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Sumry Xeport on i Cytogenetic Pilot Study for DOW Chenical Co., Rocky Flats Division, University of Denver fron
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Human radiation cyto g cnet ics studies have been reported from research on several population al categories : individuals with external irradiation forth2rape u tic and diagnostic irradiation (18-24); and, chronic internal occupational irradiation expo sure s(24 - 30). This compilation of huran radiation cytogenetics is not meant to be con ple te ; further citations are included from two symposia (31, 3 2 ) and two e x c e l l e n t reviews by Bender (33) and Bloom ( 3 4 ) . To paraphrase Sender (33, pp. 216-217), the rgcent rapid develop of human radia - tion cy co genetics has ledto some researchers ignoring: a larger background of in for nation on the radiation cytogenetics of Eon-human material extending back t o 1933. An exhaustive review is avail abl s by Evans (35) on biological cyto genetic infomation. The body of data incorporated ir,to the above cited works can b e summarized for the report by the following generalizations. These works contribute to an understanding of the somatic consequences of radiation in man, and in those studies of sufficient populat ional size have provided dcse-response indicators of human e qo sure to radiation. It, should, however, be cautioned that in man, ifnot most, human retrospective studies,occupational human radiation cytogenetics shares certain limitations. These limitations include errors inherent in the physical dos ime try,radiation biological changes since esp o sure(or during expo sure, when chronic) sampling errors, and individual differences in response to irradiation. This laboratory would b e remiss i f it was not further pointed out that proper conditions within the methodology of peripheral lymphocyte chrornosorr.e s rudi es is essential to proper interpretation of the
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resulting data. Furthermore the biological or clinical significance of chromosomal aberrations are not knorm. However, chrorosome aberration levels from peripheral blood lymphocytes offer a practical means for establishing biological dose-response re let ions(11,33,34) and I do not mean to detract from the potentialities of radiation cytogenetics, but merely t o make apparent the recognition of certain limitations. Since this is a report, and not meant tobe an exhaustive survey for the specialist, it nay be instructive at this point to compare sore studies on irt or nal emitter affects on hunan chroaosomes (Tables 1 and 2). These citations are selected because the method of reporting the prekalence of the structural chromosomal aberrations ar2 adaptable for comparis on with our work. These works includeonly internal .emitter, occupational exposure and, as can b e observed, include a variety of What can be said about these internal emitter, occupation ally in - curred, peripheral lymphocyte chronosone aberration studies ? First, w e are obviously conparing different nuclides with different doses and dose rates, different particulate irradiation,different modes of body entrance, and different organ and tissue seeking capabilities.In severa l of the studies(24,24,29)population al size was not sufficient to detect dose-reflponss rel at i or ships, and in atleast one ic stance,cell sample was also insufficient.In the studies wherein the population al size and cell sample is more ample(26,31))a dose-response relationship is apparent. It w ~ u l dappear that none of these studies deviate excessivel y from accepted base - point,control levels of structural chromosonal aberrations (0.21-1.72 for " unstable cell" aberrations and 0.00-1.17% ceil " aberrations in the controls of thc study by 3oyd, e t a1 (26).
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Prior tothe Pu-worker pilot study,I would have conjectured that the Tuscanp and F!Lller study (25) might be excessively high in the prevalence of both major cell aberration categories in their workers (11.9 and 3.1X); 5ut we observed 12.3% of di centric,rings, and deletions in I think it might b2pertinent to comment on the u rani un miner studies done a t the University of.Denver and Grand Junction laboratories. In al in it ed pilot study on the miners (29) w e observed significant d if - ferences in the pre valance of structural chromosomal aberrations between the miners and controls as groups. A subsequent PHS-supported grant has lead to analyses of the chromosomes on 157 add i tonal niner s (187 including 30 in the pilot study). To my kcowledge, exclusive of the atom bomb survivor cyto genetic study,this will comprise the nost sizeable, mutagcn-induced cyto genetic study to date. Tl;is excludes, of course, studies on general pop ul at lons, wherein only20to 10 cells are studies on each individual - Atthe very least, this is the most intensive effort on occupation ally exposed, internal emitter, populations. As w e expanded the study to 100 controls and niner s and arranged the data accordin% to different exposure groups (see Table 1 and Histogram I) a dose-response w a s evidenced by dose groups. in His to gran 1, the prevalence of the nore "unstable" a5er rations increases almost linearly from the control level through Group I V (\&!! 17402890; for reasons expressed i n our pilot study paper (29), there are problems inherent in the VLX calculations).This category of aberrations then decreases in prevalence in the highest exposure category (Group L'). As can be seen graphically
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This may b e due to the fact that we are dealing with a highly selected sub-population because of natural or induced mortality in this oldest group of men. that the "unstable" aberrations may be selected against over longer periods mining nay also be a factor in this group. category,an almost embarrasingly good linear relationship would zppear Junction and are blind - read, and the groupings deter - in ed in part by the gaps in group of individuals with particular I;TLIl estimated exposures. Although there are conceptual flaws in the designations of " stable " vs "uusta3le" cell aberrations (see 33,34), it i s thought that,in general, I hasten to add that the slides are encoded i n Grand cells with translocation s and inversions are more likely to survive division than the "unstable" cell aberrations. '€?ius, the observation that the tr stable l ' cell aberrations continue to increase from the control level through the highest exposure group (Group V), nay lend credance tothe idea that th sse cells selectively persist compared tothe more ''unstable" cell abs r rations. The significance of these findings in the uranium miners may becone apparent only after a long-term, chronic study ; they are not obvious now, except as a dose-response indicator. The data is presently in the possession of our statistician,Dr.Philip Archer of . meters, which Is hall not go into in this report (age, time-since-mining, We are looking at the data fr on several paraPu-worker population vas deemed a very in port ant potential group chromosomally. It is not essential to this report to note the
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historical evolution of this pilot study. In June of 1972, we began the study on the cytogenet:cs of 30 workers and controls which- eventually expanded t o 45 workers (5 of which were recently exposed and are not included in this report).Selection of the men to be included in the pilot study was done by Robert Bi st line,Ii adi ation Biologist at D0T.J Chemical Co., Rocky Flats Division. The 40 men included in this report include6with no hot area work experience,7hot area workers (but no detectable internal burden), anZ 27 internally exposed Pu-workers. The precautions necessary for proper standardization of the peripherally r - phocyte culture system, processing of the cells and analyses of the cells have been expressed elsewhere in saver al of the publications cited in the bib lio gr ashy.In short,we enployed a standardized nethodology used i n --s_ix laboratories in the U. S. The technique is based on minor variations for 50 and 72 h r for each individual. ._The chromosomes were blind read(approx in at ely 100 cells/case) and grouped into the categories expressed inthe Tables and the Histogram after all the data was accumulated. Our choice of grouping was soaewhat in flu zn ced by Mr. Bi st line ' s judgenent that the systemic-burdensd individuals might b e more affected by zheir exposure that the lung-burdened individuals.His estimation was borne out in certain categories.. In Histogram 2 ( and Table 2), one can observe sign if iczn t differences between the non-exposed controls,hot area workers, and the workers exposed to internal Pu-An. We have not subjected these data to statistical analysis at this tine for tvo reasons: 1) w e deem the control and hot area
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gan Medical School an2 the ABCC, Hiroshima) vievs these differences even without statistical analysis as inconsiderable escess of the general histogratis and the pluto niu n worker histogram are not the same. Note should be taken that the scales for the uranium miner -for emple,the hot area workers with no detectable internal burdens, show apr z valence of "unstable" cell aberrations approx in at ely that of the aber ratio ln s,the hot area workers exceed the aberration 'frequency observed i n > liner Grou? 11. These observation night not be apparent from ay is u al comparison of the histogram. The scale i n Histogram 3 is the same as ::-:-In Histogram 3 and Ta3le 2,we have further subdivided the internally sxpased workers, and in spite of linitec? numbers i n sone categories, we -.chromosomal aberrations(this excludes chr or at id or subchromosomal changes) appears to be almost as a linear function.This, however, should not be Tans trued as a simple dose-response relationship because w e are dealing with groups, not doses. iJe must eventually look very carefully, by indi - xi dual, and plot dose against the prevalence of aberrations, and include several parameters such as the amount and proportion of body burden, systemic burden o r combinations of these burdens. Whether a linear dose-response relationship will evolve (or a dose-square relationship3 must await further that observed in the to t21 chromosornal aberrations category. The " stable " =ell ch rano so ml aberrations show al costa straight - line relationship from the control level through the s ys tep-ic-burden-only group. \*!e cannot account for the drop intSe " stable " cell aberrations in the last group and this The " unstable " cell aberrations show as in i lar relationship to
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will require further thought and analysis. findings most interesting at this level of analysis and Dr. Bloom concurs Overall, however, I find these expectations prior to the study. ficance, if any, cannot b 2 assessed at this time. These individual and collective. . findings exceeded mY own The biological and clinical signikhat may be at hand, but can only be clarified after an increase in populational size, is a possible biological indicator of the somatic cell affects of external (hot area workers) and internal Pu-Aa exposure, at least on such radiation-sensitive cells as blood lumphocytes. * In addition to the populatiog effort (albeit rather small at this time), four of the workers have relatively high percentages of specific chrornosomal As=.#' Burden Y r s . Since Type of Prevalence of the These are summarized as follows: XPaS ZIPLB Esposure Aberration aberration per 100 1 3.44 0.50 15.4 Deletion of part 100% of the long arm of the long arm of a 13 0.10 3.30 This individual is a 79% aLe 47,SYY (High Axzericium chromosomal mosaic 4 6 , Exposure) XY/47,XYY). The double ated by C banding technique. 17 0.58 0.00 13 Translocation to t h e short 85% Too small to detect vhere the added chrormtin came from without further banding
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me nuder of individuals with these high propDrtion of cells with unusual coq a red with general populadonal studies (see 34). But t o demonstrate,in part ; why one should not jump t o prcmature conclusions, a paper is now in preparation on individual iil, i n which we conclude that the origin of the deletion of theY chromosone is congenital and not radiation induced. The justification for this conclusion will be presented in a paper that will be sub n it ted through DOWAEC in the near future. In- this paper, we have studied the pro spit us and his only syrIn divi d cal E13 (46, XY/47, XYY) is also likely to be a case of congenital origin for his aber rail t cells. However, ifwe are able to obtain the cooper at on of these in divi hals,future analyses will be Fade employing the new chromosone bandicg techniques. So w e are left with the p ossi - bil it y of 2 / 4 5 with. unusual, high frequency aberrations in this pilot In short,this is a scientifically interesting population of nen t o study, and worthy of a furt hsr, expanded effort. As noted earlier, the cytogenetics of peripheral blood lun p hory tes have proven useful as a biological indicator of radiation exposure. Suggestions for future action must b e based on the magnitude of support for this and other biomedical research efforts.Atthe very least, an3 perhaps as one phase or level of effort,the pilot study should be exTanded t o 20 cold area controls and 20-25 hot area workers (27 t o 32 additional nen). Sut this should be considered as minimal and I think this population in port ant enough t o response to the question of whether he would be interested in doing sputum 10990 r311 cytology on th2 wor!:srs, w e would also need a n ' additional technician
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or two i n our Denver laboratory. ‘ We at the University 6f Denver laboratory and Dr. Bloom, believe that,at present,there are no proper shortcuts to careful eye analyses and karyotyping of a t least 100 cells on each individual.Dr. Bloom has reviewed the present state of the art of automation i n chromosomal detectable,aberrations are not amenable t o automation. However, we shall keep abreast of current developaents in the various laboratories around the country attempting to achieve automztion i n chronosonal analysis and wh n the sensitivity of auto nation reaches the desired level of accuracy, we shall welcome and en ploy auto nation. Returning to the issue of the nurn3er of cells to sample per in divi - dual, we Selieve that atleast a hundred cells should be analyzed per case. We shall probably achieve a sufficient base go in t with the uranium n’I iners this year, and after statistical analysis we can expedite the future miner studies by reducing the nuaber of cells analyzed per individual. desired end nay b e reachzd sooner inthe Pu-workers tSan was necessary in the miners because of their higher prevalence of aberrations.But we will not expedite this process prematurely that is, before a proper population al We believe that the Fl’s from those workers who are born after parental exposure to irradiation should be studied,if only to obtain what probably will be an important negative finding. In the study of the F1 individuals w e would probably look at only 10 cells and employ recent banding techniques With regard to the banding …
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