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juence of Testicular 1 in the teste3 of the irements were made . This contrasts with amsters produced by ium in the testes of ind increased cancer 3m this isotope. When se in abnormal meiotic posit in the interstitial average dose received kcreditation of Laboratory This study \vas designed to detrrmine the influence of the microanatomy of the testes and the microdistribution of 23gPu in the testes on the radiation dose to t he spermatogonia. This information will br useful in estimating the genetic hazard from internal deposition of 239Pu. The approach used was to determine the total body retention pattern and microdistribution of 2aePuin testes and to calculate the dose to spermatogonia in Chinese hamsters following intravenous injection of ls9Pu citrate. The relationships among dose, dose rate, activity injected, and the chromosome aberration frequency were then drterminrd. Finally, to aid in the extrapolation of the dosc distribution factor from rodrnts to man, morphometric. measurements were made to determine tht, ratio of interstitial, spermatogonial, and tubular tissue in adult mice and Chinese hamsters and in aged, adult, and immature humans. These values were used to calculatr the theoretical d o m to spermatogonia for all threr To determine the retention pattern of monomrric =9Pu citrate, Chinesc Irnmsters were injectrd in the jugular sinus with 6 X 104 pCi 239Pu/gbody wt dissolved in a total volume of 0.1 ml. T h e injection solution was prepared as outlined by Schubert ef ai. (6) and characterized as monomeric by ultrafiltration. Thc animals were sacrificed in groups of five at 0, 4, 16, 32, 64,128, 236, 500, and 700 days after injection. At sacrifice the animals were dissected; liver, bone, spleen, kidney, testes or ovaries, and carcass werc analyzed for total a activity. For the cytogenetic studirs, hamstmi were injected with monomeric 23BPucitrate at activity levels of 2 X 10-3 or 6 X 10-4 pCi,'g body wt. Control hamsters were injected with the citrate. The mitotic cells from exposed testes were prepared as prcviously described (7) at 64, 129, 256, and 500 days after injection and the aberrations recorded from coded slides. For animals sacrificed a t 500 days, one testis \vas analyzed for 239Pucontent and the other prepared for histopathologic and autoradiographic examination. .\utoradiographs were exposed for 1 year and the microdistribution of the a tracks was determined. The microdistribution \\-as charactrrizcd by the method of Green et al. (S),who divided the a tracks into three classes for scoring. The first class included tracks that were entirely in the interstitial tissue; the second class included the tracks above the peritubular membrane and tracks that entered or crossrd the sprrmatogonial layer; thc third class inrluded those that lay vntirely within the peritubular membranr. Vsing this method, the fraction of the energy deposited in spermatogonia relative to that drposited in the whole organ uns mra,cured. Calculation of th(J microdistribution of the dose is based on the rdative numbers of tracks 8 pm or greater in lrngth in each of the areas of the Histologic samples were collected from Chincse hamsters at sacrifice. Human t*.ites samples were obtained from individuals of different ages: 2, 6, 18, 33, 61, arid 77 years. Relativc amounts of tissue in the three region!: of t h r testes \\ere nwasured from projrctions of tirsuc slides using tissuv configurations as input l1lt0 a computer with a GRAF,'PES sonic digitizer. For each age group of
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humans and for th(b Chincw hamster, data from two individuals w r v used with The relative areas of each region w r e meawrc.d as iv(311 as tho diameters of the tubules. Data for mouse morphology and plutonium distribution \vert. taken from the research of Green ef al. (9). For the computation of thr dose-inhomogeneity factor, a generalization of the geometrical model of Green el al. (3) was used ; that is, the tubules were assumed to be parallel cylinders in a hexagonal array embedded in the intertubular material. The parameters of this array were computed using the above measured relative amounts of tissue in the various regions and the tubule diameters. The amount of tissue in the interstitial region is designated by f],that in thr spermatogonial layer by fs, and that in the tubule intrrior by f ~ .A s in the above model, uniform concentrations of radioactivt material over tho intertubular region, CI, and over the tubule, C T , were assumed. These relative concentrations were derived from the hamster and mouse data. Both were used in the human calculations. Monte Carlo calculations were made to dc*ttmnine f l (the fraction of the energy from sources in the intrrstitium lost in the interstitium), fs (the fraction of t h k energy lost in the spt~matogonialstrm cell region), and f T (the fraction of this energy lost in thc tubule interior excluding stem cells). For these calculations, the residual a energy of a 2391’ua from a point source as measured by Harlry el a2. (8) was used. A simulation of the autoradiographic configuration using estimated tissue and emulsion thicknesses was used to obtain the ratio of the fraction of the plutonium in the intertubular region, FI,to the fraction, T I ,of a tracks which were restricted totally to the intertubular region. This calculation was used to determine the where C is the average concentration in the testes, CI is the concentration in the interstitial tissue, and CT is the concentration in the tubules. The value of CI/C was obtained from the simulated ratio F I / T I using the measured values of T I of the plutonium located in the tubules. positioned and randomly directed simulated a tracks. These calculations were repeated five times for each animal; the mean and standard deviation of the values obtained are given in Table I. The values of CI/C and CT/C for the human data were estimated srparately from the results of each animal experiment by keeping the CI/CT ratio constant, using the values derived from mice and hamsters. The expressions for the relative doses to the three regions in the testes were derived by assuming that the lower concentration in the tubule forms a background concentration C T and that the material in the interstitium can be viewed as this background concentration plus an additional concentration CI-CT. If I’ is the volume of the testes considered, is the contribution to the energy deposited in that region by the excess concentra-
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he tubules were assumtbd ded in the intertubular sing thc above measured I V tubule diameters. The f i , that in the spcrmato- . As in the above model, mine JI (the fraction of g stem cells). For thesr boint source as measured ing estimated tissue and 'raction of the plutonium cks which were restricted s used to determine the t h e concentration in the measured values of TI 7 was obtained from the here FT is the fraction on about 3800 randomly These calculations were andard deviation of the ere estimated separately he CI/CT ratio constant, pressions for the relative assuming that the lower itration CT and that the of the testes considered, by the excess concentratribution from the back-
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FIG.1. The retention and distribution of plutonium-239 in the Chinese hamster as a percentage of the sacrifice body burden following intravenous injection with plutonium-239 citrate. ground concentration. Thus the dose, being the energy loss por unit volurnc irradiated in the region a,Do.is the sum of the doses frctin thcsr t n o sourccs. Thus, where k is a constant used to convert the energy loss per unit volume to units of dose. If C is the average concentration in the testes then the average dose D intertubular, stem cell, or tubule interior regions.. respectively) and simplifying, one obtains the relative dose equation used as listed below : T h e above derivation and equations were adapted from a n unpublished manuT h e distribution and retention pattern of plutonium in the Chinese hamster is shown in Fig. 1 and illustrates an important difference in this species relative t o mice and rats. At times greater than 30 dags, the hamster retains about 60% of the sacrifiw hot is rapidly rvrnovtac to that observed i tissue, and the spacing
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c hamster as a pcrcentagv 5 per unit voluine irasp two sourws. Thus, unit volume to units n the average dose D vely) and simplifying, i the Chinese hamster in this species relative Gter retains about 60% iiation dose t o mouse testes i is rapidly removed from the liver. The retention pattmr in hamsters i l similar to that observed in dogs and postulated for man. It is of inkrest to note that immature human (D). I i O X . Note the size of the tubules, the relative amounts of interstitial tissue, and the spacing of the tubules.
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FIG.3. Autoradiograph of Chinese hamster testes tissue and crossing the spermatogonial cell layer. showing Q tracks originating in the interstitial the activity in the liver does not influence the burden in the testes, which remains constant over the 700 days of this experiment. This information is necessary to derive a meaningful estimate of average dose to the testes. The long retention half-life of plutonium in hamster testes supports the assumption made by the ICRP that plutonium is retained in the testes with an infinite half-life. The microanatomies of a typical section of the testes (Fig. 2) of the Chinese the differences in microanatomy discussed in this paper. All photographs were taken at the same magnification. Xote the differences in the size of the tubules, ratio of tubule tissue to interstitial tissue, and the distance between tubules. The morphology of the aged human was similar to that of the adult. An autoradiograpb of a hamster testes (Fig. 3) illustrates the distribution of activity in the testes at 500 days postinjection. The activity srems to be associated with the interstitial tissue. There is some indication of a nonuniform distribution within the interstitial tissue. hlany of the tracks \\-hich originate in the interstitial
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tissue, marked b!. arrows, cross into the region of the organ, marked by dashed wer lines, that contains the spermatogonia cells. diff1 The morphometric measurements demonstrated that mice and hamsters have faUc similar fractions of interstitial, spermatogonial, and tubular tissue. The hamster higl has larger tubule diameters and tubule separation than the mouse. About 50% of the tissue in humans is interstitial-compared to 10% and 16% in the mouse and hamster, respectively. The tubules in the children are much smaller (72 pm) than those in the adults (195 pm). These differences were analyzed by morphometric measurements and are summarized in Table I. Also included in the table are parameters necessary t o calculate a dose distribution factor. The summary of the measured values and the results obtained from these calculations are presented in Table 11. Under the assumptions given, there is no enhancement of the dose t o the spermatogonial stem cells of humans a t any age. togonia. This enhancement in the mouse, and to a lesser extent in the Chinese gen hamster, appears to be a result of the relatively small amount of interstitial ing tissue and the resulting short distance between tubules in the testes of these fact species. By contrast, humans of all ages have much more interstitial tissue, and tioi the tubules are more widely separated even in the child. This results in the lack I of enhancement for humans. dist The total number of cells scored for chromosome aberrations at each time was han relatively small, and the level of damage observed was low. The cells from each und ‘Itime were thus grouped so that 300, 357,and 327 total cells were scored for the 2 X 6 X lo-’, and control exposures. With this technique, the controls C h a had an aberration frequency of 0.027.This apparent increase as a function of ent, of damage was not significantly different from that of the controls a t the 5% sior level of confidence. I intt had 0.012 aberrationslcell, whereas animals injected with 6 X 10-4 pCi/g body injected activity may be produced by the plutonium in the testes, but the level ,i this The retention and distribution data were analyzed to estimate average dose &I to the organs of the body. By dividing the organ weight by the activity at sacrifice, a concentration was derived which can be used to calculate dose rate at the time of sacrifice. This dose rate in combination with retention data can be used to estimate the total organ dose. The percentage of the sacrifice body burden at 500 days after injection was divided by the organ weight to obtain a relative measure of concentration and dose rate. The 1.8 g of liver had the highest concentration, or 40y0 of the sacrifice body burden per gram of tissue. For a 3.0-g skeleton, the carcass value could be converted to 1l%/g of bone. The soft tissues, lung, kidney, spleen, and gastrointestinal tract weigh an average of 6.4 g and had 3.5% of the activity, or 0.5%/g. The Chinese hamster testes weighed 2.0 g and contained about 0.270 of the sacrifice body burden, or O.l%/g of tissue. In this species, the testes are very large and do not concentrate 219Pucitrate to the level seen in other soft tissues. These relative concentrations in other oreans
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ed by dashed in the mouse 1 in the table n, there is no IS at any age. zstes of these ts in the lack Sach time was but the level re at the time dy burden at le soft tissues, differ from those summarized by Richmond and Thomas (IO) for man from fallout exposures. Gonadal plutonium concentrations in t hese human studies were higher than those reported for other tissues except thoracic lymph nodes and hone. In both rodent species plutonium was concentrated in the interstitial tissue of the testes. The dose estimated to spermatogonial cells was higher than would be calculated using a uniform distribution of activity or an svcrnge concentration in the testes. However, since the rangr of an a particle in soft tissue is limited (40 pm), the microariatomy or micromorphology of the organ is as important in estimating the dosc to any cell type, such as the spermatogonia, as is the microdistribution of the radioactive material. As the ratio of interstitial tissue to spermatogonia increases, the dose differential between spermatogonia and the whole organ decreases since many of the CY particles deposit all their energy in interstitial tissue and never intrract with genetically important spermatogonia. This points out the difficulty in rxtrapolating a dose-distribution factor, since the microanatomy is specic.s-dcpcbndent and factors such as relative size of the testes, amount of interstitial tissue, and separation between tubules vary greatly among species. If plutonium were Concentrated along the tubule walls in man and not randomly distributed in the interstitial tissue, as was assumed in these studies based on hamster data, an enhanced dose to the spermatogonia could result. Experiments under way on primates will provide information about this possibility. …
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