Biological Effects of Ionizing Radiation at the Molecular, Cellular and Human Level

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BIOLOGICAL EFFECTS OF IONIZING RADIATION AT THE MOLECULAR,CELLULAR AND HUMAN LEVEL Louis H.Hemp e lmann and Christopher S. Lange University of Rochester School of Medicine and Dentistry PREPARED FOR THE U.S.ENERGY RESEARCH AND DEVELOPMENT ADMINISTRATION

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The DNA of mammalian cells (~5178Y)has been found to occur in units of 1.7 x 1010 daltons. This size has been determined both by sedimentation and by viscoelastometric methods. These DNA units are composed of about 21 subunits of 8 x 108 daltons each, linked together in tandem by protein linkages. Both DNA double-strand breaks and linkage breaks are repaired. The hormone or differentiation substance which inhibits brain formation and probably maintains polarity control in the planarian has been characterized. It is a protein of ~3 x 105 This report was prepared as an account of Government-sponsored work. Neither the United States, nor the Energy Research and Development Administration nor any person acting on behalf of the Commission, A. Makes any warranty or representation, expressed or implied. with respect to the accuracy, completeness, or usefulness of the information contained in this report, or that the use of any information, apparatus, method, or process disclosed in this report may not infringe privately owned B. Assumes any liabilities with respect to the use of, or f o r damages resulting from the use of, any information, apparatus, method, or process disclosed in this report. As used in the above, 'person acting on behalf of the Commission' includes any employee or contractor of the Administration or employee of such contractor, to the extent that such employee or contractor of the Administration or employee of such contractor prepares, disseminates, or provides access to, any information pursuant t o his employment or contract with the Administration or his employment with such contractor.

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Technical Progress Report I. Scope of Investigations Undertaken and Results Obtained. A . The Measurement of DNA Size and o f Double-Strand Breaks in the DNA of Mammalian Cells. 1. The Semi-Automated Gradient System. This system which we had developed is now in routine use and has been described in detail in our paper in Analytical Biochemistry (1). This system, based on the SW40Ti rotor, was made to produce and analyze linear sucrose gradients ; however, a few additions now enable us to handle No ll - type 2.Calibration of the Gradients. Both the linear and the No ll - type gradients were calibrated with T4 and T 7 DNA and were found tobe iso kinetic for both DNA s.The No ll - type gradient yields the expected ratio of,520, ~T4/S2oYwT7 when The linear gradients, however, show a greater sens i - t iv it y to DNA concentration and still lower concent rations(N 0.35,cig/rnl) are necessary to obtain the expected S-value ratio. a Burg i and Hershey equation exponent(3)value of whereas the linear gradient gave a value of - 0.416 rate E. concentration line for data between .35 and .73.hg / ml to zero concentration gave a n exponent value of 0.450for DNA in infinite dilution. However, data obtained at a concentration of 8,c, g/ml did not then fit this line.At this higher Concentration an exponent of0.308 was obtained. The No11 gradients yield 3. The Estimation of DNA Molecular Weight (or Size). An exponent appropriate to the sedimentation of DNA at infinite dilution is theoretically required to justify the application of the Burg i and Hershey equation to unknown DNA molecules. However, inthe swinging bucket rotor, it is not possible to detect the larger intact mammalian DNA s at concentrations so low that their sedimentation is comparable to that of the smaller phage DNAs.This is reflected in the increased contribution of the intrinsic viscosity [ q ] would be solved. However, the commercially available

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(1960's version) with a surface floating rotor is neither sufficiently sensitive nor gentle enough to measure [?I for the much longer mammalian DNA molecules. It shears them within its first revolution. Nevertheless, if we use the viscoelastometric method DNA size estimates (see (4) below) to calculate values based on the exponent appropriate f o r DNA at infinite dilution, then the size estimate is consistent with the viscoelastometrically derived size estimate. Without the correction for [ V I , the size is severely underestimated. The value also agrees with the size estimate based on the uncorrected sedimentation rate if the exponent value 0.308 is used, i.e. , at 8 Ag/ml, T4 DNA mimics the much larger mammalian DNA at 0.2 - 0.4 -!-lg/ml. The size estimate is 1.7 x 1010 daltons, or about 1/8th of the DNA content of the average chromatid, when the cells are lysed in 1% sarkosyl After the successful week of collaboration between arrangements have been made for Dr. Lange to spend the 1975-76 academic year in D r . Zimm's laboratory, building, standardizing, and calibrating a viscoelastometer to be brought back to Rochester f o r measurement of bacterial and mammalian DNAs. The relaxation time based DNA size estimate obtained f o r ~ 5 1 7 8 YDNA was 1.3 (0.9 - 2.0) x 10 daltons and the recoil yielded an estimate of 481 k 8% such molecules per log phase cell (or 321/G1 phase cell). This number of such large molecules per cell when divided into the fluorometrically determined G 1 cell DNA content yields an average size estimate of 1.6 x 1010 daltons. We hope shortly to submit for publication a series of three papers covering the entire problem of DNA size estimation by sedimentation in sucrose gradients, and our method for obtaining accurate size and uncertainty estimates (see (5) and publication list). The results of this work (for asynchronous, log phase cells) were reported at several international congresses and national meetings last year and are published in our FEBS Letters paper (5) (see appended list of publications). Our qualitative conclusions, i.e., that DNA double-strand breaks and linkage rejoining take place in mammalian cells and that almost all such breaks are repaired, still hold true in the light of our findings reported in Sections 2 and 3 above. However, our quantitative estimates of rate may have to be altered somewhat.

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B.Sub lethal Damage Experiments. The Taland ic Research Company cell cycle analyzer has been purchased and is in operation. We are now trying to determine optLmal cond t ions for cell growth and mitotic selection on this apparatus. Further experiments with cy clo hex imi de have been performed and a paper describing the metabolic effects of this drug as functions of dose and cell cycle stage is in preparation. Mr.V.Steele(Dr.Lange ' s student) has submitted his doctoral thesis(6) on the purification and characterization of an organ - specific differentiation control hormone of plan arians. The results of this work, which also yield a polarity - control model for the organism,are summarized inthe appended abstract of his thesis. We expect to publish three papers based on this work. D . Study o f Plutonium Workers. These studies are being carried out in collaboration with the Los Alamos Scientific Laboratory. The following three groups of plutonium workers are being surveyed : Group I, 25 subjects heavily exposed to plutonium during World War 11, each with a body burden of plutonium measurable by urine assay methods of that time. These persons were thoroughly studied at LASL i n 1971-72 (7). Almost all of these subjects have submitted additional urine and sputum samples and they will be examined by their local physicians in the next fiscal year (1976-77); Group 11, 42 subjects also exposed during World War I1 with body burdens measurable by modern urine assay methods but not by World War I1 methods. Twenty-seven of these persons have responded to a mailed questionnaire and have submitted urine and sputum samples. They will be re-examined by their local physicians and will have roentgenograms taken in the coming year.Additional efforts will be made to trace the15un located subjects ; Group 111, 181plutonium workers exposed a t LASL and with atleast one positive urine sample. The 86 workers currently employed a t LASL are being examined a t LASL b y Dr.Paul Flynn who should complete this study in the 1975-76 fiscal year.In addition to urine as says and bronchial cytology, roentgenograms, chromosome studies and chest counts will be done. Fifty - one of the95terminated employees have responded toa mailed questionnaire.Efforts will be made to trace the remaining 44 terminated employees. Physical examinations will be carried out by local medical doctors and appropriate tests made during the 1975-76 contract year.

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The expenses of laboratory tests for all subjects of Groups I1 and I11 will be charged to our budget as will be the physical examinations of all terminated employees. 1. C.S. Lange and D.F. Liberman, Anal. Biochem., -54, 129 Hanawalt and Setlow, Eds., Plenum Press, in press. 4. C.S. Lange and M.A. Resnick, Fed. Proc., 33, 1481 (1974).* 7. V. Steele and C.S. Lange, Rad. Res., 59, 47 (1974)a of ~ 5 1 7 8 YCell Growth by Cycloheximide. 2. C.S. Lange and D. F. Liberman. The Organization and Repair of DNA in the Mammalian Chromosome. I. Calibration Procedures and Errors in the Determination of a Molecular Weight of a Native DNA. 3. C.S. Lange, R.W. Clark and D.F. Liberman. The Organization and Repair of DNA in the Mammalian Chromosome. 11. Determination of the Molecular Weight of a Mammalian Native DNA. Organization and Repair of DNA in the Mammalian Chromosome. 111. A Gradient Shape-Induced Speed Dependence Affecting DNAs Larger than the Subunit Size. .The Organization and Repair of DNA in the Mammalian Chromosome. IV. The Induction and Repair of Double-Strand Breaks in the DNA of the L5178Y Mouse Leukemia Cell. *denotes reprint or abstract appended to this report.

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The Organization and Repair of DNA in the Mammalian Chromosome. v . A Model and Supporting Evidence for the Organization of DNA in the Mammalian Chromosome. Specific Differentiation Control Substance in the Planarian in Regenerating Planarians. V. Steele and C.S. Lange. Irradiation Effects on the Differentiative Integrity of Stem Cells in the Regenerating Y.K. Fu, C.S. Lange and M.W. Miller. The Absence of an Effect of Photoreactivation on Sublethal Damage Repair in a Photoreactivating Wallaby Cell Line. C.S. Lange. On the Relationship between Post-Irradiation Loss of Reproductive, Morphological and Differentiative Integrity by the Planarian Stem Cell (Neoblast). The principal investigators have devoted the following percentage of their time to the project: They expect to continue to do so for the duration of the existing

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Financial Statement of Estimated Costs University of Rochester For the Period July 15, 1974 through July 14, 1975 Estimated Total Project Cost f o r the Current Peri od $49,500.00 Total Amount Chargeable t o ERDA (Approved Support Percentage 100% of Item 1 ) Cumulative Support Costs Estimated Total cumulative Support Costs Cumulative Support Ceiling *Includes expenditure of outstanding commitments f o r property a t the end of

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Financial Statement o f Estimated Costs For the Period July 15, 1974 through July 14, 1975 1. Estimated Total Project Cost f o r the Total Amount Chargeable t o ERDA (Approved Support Percentage 100% of Item 1 ) 3. Cumulative Support Costs 4. Estimated Total Cumulative Support Costs 5. Cumulative Support Ceiling *Includes expenditure o f outstanding commitments for property a t the end of

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1. C.S. Lange.and D.F. Liberman, Anal. Biochem., 9, 129 (1974). 3. E. Burgi and D. Hershey, Biophys. J., 3, 309 (1963). 4. J. Eigner, Molecular Weight and Conformation of DNA (Chapter 128) L. Grossmanand K. Moldave, Eds., Academic Press, New York, 1968. 5. C.S. Lange, FEBS Letters, -44, 153 (1974). 6. V. Steele, Ph.D. Thesis, University o f - Rochester (1975). Year Study of Selected Los Alamos Plutonium Workers, LA-5148-MSY Los Alamos Scientific Laboratory, Jan. 1973.

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Reprints of articles published and abstracts of theses and of papers presented at national and international meetings and congresses during FY 1974 (July 1973 - July 1974).