiated solution of any one of 20 different carbohydrates and 3 carbohydrate derivatives. Baumgartner (1936) confirmed the former workers' observation and also stated that such radiation of carbohydrates is accompanied by marked production of acid, mainly formic acid. He also observed that neutralization of this acidity restores the ability of the culture media to support growth of the bacteria. Stone, Wyss and Haas (1947) obtained initial evidence that ultraviolet light produced chemical mutagens in bacterial media capable of inducing mutations in unirradiated organisms placed in these media. In the past few years, several articles dealing with cell growth and chromosomal aberrations induced by irradiated media have been published. Molin and Ehrenberg (1946) observed a bacteriostatic action exerted by irradiated glucose solutions on Pseudomonas species. An effect on the incorporation of uracil or thymine in the cells of Escherichia coli as a response to irradiated medium was reported by Pollard et al. (1965). The inhibitory action of irradiated sucrose solutions on Salmonella typhimurium was studied by Schubert and Watson (1969). They attributed this effect to hydroxyalkyl peroxides and hydroperoxides resulting from sugar hydrolysis. Cytological aberrations in higher plants produced by irradiated carbohydrates have been reported by several investi- gators. . Holsten etil. (1965) found that irradiated sucrose solution and certain fractions derived therefrom inhibit the growth of carrot cells and Vicia faba root cells, leading to chromosomal aberrations and impairment of cell division. Chopra e131. (1963) reported Chromosome breakage in barley and onion seeds grown on orange and apple juice which had been irradiated with 200 Krad of gamma rays. A nucleotoxic effect due to irradiated glucose on onion and barley root tip cells was observed by Moutschen and Matagne (1965). The inhibitory effects of irradiated sucrose on the germination and growth of pollen of Tropaeolum majus was reported by Kesavan and Swaminathan (1967). Bajaj (1970) studied the effects of irradiated (2 Mrad) sucrose, glucose‘and fructose on the growthand develop- ment of callus tissue cultures, excised roots, ovules and embryos of Glycine max, Nicotiana tabacum, Pelargonium hortorum and Phaseolus vulgaris. He observed growth inhibition by 30 to 50%. The growth of immature ovules and embryos of Nicotiana tabacum and Phaseolusvulgaris, respectively, was consideraly inhibited by irradiated sucrose. . However, this growth inhibition was markedly reduced in the roots of Glycine max, if these roots were grown on an irradiated glucose medium stored for six months. Several investigators, Swaminathan et a1. (1963), Rinehart and Ratly (1965), and Prakash (1965), have demonstrated an increase in mutations when Drosophila melanogaster are reared on irradiated foods. Nevertheless, these results failed to be reproduced by other workers (Chopra, 1965; Khan and Alderson, 1965; and Reddi e_t_a_l. , 1965). The cytotoxic effects of irradiated culture media containing carbohydrate (glucose and fructose) to human and animal cells were first observed by Berry and co-workers (1965), who studied the reproductive survival of mammalian cells. They attributed the toxicity to the production of glyoxal in the solution. Chromosome aberrations were observed when human lymphocytes were exposed to irradiated sucrose (Shaw and Hayes, 1966). Kesavan and Swaminathan reported cytotoxic effects of irradiated culture medium on human leukocytes. Scott {is}; (1966) observed growth inhibition of L517 8 Y lymphoma cells when these were in contact with irradiated medium. Kellner and Kaindl (1967) studied the influence of glyoxal on the growth of human fibroblasts. They found that glyoxal could be degraded by the cells and thus no toxicity was apparent after 24 hours of contact between cells and the glyoxal containing medium. De, Aiyar and Sreenivasan (1969) investigated the effect of irradiated sucrose on rats. When rat liver slices were exposed to irradiated (0. 5 Mrad) sucrose solutions, inhibition of succinate oxidation and phosphorylation as well as the synthesis of lipids, proteins and DNA was observed. However, no deleterious effects could be observed in the 12 rats fed irradiated sucrose solutions for a period of 8 weeks. Articles, books and monographs dealing with the effects of irradiated media on living organisms have been pub- lished. These include the proceedings of a symposium on implica- tions of organic peroxides in radiobiology (Feenstein, 1962), a review article by Stone (1955) on the general effects of medium irradiation on genetic material, a review by Scarascia -Mugnozza §t_a1. on the genetic effects produced by irradiated food and food components (1965), and Latarjet' 3 discussion on the viral and bacterial effects using growth as a criterion (1956). The products of radiolysis of carbohydrates have been studied in great detail by Phillips and co-workers (1965, 1969). Their work has served as the basis for many of the studies referred to in this manuscript. MATERIALS AND METHODS Culture Human amnion AV 3 cells obtained from the American Type Culture Collection (Rockville, Md.) were cultured to form a mono- layer in Roux bottles at 37° C in a humidifed 5% C02 atmosphere in Eagle' 3 Minimum Essential Medium (MEM) supplemented with 10% calf serum. Fifty ml of MEM were used per bottle of culture. After several days of incubation a monolayer of cells was formed, the growth medium was discarded, and 10 ml of 0.05% trypsin prepara- tion in Puck's Saline "A" was added to the culture bottle in order to detach the cells from the glass. The cells collected from several bottles were pooled in a flask containing MEM and their concentration was determined with a hemocytometer. Falcon disposable plastic tissue culture plates (60 mm in diameter) were used to inoculate media for the experiments. Equal amounts of cells were pipeted into each plate (2 X 105 cells/plate). For any given experiment the cells were incubated for at least 18 hours at 37° C to allow the cells to attach before any treatment was applied. The pH of the culture medium was 7. 2. Irradiation Sugar solutions containing 4% of either glucose or fructose or sucrose-were prepared in distilled -demineralized water and filter sterilized using Millipore filters, HA -0. 4511.. The solutions were transferred into sterile 25 ml vials, screw capped and placed at the calculated distance from the 60Co source in order to absorb the desired dose. The 50, 000 Ci cobalt-60 irradiator installed in July 1967 in the Department of Food Science of this University was used for all the experiments described in this work. Irradiations were performed at constant temperature (20° C) for a period of 16 hours. Labeled Compounds Tritiated thymidine (3H- Tdr), tritiated uridine (SH-Udr), and tritiated leucine (3H-leucine), specific activity 2.0 Ci/mM, were obtained from New England Nuclear. 3H-—Tdr was used as a labeled precursor for DNA synthesis, 3H-Udr for RNA synthesis, and 3H-leucine for protein synthesis in AV cells incubated with MEM 3 containing irradiated sugars. 10 DNA, RNA, and Protein Synthesis Measurement of DNA synthesis was performed by following the incorporation of tritiated thymidine into trichloroacetic acid (TCA) - insoluble material as described by Chu and Regan (1966). 3H- thymidine, specific activity 2.0 Ci/mM, ZlLCi/ml in MEM- sucrose was used for the experiments. Three ml of MEM containing 3H- Tdr and 5. 8 X 10-2M irradiated sugar-were added to culture plates of AV3 cells in a mono- layer. The plates were incubated at 37° C for 2, 4, and 6 hours'to allow for the incorporation of the labeled precursor into DNA. At the end of each period the plates were placed over ice to stop the incorporation of thymidine. The MEM containing 3H- Tdr was discarded and replaced by 3 m1 of Puck' s Saline D solution. The monolayered cells were disrupted by sonication for 10 seconds with a Branson 140C sonnifier and 0. 1 m1 of well—agitated sonicate was applied to each of two Whatman 3 MM 2. 3 cm diameter filter discs. After being washed three times in cold 5% TCA, twice in absolute ethanol and once in acetone, the discs were dried and placed in liquid scintillation vials. Five ml of a solution containing 4g of 2, 51diphenyloxazole (PPO) and 0. 1g of 1, 4-bis[ 2 -(4-methyl- 5- phenyloxazole)] benzene (dimethyl POPOP) per liter of toluene were 11 used. The radioactivity was measured in a Packard Tricarb Scintillation Spectrometer. Each data point consists of the average of the counts per -minute (Cpm) from duplicate plates and duplicate samples from each plate. A similar procedure was used to determine RNA and protein synthesis in AV cells, only the labeled precursors 3 were changed. Unirradiated sugar controls were run simultaneously with each treated sample throughout all the experiments described in this work. The pH of the media was kept at 7. 2 unless otherwise stated. A series of experiments was conducted in order to study the effect of irradiated glucose, fructose and sucrose on the DNA, RNA and protein synthesis of human amnion (AV3) cells in culture. The radiation doses used were 1, 2, and 3 Mrad. These doses were chosen because it was considered desirable to work within levels below those recommended for the complete radiation sterilization of foods. The molar concentration of the sugar solutions added to the growth media was 5. 8 X 10_2M. This concentration was used for all the studies reported in this manuscript. The duration of the period of incorporation of the particular labeled precursor (thymidine, uridine or leucine) was 6 hours. WW“- II". In . 12 Cell Viability Study Tissue culture plates containing 1. 5 X 106 cells/plate were pre -incubated to form a monolayer in 2% sucrose -MEM. The medium was discarded and 3 ml of 2% 3 Mrad irradiated sucrose - MEM at pH 7. 2 were added to the monolayered cells. The plates were then incubated at 37° C for 6, 12 and 24 hours. The MEM containing unirradiated 2% sucrose was added to the control plates. After each incubation period the media were removed from the plates, new MEM-2% unirradiated sucrose was added, and the plates were placed back in the incubator. Cell counts were made with a hemo- cytometer at 2, 4 and 6 days after the removal of the irradiated sucrose medium. Activated Carbon Activated carbon was used to try to remove the slight yellow pigment and possibly toxic fraction of the irradiated sugars. 2. 4g of activated carbon were added to 60 m1 of 4% solutions of sucrose irradiated at 1, 2 and 3 Mrad. This amount of carbon represents a 1:1 proportion by weight with the irradiated sugar. The mixtures were continuously agitated in 250 ml conic flasks for one -half hour. Filtration through Whatman No. 1 paper was done to remove the carbon. The clear filtrate of the 4% irradiated sucrose 13 was then filter -sterilized through a Millipore HA-O . 45M filter and mixed with equal volumes of 3H- Tdr-MEM 2'11. Ci/ml. The incorporation of 3H-Tdr into DNA of AV3 cells was determined at 2, 4 and 6 hours of incubation at 37° C with the monolayered cells. DNA Polymerase Activity DNA polymerase was obtained from an AV3 cells extract. The cells were grown in Roux bottles until a monolayer was formed. They were harvested using 5 ml of 0.02% EDTA per bottle of culture. A pellet was collected in a 15 m1 centrifuge tube and 5 ml of 10 mM Tris buffer at pH 8. 1 and 1 mM in Mg C12 were added to each tube. The pellet was reduced to a suspension by shaking, and the suspension was allowed to stand for 1 hour at 37° C. The cells were spun at 6, 000 rpm in a refrigerated International Electric Centrifuge, and the supernatant was called extract #1. Extract #1 was centrifuged for 1 hour in a Sorval high speed refrigerated centrifuge at 30, 000 rpm to collect extract #2. This was the extract used for the DNA Polymerase activity assay. The DNA polymerase activity assay was Conducted according to the method described by Zimmerman (1966). The requirements for maximal activity of the enzyme system which has been called DNA polymerase are the presence of all the four deoxyribonucleoside - 5' -triphosphates, Mg ions and DNA primer. 14 Catalase Activity ——'-—v Stock solutions of catalase were prepared from beef liver catalase solution obtained from Sigma Chemical Company, Inc. (33, 000 Sigma units/ml or 20 mg/ml). One unit decomposes 1“. mole of hydrogen peroxide per minute at pH 7.0 at 25° C. Two con- centrations were used for the experiments: 20/.Lg/ml and 40;.Lg/ml. These concentrations of catalasewere tested in two experiments in . .1 which the catalase was incubated at 25° C at pH 7.0 with 3 Mrad irradiated 5. 8 X 10-2M sucrose solution from 20 minutes to 3 hours. MEM containing unirradiated sucrose was used as control as well as heat inactivated catalase. At the end of the catalase incubation period 3H- Tdr MEM was mixed, 1:1 by volume, and the synthesis of DNA in AV3 cellswas measured by following the incorporation of the DNA labeled precursor into TCA- insoluble material for a period of 4 hours. Each count recorded represents the average of duplicate counts of duplicate samples. Ig'adiated Sucrose in Storage Sucrose solutions prepared 4% by weight in distilled- demineralized water-were filter -sterilized and transferred with a sterile-syringe into 20 ml sterile ampules. The ampules were sealed and placed at the calculated distances from the 60Co source 15 to absorb 3 and 4 Mrad respectively. The irradiated solutions were kept at room temperature until they were assayed for toxicity on AV3 cells. The effect of the presence of 3 and 4 Mrad irradiated sucrose on the synthesis of DNA in AV3 cells was determined at 0, 2, 4 and 7 months of storage. RE SULTS AND DISCUSSION Cell Viability The ability of human amnion AV cells to multiply after 3 having been in contact with irradiated sucrose was tested. AV3 cells in their exponential growth phase‘were transferred to minimum essential medium (MEM) containing 2% sucrose irradiated at 3 Mrad. The system was incubated at 37° C for 6, 12 and 24 hours. After these periods the medium containing irradiated sucrose was replaced by medium containing non- irradiated sucrose and cell counts were made with a hemo- cytometer at 2, 4 and 6 days from the time of removal of the irradiated sucrose. The results are presented in Figure 1. 1 The growth inhibitory action of 3 Mrad irradiated sucrose is time dependent. The findings suggest that the damage caused to the cells by the presence of radiolysis products of sucrose is not repaired even after removal of such compounds from the growth medium. 16 17 6 hr Control 6 hr 3 Mrad © 12 hr Control 0 12 hr 3 Mrad O 24 hr Control 0 24 hr 3 Mrad Days Figure l. -- Effect of duration of incubation on the growth of AV3 cell exposed to 3 Mrad irradiated sucrose solution 5. 8 X 10' M in MEM. 18 Synthesis of DNA According to Edmunds (1964) the doubling of DNA is not the only requirement for cell division, but inhibition of DNA ’ synthesis will generally result in an inhibition of cell division. A series of experiments was performed in order to study the effects of irradiated glucose, fructose and sucrose solutions on the synthesis of DNA in AV3 cells. 1. Effects of irradiated water and dose rate Since all the sugar solutions used for this study were prepared with distilled and demineralized water, it was important to determine if there is an effect on the DNA synthesis in AV3 cells due to the radiolysis products of water. The results presented in Figure 2 show no difference between the water irradiated at 3 Mrad and the unirradiated water. Two dose rates were used in this experiment: 187 Krad/hr for 16 hours and 1574 Krad/hr for 1 hour and 54 minutes. These were found not different from the control (Figure 2). (3H-Tdr) counts per min x 10"3 19 E] 187 Krad/hr/16 hr D 3 2 __ A 1574 Krad/hr/l. 9 hr 0 Control 0 24 .. 'l 6 .. A 8 t O l 1 J o 2 4 a incubation time (hr) Figure 2. --Effect of water irradiated at different dose rates on the H-thymidine uptake in DNA synthesis by AV3 cells. 20 Effect of irradiated sucrose on DNA synthesis --preliminary experhnent A preliminary test on the synthesis of DNA was done on AV3 cells in contact with 2% sucrose-MEM for 2, 4 and 6 hours using irradiation doses of 0, 1, 2 and 3 Mrad I (Figure 3). Inhibition of DNA synthesis was most marked : l '5 at 3 Mrad. The effect of activated carbon An attempt to remove the toxic compounds from the irradiated sucrose solutions was made by using activated carbon. A 13% increase in the DNA synthesis of the cells was observed for the 1 Mrad treated sucrose (Figure 4). No significant difference was found between the 2 and 3 Mrad carbon treated samples and the nontreated ones. Thus activated carbon does not seem to be effective in removing the compounds responsible for the toxicity of irradiated sucrose. The effects of 3 and 4‘Mrad irradiated 2% glucose or fructose in MEM medium on the DNA synthesis of AV3 cells were compared to nonirradiated controls. A higher inhibi- tion of DNA synthesis by fructose was observed (Figures 5 counts per min x 10'3 (3H-Tdr) 21 o 0 Control A l Mrad o O 2 Mrad ‘ 3 Mrad A A O o A 1 J J 2 4 6 incubation time) (hr) Figure 3. "3H -thymidine uptake in DNA synthesis by AV cells incubated with 5. 8 X 10" M irradiated sucrose solutions (preliminary experiment). counts per min x 10‘3 (3H-Tdr) 22 Control 0 4 _ 1 Mrad A O 3 .. 2 " 2 Mrad . A 3 Mrad 1 .. A 1 1 1 O 2 4 6 incubation time (hr) Figure 4. --3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 2% irgadiated sucrose solutions treated with activated carbon post -irradiation. 23 and 6). Berry, Hills and Trillwood(1965) were the first to report on the toxicity of irradiated glucose and fructose solutions to mammalian cells in vitro. For 1% solutions of glucose or fructose added to the growth medium the toxicity was maximal at 105 rads. They also found that thereffect persists for more than 6 months. The toxicity of irradiated fructose was recognized by Blank and Arnold as early as 1935. They observed that the irradiation of a ketose, or of a carbohydrate which hydrolizes to produce a ketose, brings about complete inhibition of the growth of Bacillus subtilis in a shorter period of irradiation than is required for any aldose in that class. Effect of pH When a 4% sucrose solution is irradiated to 3 Mrad, its pH falls from 7.0 to 3. 2. If this solution is added to MEM in a 1:1 proportion, the resulting medium is 2% in the irradiated sucrose and its pH is raised by the buffering capacity of the MEM to around pH 6. 40. It was observed that an unirradiated sucrose medium at pH 6. 4 was as inhibitory as a 3 Mrad irradiated sucrose added to the medium when the synthesis of DNA was followed under counts per min x10"3 (3H-Tdr) 24 0 Control water . Irradiated water A Control glucose D Control fructose 1 Irr. Glucose Irr. Fructose L. incubation time 4 (hr) Figure 5. --3H -t.hyrnidine uptake in DNA synthesis by AV3 cells incubated with 5. 8 X 10 ’2M glucose and fructose solutions irradiated at 3 Mrad. 28 24 —l d N M O 0 counts per min x10"3 (3H-Tdr) 25 0 Water A A Glucose O D Fructose . Irradiated water CI 0 O Irr. Glucose ’ A 'A‘ O In. fluctoss l l 1 2 4 6 incubation time (hr) Figure 6. -- 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 5. 8 X 10 '2M glucose and fructose solutions irradiated at 4 Mrad. 26 similar pH conditions (Figure 7). Similar observations were made by Baumgartner (1936), who found that irradiation of 1% sucrose solution resulted in a drop in pH from 7.0 to 3. 5- 4.0. The use of such solutions as a base for nutrient media without subsequent pH adjustment was naturally followed by inhibition of the cell normal processes. Equimolar concentrations (5.8 X 10-2M) of 3 Mrad irradiated glucose, fructose or sucrose contained in growth medium were used to study the synthesis of DNA in AV3 cells. The pH of the media containing irradiated sugars and nonirradiated controls was adjusted to 7.2 prior to incubation. No change in pH was observed at the end of the test period. Most interesting were the results obtained with irradiated glucose. A highly significant reduction in the inhibition of DNA synthesis (a drop from 60% to 12%) was observed when the pH was adjusted to 7.2 (Figure 8). It seems that these cells can incorporate thymidine into their DNA molecule at a rate comparable to the control even in the presence of 3 Mrad irradiated glucose (2%) solution. Berry, Hills and Trillwood (1965) found that irradiated glucose powder reconstituted with irradiated water proved toxic to Strain L fibroblasts, but not to the He La cell -lines. 27 Control pH 7.10 T) 14 .. A l 2 __ L 'c ‘T I m 10 r- “? O o H 8 t. x C E 6 __ 3 Mrad L pH 6. 40 a: q I m *-' 4 A g P Control A . o I pH 6. 40 u 2 .. . I l t O 2 4 6 incubation time (hr) Figure 7. -- 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 5. 8 X 10 '2M sucrose solution irradiated at 3 Mrad and unirradiated sucrose solutions at pH 6. 40 in MEM. 28 2 4 t. Controls I A O 20 t. C Irr. 'c '_'_ lucose I m I 6 A A. I. . m .- I o Irr. H Fructose x 1 2 __ E rr. E Sucrose L 0 0 a. 8 ._ s U) .s—o C 3 A o o l 1 4 O 2 4 6 incubation time (hr) Figure 8. -- 3H —thymidine uptake in DNA synthesis by AV3 cells incubated with 3 Mrad irradiated sucrose, glucose and fructose solutions (5. 8 X 10 '2M in MEM at pH 7. 2). 29 These findings suggest that the toxic products can be handled in different ways by different cell types. No appreciable reduction in the toxicity produced by irradiated fructose or sucrose solutions was obtained by adjusting the pH to 7. 2 (Figure 8). Irradiated glucose vs. DNA synthesis The results of the experiments designed to determine the effect of irradiated glucose on the DNA synthesis of human cells are shown in Figures 8 and 9. It is clearly seen from these results that irradiated glucose does not significantly inhibit the synthesis of DNA of these cells. No difference was found between 1, 2 and 3 Mrad treatments at the end of 6 hours of incubation. The percentage of inhibition of the DNA synthesis was 33 for all 3 doses (Figure 9). Irradiated fructose vs. DNA synthesis The results shown in Figure 10 clearly demonstrate that 3 Mrad irradiated 5. 8 X lO-ZM fructose induces severe inhibition (63%) of the synthesis of DNA when in contact with human cells in a rapid growth phase. A 30 to 35% inhibition was observed for the doses of 1 and 2 Mrad. counts per min x 10'3 (3H-Tdr) 18 16 l4 12 10 30 0 Control I 1 Mrad A 2 Mrad ‘ 3 Mrad O _1 4 m1 2 4 6 incubation time (hr) Figure 9. -- 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated glucose solutions (5. 8 X 10'2M in MEM at pH 7. 2). counts per min x10.3 (3H-Tdr) 16 u... L —s N _s O 31 0 Control . 1 Mrad A 2 Mrad A 3 Mrad l 1 4 incubation time (hr) Figure 10. --3H —thymidine uptake in DNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated fructose solutions (5. 8 X 10‘2M in MEM at pH 7. 2). 32 7. Irradiated sucrose vs. DNA synthesis The results of experiments conducted to determine the effect of 1, 2 and 3 Mrad irradiated 5. 8 X 10-2M sucrose solution on the incorporation of tritiated thymidine into DNA of AV3 cells are well represented by Figure 11. The decrease in DNA synthesis is dose dependent. At the 1 Mrad dose level only 20% inhibition was observed, while 51 to 53% inhibition were obtained for the 2 and 3 Mrad irradiation doses respectively. Synthesis of RNA Since tritiated uridine is only incorporated in the molecule of RNA, it represents an excellent precursor to study the synthesis of RNA. 1 . Irradiated glucose vs. RNA synthesis Figure 12 shows the relation of the incorporation of uridine into RNA of human cells in contact with 5. 8 X 10-2M glucose irradiated at 1, 2 and 3 Mrad respectively. It was demonstrated that irradiated glucose exerts only a moderate inhibition in the synthesis of RNA of these cells. Thirteen to 15% inhibition was obtained at the 3 Mrad dose level. counts per min x10"3 (3H-Tdr) 20 18 16 14 12 10 33 0 Control 0 O l Mrad A 2 Mrad ‘ 3 Mrad O O l L 2 4 0L incubation time (hr) . 3 . Flgure ll. -- H-thymldine uptake in DNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated sucrose solution (5. 8 X 10 ’2M in MEM at pH 7. 2). l,“ counts per min x10'3 (3H-Udr) 34 18.. s 16... O 14.. . A 121.. s ‘o— b.‘ 3r- 6L— 0 Control O erad At. A 2Mrad A 3Mrad 21- l l J O 2 6 incubation time (hr) Figure 12. -— 3H -uridine uptake in RNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated glucose solutions (5. 8 X 10 '2M in MEM at pH 7. 2). 35 Irradiated fructose vs. RNA synthesis It was found that under similar conditions of radiation doses, temperature, duration of exposure, pH of the medium and cell concentration, irradiated fructose reduced RNA synthesis in AV 3 cells to a degree slightly higher than that 71 observed for irradiated glucose (Figure 13). Irradiated sucrose vs. RNA synthesis A very similar response to that obtained with fructose was apparent when irradiated sucrose solutions were added to the growth media to determine the synthesis of RNA in human cells (Figure 14). Protein Synthesis Tritiated leucine was used to follow the synthesis of protein in AV cells exposed to irradiated sugars in their growth 3 medium. Irradiatedfiglucose vs. protein synthesis Glucose solutions irradiated at 1, 2 and 3 Mrad and added to the growth medium of AV 3 cells at a concentration of 5. 8 X 10-2M proved not to be very different from the counts per min x 10'3 (3H-Udr) 18 16 14 12 10 36 O A O A A 0 Control O 1 Mrad A 2 Mrad ‘ 3 Mrad i 1 2 4 incubation time (hr) Figure 13. -- 3H -uridine uptake in RNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated fructose solutions (5.8 x lO‘zM in MEM at pH 7.2). counts per min x10"3 (3H-Udr) 37 20... 18.. 16... 0 ‘4h 0 121. ‘ 10- A 8i- 6.. 0 Control O erad 4a AzMrad A 3Mrad 2L. 1 l 1 0 2 4 6 incubation time (hr) Figure 14. -- 3H -uridine uptake in RNA synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated sucrose solutions (5. 8 x 10'2M in MEM at pH 7. 2). .‘J 38 unirradiated glucose when tested for toxicity related to protein synthesis. Only 20% inhibition of the protein synthesis was observed at the 3 Mrad dose level (Figure 15) after 6 hours of incubation. Irradiated fructose vs. protein synthesis The presence of irradiated fructose in the growth medium of AV3 cells exerted a reduction on the synthesis of protein of these cells. A 41% inhibition in protein synthesis was observed at the 3 Mrad dose level (Figure 16). Irradiated sucrose vs. protein synthesis The observations from experiments performed to test the influence of irradiated sucrose on the growth medium of human cells in terms of protein synthesis are summarized in Figure 17. The inhibitory effect of irradiated sucrose on the protein synthesis of human AV3 cells is clear at the 3 Mrad dose level. It appears that the levels of 1 and 2 Mrad are too close to a threshold effect for any definite conclu- sions to be derived regarding the inhibition of protein synthesis in these cells. counts per min x10'3 (3H-leucine) 26 24 2O 18 16 14 12 10 39 \I Control 1 Mrad 2 Mrad O O A A 3 Mrad J l l 2 4 6 incubation time (hr) Figure 15. "3H -leucine uptake in protein synthesis by AV3 cells incubated with l, 2 and 3 Mrad irradiated glucose solutions (5.8 x 10‘2M in MEM at pH 7. 2). counts per min x 10'3 (3H-Ieucinei 30 28 26 24 22 20 18 16 14 12 10 40 O O A O A 0 Control O erad A 2 Mrad A 3Mrad l L m 2 4 6 incubation time (hr) Figure 16. -- 3H ~1eucine uptake in protein synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated fructose solutions (5.8 x lo-ZM in MEM at pH 7. 2). counts per min x10'3 (3H-leucine) 16 15 14 13 12 11 10 41 o 0 Control O 1 Mrad A 2 Mrad I 3 Mrad A O A O A A L 1 2 4 o incubation time (hr) Figure 17. "3H -leucine uptake in protein synthesis by AV3 cells incubated with 1, 2 and 3 Mrad irradiated sucrose solutions (5. 8 X 10 '2M in MEM at pH 7. 2). 42 The Effect of Ageing of Irradiated Sucrose Solution on the DNA synthe sis The ageing or storing of irradiated sucrose (3 and 4 Mrad) did not minimize its capacity to induce inhibition of the synthesis of DNA in AV3 cells (Table 1, Figures 18 to 22). These results are in agreement with Schubert, 1967; Steward, 1967; Molin and Ehrenberg, 1964; Berry, Hills and Trillwood, 1965; and Holsten e_t_a_l_. , 1965, who reported inhibitory effects of irradiated sugars (on bacterial and plant growth) after storage for long periods at low temperature and low pH. Table 1. --Percentage of DNA synthesis inhibition in AV cells observed using irradiated sucrose stored from 0 to 7 months at room temperature. "/0 Inhibition Dose 0 2 4 7 Months Months Months Months 3 Mrad 52 60 46 75 4 Mrad 68 75 53 87 F. EVA Polymerase Activity Since the DNA synthesis was shown to be impaired by the Presence of irradiated sucrose in the growth medium, an 20 18 16 14 12 10 counts per min x10'3 (3H-Tdr) 43 Control '0 . L 3 Mrad A A 4 Mrad O A l 1 1 2 4 6 indubation time (hr) Fi8'1-lre 18. -- 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 and 4 Mrad irradiated sucrose solutions (5. 8 X 10 '2M) immediately after irradiation. 44 counts per min x10"3 (3H-Tdr) 26' Control 24... . 22.. 20.. 18.. 16.. 14.. O 12.. 10_ 3Mrad 8.. 0 4Mrad 6s. A 4' A A 2- / l 1 ml 0 2 4 6 incubation time (hr) Figure 19. -- 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 and 4 Mrad irradiated sucrose stored for 2 months at room temperature. Sucrose solutions (5.8 x 10’2M). counts per min x10 '3 (3H-Tdr) 18 16 14 12 10 45 Control pH 7. 15 O O 3 Mrad pH 7. 15 O O 1 i l 2 4 6 incubation time (hr) Figure 20. -— 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 Mrad irradiated sucrose solution stored for 2 months at room temperature. Sucrose concentration was 5. 8 x 10‘2M in MEM at pH 7.15. counts per min x10.3 (3H-Tdr) 46 36L- . 0 Control A 3 Mrad pH Adj. 32 ,_ A 3 Mrad pH Not Adj. C1 4 Mrad pH Adj. . I 4 Mrad pH Not Adj. . .. t 20 ,. AL.” A 16 t' I A A 12L I O 8 .. I ,. 4 . ‘ l m L l O 2 4 6 8 incubation time (hr) Figure 21. -- 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 and 4 Mrad irradiated sucrose stored for 4 months at room temperature. Sucrose concentration was 5. 8 X 10‘2M in MEM at various pH' 3. counts per min x 10’3 (3H-Tdr) 47 34 r- Control O 32* pH 7.20 30- . 28.. 26.. 24. 22t- 20, 18_ 16. 14.. 12. 0 10.. 3Mrad 8 A I pH 7.20 6. 3Mrad pH 6.80 I 4Mrad pH 7.20 4Mrad 1‘ pH 6.65 incubation time (hr) ’ Figure 22. -- 3H -thymidine uptake in DNA synthesis by AV3 cells incubated with 3 and 4 Mrad irradiated sucrose stored for 7 months at roam temperature. Sucrose concentra- tion was 5. 8 X 10" M in MEM at various pH' 8. 48 experiment was conducted to test whether the action of DNA polymerase is affected by the irradiated (3 Mrad) sucrose. The assay for the activity of DNA polymerase was performed using the method described by. Zimmerman (1966). Theresults are presented in Table 2. No significant difference in activity of the DNA polymerase was observed when 3 Mrad irradiated sucrose was present in the substrate. The inhibition of the synthesis of DNA in AV 3 cells might be caused by an impairment in the production of its precursors. The Influence of Catalase on the Medium Containing Irradiated Sucrose Molin and Ehrenberg (1965) found that the antibacterial effects of irradiated glucose was abolished by adding catalase to the irradiated solution 60 minutes prior to inoculation. Schubert (1969) provided evidence of the cytotoxicity of irradi- ated sucrose solutions for Salmonella typhimurium. He reported that the inhibitory action of sucrose is attributed to organic'peroxides. The addition of catalase prior to or shortly after inoculation of the organism into the growth medium con- taining irradiated sucrose eliminated most of the inhibitory 49 Table 2. --DNA polymerase activity in extracts of AV 3 cells incubated with 3 Mrad irradiated sucrose for 1 hour at 37°C.a’ Tube H E) 3 Mrad Control Counts per Number 2 Sucrose Sucrose Minute ; 32 :: :: 2, 077 Z I :: g: 2, 201 2 2: :3 :: Z. 22 :: 33 2, .3 :2 :8 :: 2, i; if: :: :8 ii 2: :: :: :2 2 :3 :: ii 2 :: 38 2, 187 :3 3: :: :: 2. 3; :: :: 3: 2.199 aEach tube contained 25>\ Tris buffer 1M pH 7. 6; 5A 0.1M MgClz; 10A 40mM Mercapto ethanol; 50A denatured DNA primer; 50>\ 3H-dNTP and 25>\ extract from AV3 cells. The total volume per tube was 250A . bActivity measured as counts/ min of incorporated labeled precursors into DNA. 50 action produced by the irradiated sucrose solutions. Berry, Hill and Trillwood (1965) added catalase to a growth medium for mouse Strain L fibroblasts containing irradiated glucose. Incubation with catalase (5 mg/ml) for 30 to 60 minutes prior to the addition of cells to the media failed to protect against toxic effects of irradiated glucose. Two experiments-were performed to determine-whether catalase could protect AV cells against the deleterious effects 3 already observed when these cells were in contact with 3 Mrad irradiated sucrose. A beef liver catalase preparation (Sigma Chemical Company, Inc. , St. Louis) containing 33, 000 Sigma units/ml, 20/..Lg/m1 (1 unit decomposes 1 millirnole H202 per minute at pH 7.0 at 25° C) was used for the experiment. This preparation‘was diluted to a final concentration of 201Lg/ ml. The incubation for the different treatments was done at 25° C. At the end of the incubation periods (from 20 minutes to 3 hours), MEM contain- ing tritiated thymidine was mixed in 1:1 proportion and the synthesis of DNA was followed for 4 hours. The values in Table 3 show the averages of duplicate counts for each experiment. The presence of catalase in as much as 40 [Lg/ml for 3 hours of pre -incubation only partly (50 to 67% of control) 51 eliminated the inhibitory effect of 3 —Mrad -irradiated sucrose on the synthesis of DNA in human amnion cells. It seems that peroxides are not the only toxic radiolytic product of irradiated sucrose for these cells. Table 3. --The effect of catalase on 3 -Mrad -irradiated sucrose, measured by the incorporation (in counts/ min) of tritiated thymidine into the DNA of AV3 cells. a Treatment Counts per Minute Experiment 1 Catalase = 201Lg/ml Control + catalase 12, 744 Heat inactivated catalase 5, 835 20 minute incubation + catalase 5, 435 3' hour incubation + catalase 6, 487 Experiment 2 Catalase = 40 jig/m1 No catalase, no irradiation 17, 985 No catalaseh+ irradiation 8, 057 Catalase, no irradiation 15, 105 Heat inactivated catalase 10, 250 20 minute incubation + catalase 9, 146 3 hour incUbation + catalase 11, 779 aCounts per minute at 4 hours after inoculation of the media. 52 Reducing Sugar Content of Irradiated Sucrose The amount of reducing sugars obtained from irradiated sucrose has been reported by several investigators (Phillips, 1960, 1963; Steward gt_al. , 1967; Schubert, 1969). The figures vary depending on the physical state of the sugar, the tempera- ture, pH and ultimately the radiation dose. It was considered important to know how much total reducing sugars (glucose and fructose) are produced by the radiation doses in this study. The determination of reducing sugars was performed by the colorimetric method using 3, 5 dinitrosalicylate reagent as indicated by Clark (1964). The results shown in Table 4 are expressed as percent total reducing sugar. The values are in agreement with the findings of other investigators (Schubert, 1969; Steward _e_t_a_l. , 1967). The analyses were performed 5 days after the irradiation of the sucrose solution. Table 4. -—Percent of total reducing sugars from 1, 2 and 3 Mrad irradiated sucrose solution. Dose % Reducing Sugars Average 2. 35 1 Mrad 2.76 2. 56 5. 00 2 Mrad 5. 34 5. 17 6. 32 3 Mrad 7. 56 6. 94 53 Table 5 summarizes the results obtained from all the experiments performed in this study in which AV3 cells were incubated with 3 Mrad irradiated sugars for 6 hours. Table 5. --Average percent inhibition of DNA, RNA and protein synthesis for AV3 cells in contact with 3 Mrad irradiated sugars. (Duration of experi- ment = 6 hours) DNA RNA Protein Sticrose 49 26 38 Glucose 20 14 24 Fructose 51 26 34 Discussion The interpretation of results obtained from the action of irradiated sugars on biological systems is a rather difficult task due to the complexity of the reactions involved. It is well established that irradiated sugar solutions are toxic to bacteria, plants and mammalian cells in culture. The results of the studies performed using AV3 cells as test subjects for the action of irradiated sugars are in agreement iwith the behavior of most of the biological material tested previously. The inhibitory effects on growth and reproduction of the cells were more pronounced when the time the cells were in contact with the 54 irradiated sugar-was longer. The removal of the toxic medium and the addition of fresh normal growth medium did not improve the condition of the already damaged cells. Appreciable impairment of growth was observed even after a 6 hour incubation period. The fact that the percentage of growth with the control as 100% was almost constant from 2 to 6 days after the removal of the irradiated sugar indicates that a certain fraction of the original cell populationwas damaged and/ or probably killed. Those cells that for some reason survived the treatment continued to grow and multiply at the normal rate. Repair processes involve enzymes, and their efficiency depends on the time available between the production of the chemical change and the next replication of DNA. The mutagenicity of irradiated media containing carbo- hydrates has been suggested by some investigators (Stone, 1955, and Swaminathan, 1953 ). It is well known that organic peroxides pro- duced in the irradiated medium, together'with formaldehyde and hydrogen peroxide, are quite potent mutagens. Peroxides have been found responsible for the mutagenic action of bacterial medium that has been exposed to heavy irradiation with ultraviolet light or ionizing radiation. In recent years, the sterilization of human food with high doses of ionizing radiation has given concern about the possible introduction of mutagens into human consumption. 55 Evidence for severe inhibition of the synthesis of DNA was obtained in the present work, especially when irradiated sucrose or fructose solutions were introduced to the growth medium of human amnion cells. DNA and protein synthesis were demonstrated to be greatly diminished when rat liver slices were exposed to irradiated sucrose solutions by Aiyar and Sreenivasan (1969). They attributed the impairment of DNA synthesis to uncoupling of oxidative phosphory— lation with a subsequent decrease in the formation of ATP. The radiolysis of simple sugars has been extensively studied by Phillips and co -workers (1960, 1963). Among the products of radiolysis derived from sucrose in aqueous solution they reported: glucose and fructose as primary products, together with smaller amounts of glyoxal, glucosome and gluconic acid. Glucuronic acid, 2 -oxogluconic acid, arabinose and 2 - and 3 —carbon,aldehydic frag- ments arise in secondary processes. In addition, these authors have also reported the following compounds from the radiolysis of glucose: D-erythrose, formaldehyde, saccharic acid and 1:3—dihydroxyacetone. Hydrogen peroxide was also formed. The presence of deoxycompounds in irradiated carbohydrates has recently been demonstrated by Scherz (1968). ~ He has determined deoxycompounds originating from irradiated 1% solutions of sucrose 56 and glucose. The formation of polymers from glucose supposedly through gluconic acid has been reported by Barker e_t_tfl. (1959). Hydroxyalkylperoxides derived from the interaction of radiolytic H202 with carbonyl compounds produced in the radiolysis of sucrose have proved to be toxic and mutagenic (Schubert, 1969). Perhaps a route different from that taken by Aiyar and Sreenivasan (1969) may lead to finding an answer for the marked inhibition of DNA synthesis observed in human cells under the effects of irradiated sucrose solutions. The biosynthesis of DNA may be divided into three phases (Kihlman, 1966). The first of these is the _d_e go_v_gsynthesis of uridilic acid (UMP), and inosinic acid (IMP). The second phase is the synthesis of the fourdeoxyribonucleoside triphosphates, which are the immediate precursors of DNA, and the third phase is the polymerization of these deoxyriboside triphosphates in the presence of DNA polymerase and a suitable DNA primer or template. From the second phase of the DNA synthesis, the formation of deoxycytidine - triphosphate (dCTP) is of importance for this discussion. The intermediate step of the reduction of cytidinediphosphate (CDP) to dCDP by CDP -reductase is strongly inhibited by a phosphorylated derivative of cytosine arabinoside, probably cytosine arabinoside diphosphate (Chu and Fischer, 1962). The product of the 57 CDP -reductase reaction, deoxycytidine diphosphate (dCDP) is then phosphorylated by a deoxyribonucleoside diphosphate kinase to the immediate DNA precursor dCTP. Arabinose is one of the radiolysis products of irradiated sucrose (Phillips, 1960). Cytosine arabinoside (CA) is the structural isomer of cytidine from which it differs by the in l configuration at carbon 2 in the pentose sugar. Kihlman gt_al. (1963) j "‘ "i found that cytosine arabinoside/induces chromosome breakage in human leukocytes at a concentration of 10-6M. Although adenine deoxyribo- side (AdR), like CA, also inhibits the formation of deoxyribonucleotides, Chu and Fischer (1962) found that CA acts more specifically since apparently it is only the formation of deoxycitidine diphosphate from cytidine diphosphate which is inhibited by CA. These reactions are of considerable interest, since they may provide a mechanism by which the supply of precursors needed for DNA biosynthesis is controlled. SUMMARY AND CONCLUSIONS Summary m 1. The synthesis of DNA, RNA and protein in AV3 cells was inhibited when the cells were incubated with 3 Mrad irradiated sucrose, glucose and fructose solutions. The j —--'- average percentages of inhibition of DNA, RNA and protein synthesis (in that order) were: 49, 26, and 38 for sucrose; 20, 14, and 24 for glucose; and 51, 26, and 34 for fructose. 2. The synthesis of DNA in AV cells was impaired most by 3 sucrose and fructose. 3. The toxicity induced by the radiolysis products of sucrose and fructose was most accentuated at the dose level of 3 Mrad. 4. The synthesis of RNA and of protein in AV3 cells was less affected than that of DNA in the same cells. 5. Irradiated glucose was less harmful to AV3 cells in culture than sucrose or fructose. 58 59 6. Sucrose solutions irradiated at 3 and 4 Mrad and stored for 7 months at room temperature did not reduce the inhibitory effects on the synthesis of DNA in AV3 cells. 7. A concentration of 40 [1 g/ ml of catalase only partly eliminated the deleterious effects of 3 Mrad irradiated sucrose to human amnion cells. 8. The activity of DNA polymerase from an AV3 cells extract was not affected by the presence of irradiated sucrose in the substrate. 9. The treatment of irradiated sucrose with activated carbon did not remove the toxic material from the sugar. 10. Cell growth and reproduction was severely affected by incubation of the cellswith 3 Mrad sucrose (5.8 X 10-2M). The percentage of growth inhibition for the incubation periods of 6, 12 and 24 hours were 40, 60 and 90, respec- tively. Conclusions Underthe conditions of these experiments it is apparent that the damage caused to human amnion cells by the radiolysis 60 products of sucrose and fructose cannot be repaired. The duration of the incubation of the cells with the irradiated sugar, the radiation dose applied, the pH of the medium and the stage of development of the cells are critical for their survival. It seems that only the fraction of cell population specifically involved in DNA synthesis .4 _2';_‘."‘l.‘} (the S phase of development) was affected by the radiolysis products of the sugars. This effect is time dependent; the longer the incuba- tion period, the higher the number of cells rendered unable to grow , J b and multiply. LIST OF REFERENCES LIST OF REFERENCES growth and development of plant tissue cultures. Unpub- lished manuscript. t Bajaj, Y. P. S. 1970. 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Purification and properties of deoxyribo- -'-‘= nucleic acid polymerase from Micrococcus lysodeikticus. T! J. Biol. Chem. 241, No. 9, 2035-2041. p Int'rr.- _ . APPENDIX Table 6. --Effect of duration of incubation on the growth of AV3 cells 66 incgbatedwith 3 Mrad irradiated sucrose solution (5. 8 X 10' M). ) Incubation Cells/ ml % tlme Control 3 Mrad Outgrowth Enumeration of cells after 2 days 6 6 6hr. 2.31X 10 1.41X 10 61% 12 hr. 2.39 x lo6 9. 48 x lo5 40% 24 hr. 2. 42 x 106 2. 68 x 105 11% Enumeration of cells after 4 days 6 6 6 hr. 3. 51 X 10 1. 98 X 10 56% 12 hr. 3.32 x lo6 1.31 x lo6 39% 24 hr. 4.09 x 106 3. 24 x lo5 8% Enumeration of cells after 4 days 6 6 6 hr. 4. 43 X 10 2. 68 X 10 60% 12 hr. 4.00 x 1‘06 1. 91 x lo6 48% 24 hr. 3.79x lo6 3.83X lo5 10% 67 Table 7. -- Percent inhibition of DNA synthesis in AV cells incubated with 5. 8 X 10‘2M sucrose, glucose and fructose solutions irradiated at 1, 2 and 3 Mrad (average; individual in parentheses). Incubation time Dose Mrad 2 Hours 4 Hours 6 Hours Sucrose 1 20 10 20 (18,16,21,25) (12,12,7,9) (20,25,17,18) 2 22 32 51 (17,21,25,25) (30,34,36,28) (47,46,53,58) 3 68 51 53 (61, 66, 68, 77) (52, 55, 48, 49) (48, 50, 55, 59) Glucose 1 8 8 32 (7,9,11,5) (8,8, 10,6) (29,35,33,31) 2 20 11 33 (21,23,17,19) (14,8,10, 12) (31,30,38,33) 3 25 12 33 (19,27,29,25) (9,11,17,11) (32,33,31,36) Fructose 1 20 33 39 (17,21, 18,24) (30,28,36,38) (34, 37,41,44) 2 33 40 41 (29,31,37,35) (46,39,37,38) (38,40, 40,45) 3 52 50 63 (48, 51, 56, 52) (52, 57, 46, 45) (63, 68, 61, 60) 68 Table 8. --Percent inhibition of RNA synthesis in AV cells incubated with 5. 8 X 10 '2M sucrose, glucose and fructose solutions irradiated at 1, 2 and 3 Mrad (average; individual in parentheses). Incubation time Dose Mrad 2 Hours 4 Hours 6 Hours Sucrose 1 _ 12 . 5 9 (13,8,10,16) (3,7,5,5) (8,13,6,10) 2 24 17 18 (30, 25, 28, 13) (16, 18, 14, 20) (20, 23,14, 15) 3 29 35 24 (30,25,27,34) (30, 40,37,33) (21,20,25,30) Glucose 1 8 7 10 (7, 10,8,7) (9, 10,5,4) (8, 12,9,11) 2 8 15 9 (6, 6, 9,11) (20,15,13,12) (13,10,9,5) 3 22 29 13 (18,20,26,24) (31,35,26,24). (10,10,17,15) Fructose 1 2 16 4 (3,2,1,2) (14,18,20,12) (3,2,4, 7) 2 5 30 27 (4, 5,7,3) (39,40,26,35) (24,21,33,30) 3 19 32 27 (18,15,20,23) (33,31,28,36) (20,27,30,31) _afifi' 69 Table 9 . --Percent inhibition of protein synthesis in AV 3 cells incubated with 5. 8 X 10' M sucrose, glucose and fructose solutions irradiated at 1, 2 and 3 Mrad (average; individual in parentheses). Incubation time Dose Mrad 2 Hours 4 Hours 6 Hours Sucrose 1 5 29 16 (4. 4. 7. 5) (30, 34.27.25) (13. 17.20. 14) 2 26 45 27 (26,29,25,24) (40,41,49,50) (22,26,28,32) 3 46 52 37 (41,44, 49, 50) (51,47,54, 56) (32,35,37,44) Glucose 1 7 23 12 (8, 10, 5, 5) (20, 19, 24, 29) (11, 10, 10, 16) 2 12 30 8 (10,12,16, 10) (29,26,31,34) (7,6,8,11) 3 19 36 20 (15,16,21,24) (31,33,37,43) (18,16,23,25) Fructose 1 15 15 so (19, 16, 12, 13) (21, 15, 12, 12) (37, 35, 26, 22) 2 20 16 3O (20,23,21,22) (10, 12,21, 19) (31,36,28,25) 3 22 18 41 (18, 20, 25, 25) (18, 23, 16, 15) (39, 44, 46, 41) ‘w ‘L'd 2!.”me ' l f n I i "IIIIIIIIIIIIIII