Biological and Medical Research Group (H-4) of the Health Division - Semiannual Report, January Through June 1961 (Redacted)

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BIOLQGY AND MEDICXNE LxlS ALAMOS SCIENTIFIC LABORATORY OF THE UNIVERSITY OF CALIFORNIA LO5 MAYOS NEW MEXICO BIOLOGICAL AND MEDICAL RESEARCH GROUP (H-4) OF THE HEALTH D-ON - SEMIANNUAL REPORT JANUARY THROUGH JUNE 1961 Dlvirlon Leader, 1. L. shipman

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2 - MOLECULAR RADIOBIOLOGY SECTION Kinetics of the Degradation of Polymer. by Successive Enzymatic Hydrolysis to Monomer Nucleic Acid Chromatogrrphy 33 ~ ethod sfor Routine Phoaphorua Analy8im of Effluents from Chromatographic Columns 30 .Determination of Beta Activity in Nucleic Preparation and Purification of TritiumLabeled Thymidine, Deoxycytidine, and On the Significance of Radiolytic Degrach; tion of Purine and Pyriridlne Nucleoside8 57

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7. Neutron Dome Eetimates inthe SL-1 Accident Utilizing Biological Sulfur Activation 66 Molecular Radiobiology Section Publication71 CEAPTER 3 - CELLULAR RADIOBIOLOGY SECTION 73 1. Uptake of Tri tia ted Deoxynucleo8lde Pro2. The Influence of Intracellular Tritium Beta Irradiation on the Growth of Hemophilue Studies on Detection of Biological Effect 83. of Magnet ic Fields 94 ing Principle (DNA) of Hemophilu8 Influenzae 99 Cellular Radiobiology Section Publication 102 CBAVTER 4 - MAMMALIAN RADIOBIOLOGY S1DcTION 103 1. Reduced Life Expectancy in D@8COndrmt8Of 2. Effect of Ago on Ability of Mice to Survive Fraction a ted Radiation Exposure 110

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Protection of CFW Swiss Mice from Post Irradiation Transplantable AK Leukemia by The Effect of Dose Rate on t h e Xncidence of Leukemia in Several Strains of Mice Maintenance at Low Temperature of Several Preliminary Data on Neutron and Gama Depth Dose Relations with Fission Neutron8 Using a Plastic Man Phantom Calculation of the Uncertaintiem Involved in the Evaluation of Neutron Tis8ue Dose by the Threshold Detector Technique Advancements in the Electronic Circuitry for the Large H - 4 Fission Gamma Counter Neutron Calibration Procedures Employed by t h e Biomedical Research Group Ma mullan Radiobiology Section Publicat ions CEUPTER 5 - YAYYALIAN METABOLISM SECTION

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Estimation of Maximum Permissible ConcentraInterspecies Correlations. 11. Comparison of Eatimated and Measured Values for Cesium 13’ Retention and Excretion of Iodinel3l. In Mice after Oral and Intraperitoneal Retention and Excretion of Iodine131. In Rats after Oral and Intraperitoneal Retention and Excretion of Iodinel3l. In Monkeys after Oral Admini8tration Retention and Excretion of IV. In Dogs after Oral Administration Retention and Excretion of Iodine131. Interspecific Correlations and Maximum 8. -. Retention and Excretion of Ruthenium Mammalian Metabolism Section Manuscripts Submitted

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1. Fallout Levels of Cesium137 i n United States Milk as Influenced by Feeding Variation of the Ceaium137 Content of New Mexico Residents as a Function of Tire 223 3. Radioactivity of Nevada Cattle: 1959 226 4. A Double Sodium Iodide (Thallium) Cry r tal Gamma Ray Detector 234 5 . Radiocarbon in Contemporaneous Plant Lor-Level Counting Section Publication a CfUPTER 7 - CLINICAL INVESTIGATIONS SECTION 263 Meta6ollsm of Zinc" i n a Terminal Leukemia 2. Clinical Application. of Whole Body Sc in til - . lo me try. V. E qui lib ration Rates of Rat and Main with Environmental Iodine 270 3. Clinical Application8 of Whole Body Sc in til - lone try. VI. De tern in rti on of Cob8lt Vitamin Bla Absorption and Retention

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Experimental Iodide Blockade of Iodine131 Thyroid Uptake in Rats 284 Progremm in Refinement of the Whole Body Counting Technique for Determining Thyroid Standardizit ion of Water Bot t lam Yechrnical Bottle Wa8her and Filler Animal Production and Inventory

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Re orientation of the biological and medical re8earch program in the direction of more fundamental and long-term ophy of the Laboratory Director and the Laboratory Policy Committee and ha8 been di8cusmed with varioum repre8ent at ives of the AEC's Diviaion of Biology and Medicine. AE an initial T. R. Willi arm were transferred on January 1, 1961, from P. 9. Harris as Group Leader. The new group is t a k i n g over all programmatic project. related to field tests of nuclear This change is in keeping with the general philo s - devicea, thereby relieving Group H-4 of the interrupt ions inherent in test operation8 and allowing full time to be devoted t o more fundamental re8earch.

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A 8 of the end of this report period, re or gr niz ation of the Biological and Medical Research Group and general reorientation of the program are e an ent i ally complete. The Organic and Biochemistry Sections have been combined into a Section of Molecular Radiobiology, oriented toward the application of the per1onne1 ~ ~rather ride experience in the field8 of isotopic labeling and tracer methodology t o inV08tiga tion of radiation effect8 atthe molecular level.A Section of Cellular Radiobiology has been formalized, and it8 program i r being oriented toward rt u die a of the effect. of radiation on growth and aetabolfu of cell8 grown in i8 being8tu died jointly with the Yolmcular Radiobiology and C1*-labeled precursors by cell8 grown in agitated fluld The technique employed is based on uptake of tritium medium. The Mammalian Radiobiology Section's progrra is 5 of chronic radiation exposure and genetic effect8 of radiation a8 manife8tlbd by the general viability and reproductive potential of offspring of succes ~ i v eg ene ration r of lrradirtod mice. Theme Studi e8 have been under ray for about 3 year8 and probably will continue for r ever al more. A Section of Mammalian Metabolism h88 been ertablished from that portion of the old Bio cheri u try Section ' s program concerned

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with radioisotope metabolism. Its program con818te prin - ci pally of using whole body counting technique8to investi gate radioisotope uptake and retention in mice, rats, dogs, m~nkeys, and man. The prime objectives of.the see tu die8 are t o look for inter species metabolic car relations,to collect metabolic data applicable to man, and to study the effect of various physiological and dietary factors on the normal metabolic pattern of mammals. The Lor-Level Counting Section, which in the past has been concerned with development and study of whole body counters, is dropping development work and being oriented toward the research uses of low level counters and toward measurement of space radiation.. The Radiopathology Section has been changed to a Clinical In ve o tig at ions Section.This Section ’ s program will continue t o be one of lnve8ti gating the use of radiO i8OtOpe8 and whole body counters in clinical and di agno8tic medicine. Largely for budgetary reasons,the Veterinary Section was abolished by dividing the responsibility for animal ca&eand Mammalian Metabolism Sections on the bmir of animal urage. Ren pon8lb ili ty for the mouse and rat colonies was cr88igned tothe Mammalian Radiobiology Section, and care and nu in ten ance of dogs and monkeys was asnlgned t o t h e Mammalian Metabolism Section.Personnel of the Veterinary Section were rear rig ned

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to the respective Sections on the bas28 of work load. Rearrangement and renaming of Sections during thi8 report period naturally have resulted in change8 in chapter report. Occa ~ ioa all ya progresa report may appear t o be out of plrcm with regrrd to section and chrpter. Reference t o the Table of Content., however, 8hould make it er8y t o gard t o chapter heading.. Past i 8 ~ u e 8of tho Biological The following table of org8aiz rti on shors thm amctional dia tri but ion of group personnel 8a of June 30, 1961.

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MOLECULAR RADIOBIOLOGY SECTION A potential method of establishing the sequence of ba8.8 in a DNA o r RNA molecule may be the determination of the order In which individual nucleotides are enz yu tic rl ly hydrolyzed from the polymer. Specific enzyme8 are available which work along the polymer chain from either the 3'- hydroxyl (1-4) or t h e 5'-hydroxyl ( 5 - 7 ) end of t h e polynuThe method has been demonstrated on a synthetic 6-member polythymidylic acid (4) but has not been applied to natural DNA's because of the difficulty of obtaining a .ingle molecular applied to a mixture of DNA molecules with different ba8e

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Because of the great difficulty of 8uch a aeamurement, it i m of interest to predict i n advance as much as posmible the course of the reaction,particularly in regard tothe rate of change of concentrations of the succesmive hydroly8im product, optimum times of maapling the myster, accuracy redue to the increaming complexity of the mymtem a8 the ceaction8 proceed. A cornparimon of alternative experimental procedurem, such am continuoum stripping of the hydro lys i8 product. versus %atch" operation, etc., is alu, demirablo. Thim paper im intended to provide the mathoutical foundation for such an analysis.An rnrlp8im of tho kinetics of a 8eriem of firmt order reactionm with equal rate conmtants doem not appear t o have been porforred prevloumly. It rill be a8mured that the enzymatic hydroly8Im of the DNA polymer proceodr am I mequonce of flrmt order roactifxm. pe not m tion of the reaction barrier by an activated complex or f om tion of the complex from enzyro plu8 mub8trate. Peru non t attachment of the enzyme to them ub8tr rte during degradation is note88entla1,but it i m ammurod th8t i f recorbi nation m occur between 8uccom8ivo hydmlymI8 mtopm there

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is no preferential attachment of enzyme tosub8trate dependent on degree of previous hydrolysis. Whether or not the enzyme rei in s Attached, it is asaumed that all successive steps proceed with the same rate constant. specie. l a aaoumed t o be 8u bj ect to hydro lys io. Only one chemical There aoeumptions are neces8ary in order t o make the problem amenable t o mathematical analysis ; the amumption that the rate con at ants for all steps in the hg d roly8io are identical implies that the breaking of the phoophate bond t o a terminal nucleotide occurs among the remaining polymers a t random. This rs8umption seems not unreasonable, a t least for the initial etrges of the degradation. The reaction aequence can be represented as follows : the succes rive residue8 after removal of 1, 2, 3 ... n nuthat theae are removed in serial order along the DNA chain in 1direction only by hydrolyaim of only 3-phosphate or 5phosphate bonds. I t is also aa8umed that the polymer chain 1. long compared with the total number of nucleotide. removed.

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Tha r rte constant8 for all stagem are asmumed t o be identical and equal to a constant k; khas the dimensions of reciprocal time for Ro t o decreame by half. i m de8crlb.d by a simple exponential law, it i m not meaningfulto ume the concept of a hrlf-tire for later mtrges. The time required for the concentration of Ro t o drop Since only the first rtep Before making a formal analymlm of the rate equations, it can be noted that on the brmim of the am8urption8, the tot81 rrte of production of all hydrolymi8 product. is conmtant rnd the total amount of nucleotide8 produced will rise linearly with tire. Thir follorm mince all the original DNA is alwapm a vall able for further hydro ly8is at the same reaction rate k, regardlaom of the particular 8trge of degradatfon which It ha8 reached, 1.8. , where RZ i m the initial c once ntr rtl on of undograded polymer. . (Thim rill not be true if any of the DMA rerchem the atate of corplete degradation to ron our lc nucleotidem, o r If the eaao'Of hydrolyslm chmgem with polymer length.

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where N is the total concentration of nucleotides in the system at anytime t. The general equations for a seriea of consecutive first order reactions were derived by Bateman (8)for the case of the oimple differential equation, The disappearance of Ro is governed by which integrates to the Well known exponential law, The mecond member of the8eries ~R1, i m formed ata rate equal tothe rate of di6appearance of Ro and di88ppear. a t a rate proportional to it a own instantansourn concentration : Sub at it u ting Eq. 2in Eq. 3 and in te gr r ting, one obtain8 Thio expreoeion im indeterminate when ko - kl, 80 that the Bateman.derivation cannot be used for this and all following

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step.. The in determination in Eq. 4 , however, can be easily removed by the following method. the parenthesem gives : Factoring exp (-kat) from This e qur tion differ8 from that ordin8rlly not Involve the ha8 in rte ada -The concentration of the polymer R3 produced by tho second hydrolymi8 im given by an equation analogou8

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which integrates to The general equation for then th member of the seriem is: and the uximum concentration of then th re8idue occur. a t The family of curve. corremponding t o Eq. 10 for the range sent a t mignificant concentration by timet - 0 / k . The failure of the firm t hydrolymis product t o di88pWW before the app err - prement Is constant,it m division through a number of stagee of degradation means that no one stage i 8 prement in high concent ration.All are lea. than 20 per cent after tiret = 4/k. The rapidly increasing complexity of For the purpose of analysem and mtr uc ture determination, One 1s concerned not with the remaining degraded polymer fraction. but with the individual nucleotides and the order in which they are hydrolyzed. i a proportional to the concentration of the parent polymer The rate of production of nucleotide Nn

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Fig. 1. Relativo concentrationm o f ~uccomaivopolyior romfduoa am a function of tiu in 8aym tam undergoing dogradation with oq-1 rate conmtantm for

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Therefore, Fig. 1 a160 represents the rates of production of each successive nucleotide as degradation proceeds. continuous stripping process is used so that the nucleotide n are separated from the polymers as they are formed [e.g., i n a stable-flow electrophoresis system (9) with recycling of the polymers], then these figures give the relative concent rations of the several nucleotides as a function of time. Note that since the total rate of hydrolysis of all polymer species is constant (- k RZ), the total differential concent ration of all nucleotides as observed in this type of experiment should also be constant and equal to kAtR z, where A t is the (small) time elapsed during recycling between passes through the separator.Verification of the times and orders of appearance of the first several nucleotide6 rpperrr t o be quite feasible. By the time the eighth nucleotide st it ute s about 15 per cent of the total nucleotide concent ration. If the first 8nucleotides to be hydrolyzed were identical, then 15 per cent would be the concentration change due tothe appearance of Ng. However, on the average, only 1 out of every 4nucleotides will be identical with a given one (adenine, guanine, cytosine, and thymine being the

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poa8ibi li ties from DNA), so that of the total nucleotide concent ration at thi8time, about 25 per cent rill be due toa nucleotide identical With N8. N8 Will, therefore, on the average,nearly double the ambient c once ntr rti on level of the corresponding nucleotide. ytic problem would be lema …