- Email: [email protected]
Radiation-induced clastogenic plasma factors
Radiation-Induced Clastogenic Plasma Factors Guy B. Faguet, Sherwood M. Reichard, and Dave A. Welter
ABSTRACT: Ionizing irradiation induces chromosomal aberrations in directly exposed cells and is known to have mutagenic and carcinogenic potential for the exposed host. Under controlled conditions, we examined whether such clastogenic effects of irradiation might be due in part to radiation-induced plasma factors. Irradiated cells and sera from CF-Nelson rats were used at 15 min, and 1, 7, 14, and 56-70 days after total body irradiation (250 R, n = 67 or 400 R, n = 39). Control rats (n = 44) served as donors of nonirradiated sera and cells. In addition, sara from six rats were irradiated (250 R or 400 R) in vitro. On the average, 298 metaphases from six rats were studied at each time-point. Cy~ogenetic abnormalities observed included chromatid- and chromosome-type lesions and hyperdiploidy. The frequency of abnormalities was comparable at both radiation doses. Nonirradiated cells exposed in vitro to irradiated serum (15 rain postirradiation) exhibited a 36- to 48-fold increment in hyperdiploidy (p = 0.0001) and a 2.- to 2.2-fold rise in chromatid gaps and breaks (p < 0.01), but none of the chromosome-type aberrations seen in cells exposed to radiation. The clastogenic activity of irradiated plasma persisted in circulation for the lO-wk duration of the study and was not abrogated by dilution with nonirradiated serum. Serum irradiated in vitro was not clastogenic. This study shows that irradiation of rats results in the prompt appearance of clastogenic activity in their plasma. This activity is not due to radiation-induced depletion of protective factors nor to chemical-physical changes of normal plasma components, but results from circulating factors released by irradiated cells. INTRODUCTION Chromosomal aberrations resulting from ionizing radiation are well documented. While irreparable chromosome damage results in cell death but has no long-term consequences to the exposed host, persistence of nonlethal alterations to the genome is of greater concern by virtue of its mutagenic and carcinogenic potential [15]. The exact m e c h a n i s m by which radiation and chemicals induce cancer is unknown. At present, carcinogenesis is viewed as the result of somatic mutations reflecting alterations in the genetic material of exposed cells. This hypothesis finds experimental support from data demonstrating b i n d i n g of chemical carcinogens to DNA [6], and from the associated high frequency of chromosomal aberrations and increased incidence of malignancies in animals [7] and in h u m a n s [1] exposed to
From the Departments of Medicine and Cellular and Molecular biology (G.B.F.), Department of Radiology (S.M.R.], and Department of Anatomy (D.A.W.), Medical College of Georgia; and the Medical Research Service (G.B.F.), VA Medical Center, Augusta, GA.
Address requests for reprints to Guy B. Faguet, M.D., Medical Research Service, VA Medical Center, Augusta, GA 309]0. Received May 6, 1983; accepted June 29, 1983.
73 © 1984 by Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 12, 73-83 (1984) 0165-4608/84/$03.00
74
G.B. Faguet et al. radiation. While a large body of evidence supports a direct effect of ionizing radiation on the chromosomes of exposed cells, preliminary data has shown that plasma from irradiated animals and from patients exposed to accidental or therapeutic radiation contain factors capable of inducing chromosomal aberrations in unexposed cells [6-19]. In this communication, we report that whole body irradiation of rats results in the prompt emergence of clastogenic factors (CF), which persist in circulation for at least 10 wk postirradiation. Our data show that the CF do not reflect radiation-induced depletion of protective factors nor radiation-induced physical or chemical changes of normal plasma components, but rather represent products secreted or excreted by cellular elements as a result of radiation effect, activation, or cell damage.
METHODS
Animals Young adult CF-Nelson female rats weighing 200-250 gm {Harlan-Sprague Dawley Co., Indianapolis, IN) were used in this study.
Cytogenetic Preparations Ether anesthetized rats were exsanguinated by aortic puncture, performed under direct visualization, using sterile techniques. Blood was drawn in 10-15 U/ml of preservative-free heparin (Connaught Laboratories, Toronto) immediately and on days 1, 7, 14, and 56-70 postirradiation. Nonirradiated blood was obtained at similar intervals from control rats. Twelve drops (approximately 0.6 ml) of whole blood was cultured in Gibco chromosome medium 1A (Grand Island Biological, Grand Island, NY) maintained at 38.5°C at a 30 ° angle and rotated every 12 hr. To maximize detection of unstable chromatid aberrations in first generation cells and to minimize chromosomal aberrations resulting from duplication of aberrations that were initially chromatid-type, culture time was limited to 40-42 hr [20]. Two hours prior to harvesting, mitoses were blocked in metaphase by exposure to colchicine (l~.g/ml culture medium) for 2 hr. After hypotonic shock (75 mM KC1) for 10 min, cells were fixed in a 3:1 ratio of absolute methanol:glacial acetic acid, spread on the glass slide, heat-dried, and stained with 1:50 Giemsa in Sorenson's buffer.
Cell-Serum Combinations Six cell-plasma (or serum) combinations were evaluated: Combination A, whole blood from nonirradiated animals; combination B, nonirradiated cells preincubated for 2 hr in serum from irradiated animals prior to addition to the culture medium; combination C, whole blood from irradiated animals; combination D, cells from irradiated animals preincubated in serum from nonirradiated animals; combination E, nonirradiated cells preincubated in a 1:1 ratio of irradiated:nonirradiated sera; and combination F, nonirradiated cells preincubated in serum irradiated in vitro. For cell-serum combination experiments B, D, E, and F, the cell and serum fractions were separated from whole blood by centrifugation at 300 x g for 15 min and reconstituted to generate the desired cell-serum combination, while preserving the original cell density. After incubating the cells in their new serum environment for 2 hr at 37°C, 0.6 ml of the reconstituted whole blood was processed as described herein for chromosome analysis.
Clastogenic Plasma Factors
75
Radiation Delivery Whole body irradiation was administered by h therapeutic x-ray unit operated at 200kVp and 15 mA with a filter of 0.5 mmCu and 1 mmAl at dose rates of 40-100 R/min. The half-value is equivalent to 0.9 mmCu. The dose delivered to the center of the body/area in R/min in air is measured by a Victoreen dosimeter. One hundred-six irradiated rats (250 R, n = 67 or 400 R, n = 39) were used as donors of irradiated cells and irradiated sera 15 min following irradiation and on postirradiation days 1, 7, 14, and 56-70. Nonirradiated animals (n = 44) donated nonirradiated sera and nonirradiated cells. In addition, sera from six rats were irradiated in vitro (250 R, n = 3, or 400 R, n = 3) using the same apparatus and technique described herein.
Data Collection and analysis Data at each time-point were derived from duplicate cultures from each of an average of six rats (range, 3-11). Triplicate slides were made from each culture and coded. An average of 298 metaphases per time-point (range, 35-800) were examined under the microscope by one of us (DAW) without previous knowledge of code or type of experiment. Chromatid-type damage recorded included chromatid and isochromatid gaps and breaks (Fig. 1). Chromosome-type aberrations observed included dicentrics, ring chromosomes, acentric fragments, and translocations. Because chromosome-type aberrations were not induced by exposing nonirradiated cells to irradiated plasma, however, these changes are tangential to our study and are not included in the analysis. Hyperdiploidy (>2n but <4n) noticed in initial cultures was subsequently monitored and recorded. Most gains were of one or a few chromosomes (Fig. 1). Abnormalities observed per slide were recorded and appropriately pooled to generate each time-point of each experimental series. Results are reported as a percentage of the corresponding total number of metaphases examined. Significance of difference between two proportions was determined by standard two-tailed tests [21]. Because the numbers of specific lesions are small, meaningful analysis were derived by comparing chromatid-type abnormalities. RESULTS
Assessment of Spontaneous Cytogenetic Aberrations in Nonirradiated Rat Cells As shown in Figures 2, 3, and 4, series A, hyperdiploidy and chromatid-type changes (single and isochromatid gaps and breaks) were seen, on the average, in 0.16% (range, 0%-0.52%) and 4.6% (range, 3.7%-4.9%), respectively, of cells from 44 nonirradiated control rats studied over a period of 10 wk. No chromosome-type aberrations were observed.
Assessment of Cytogenetic Aberrations in Cells from Irradiated Rats Because of the well known radiation-induced lymphocytopenia and the inhibitory effects of radiation on lymphocyte transformation and mitosis, experiments performed immediately following irradiation yielded a low number of analyzable metaphases. Experiments that yielded fewer than 10 analyzable metaphases per culture or 35 metaphases per time-point were not considered to be reliable and were excluded from data analysis. As shown in Figures 2, 3, and 4, when compared to nonirradiated blood (series A), blood cells from irradiated animals (series C, 400 R
76
G.B. Faguet et al.
A
• t
6
I Q
I
O V
c
4
Figure 1A Representative metaphases showing: (a) hyperdiploidy (43 chromosomes), (b) single chromatid break, and (c) single chromatid gap.
and 250 R) showed a marked increase in hyperdiploidy (p .< 0.0001) and chromatid-type lesions (p -< 0.01). In addition, chromosome-type aberrations (acentrics, dicentrics, rings, and translocations) were observed in these experiments, whereas they were not seen in nonirradiated cells. The radiation-induced increased incidence of hyperdiploidy and chromatid abnormalities seemed to abate 2-10 wk post-250 R irradiation, although hyperdiploidy remained significantly above base-
Clastogenic Plasma Factors
77
Figure IB Scanning electron micrograph (35) of the large submetacentric chromosome demonstrating a single chromatid gap. Marker bar represents 0.5 p.. line values (p ~ 0.0001). Whether this reflects gradual elimination of unstable types of aberrant chromosomes resulting from in vivo cell division of lymphocytes harboring abnormal chromosomes or whether it reflects experimental variation cannot be ascertained from our data. Assessment of the Clastogenic Effect of Irradiated Sera on Nonirradiated Cells Incubation of nonirradiated cells in sera from irradiated rats prior to culturing markedly increased the baseline frequency of hyperdiploidy (Fig. 2, series B) and chromatid-type lesions (figs. 3 and 4, series B) but did not induce chromosome-type aberrations. As shown, the clastogenic activity of irradiated sere on nonirradiated cells was demonstrable immediately postirradiation and persisted throughout the lO-wk study. The effect of sera from rats irradiated with either 250 R or 400 R on the frequency of hyperdiploidy of nonirradiated cells was highly significant (P ~ 0.0001) throughout the study-period (Fig. 2, series A versus B). This effect was comparable to that observed in irradiated cells (series B versus C, p : NS) at each time-point (except 14 days post-400 R irradiation, p ~ 0.001). The increased incidence of chromatid-type abnormalities induced by irradiated rat serum was less pronounced although highly significant (Fig. 3, series A versus B, p -~ 0.01) for all
78
G.B. Faguet et al. time-points except 1 and 56-70 days following 250 R irradiation (p = NS). The type and i n c i d e n c e of these changes were comparable to those seen in cells from irradiated animals at each time-point and for both radiation doses (Figs. 3 and 4, series B versus C, p = NS). Figures 3 and 4 suggest that the clastogenic effect of rat serum irradiated with 250 R d i m i n i s h e s with time, as e v i d e n c e d by its decreased capacity to i n d u c e h y p e r d i p l o i d y and c h r o m a t i d changes at 56-70 days postirradiation. Whether or not a similar reduction in the clastogenic activity of sera from animals postirradiated with 400 R occurs was not ascertained.
Assessment of the Clastogenic Plasma Factors The clastogenic activity in the p l a s m a of irradiated rats could be due to the generation of circulating clastogenic factors, d e p l e t i o n of c h r o m o s o m a l protective factors present in n o n i r r a d i a t e d plasma, or to r a d i a t i o n - i n d u c e d physical or chemical changes of normal p l a s m a components. If cytogenetic abnormalities found in cells from irradiated animals were due to d e p l e t i o n of protective serum factors, it w o u l d be expected that (a) reconstitution of the cell's normal serum e n v i r o n m e n t immediately following irradiation w o u l d preclude the d e v e l o p m e n t of cytogenetic abnormalities; (b) normal plasma should reconstitute " d e p l e t e d " (irradiated) p l a s m a and, thus, preclude its clastogenic activity; (c) depletion of putative c h r o m o s o m a l protective plasma factors by irradiation should be achievable by irradiation of plasma in vitro as well as in vivo. To evaluate these possibilities, the following studies were conducted: (a) cells from irradiated animals were p r e i n c u b a t e d in nonirradiated serum prior to culturing. As shown in Figs. 2, 3, and 4 (series D), nonirra-
Figure 2 Incidence of hyperdiploidy observed in nonirradiated cells unexposed (series A) and exposed to serum from irradiated rats [series B) compared with that seen in cells from radiated rats preincubated (series D) or not (series C) with nonirradiated rat serum. Cells and sera were obtained at the times indicated following irradiation (O = 250 R, A = 400 R).
A
B
C
0
10-
"0 °-0 ¢k
0
56-70 0
~'76
0 r '
~-76
Observation t i m e in days
0 ' r
G6-70
79
Clastogenic Plasma Factors 10] A
X
4 C I
0
ID
•~
C
p,,
o
B
1
O
l
't 10-
0 ¢
"O o~
e
J Observation time in days
Figure 3 Incidence of chromatid-type abnormalities (chromatid gaps/breaks, []; isochromatid gaps/breaks, A; total, 0) in nonirradiated cells unexposed (series A) and exposed to serum from irradiated rats (series B) compared with that seen in cells from irradiated rats preincubated (series D) or not (series C) with nonirradiated rat serum. Cells and sera were obtained at the times indicated following 250 R irradiation. diated rat serum had no protective effect on r a d i a t i o n - i n d u c e d cytogenetic abnormalities. The incidence of chromatid-type aberrations in irradiated cells obtained at various times postirradiation were not altered by a d d i t i o n of nonirradiated serum (series C versus D, p -- NS). The same is true for the incidence of h y p e r d i p l o i d y , except for cells obtained 14 days post-400 R irradiation (p < 0.01); (b) cells from nonirradiated rats were incubated with a 1:1 mixture of nonirradiated and irradiated rat sera for 2 hr prior to culturing. The incidence of h y p e r d i p l o i d y (5.9%) and c h r o m a t i d - t y p e abnormalities C8.7%) observed in these experiments (not shown) were comparable (p = NS) to that i n d u c e d by u n d i l u t e d irradiated serum on nonirradiated cells (Figs. 3 and 4, series B); (c) finally, cells from n o n i r r a d i a t e d animals were p r e i n c u b a t e d for 2 hr in rat serum irradiated in vitro (250 R or 400 R) prior to culturing. Irradiation of serum in vitro failed to generate clastogenic activity. Indeed, p r e i n c u b a t i o n of n o n i r r a d i a t e d cells in serum irradiated in vitro had no effect (p -- NS) on the i n c i d e n c e of h y p e r d i p l o i d y (0% at both radiation doses), or chromatid-type changes (5.0% at 250 R and 4.0% at 400 R), w h e n c o m p a r e d to baseline values (series A). DISCUSSION W h i l e the deleterious effects of ionizing radiation on cells e x p o s e d directly have been long recognized, the initial observation that p l a s m a obtained from irradiated animals (and, subsequently, from i n d i v i d u a l s exposed to accidental or therapeutic
80
G.B. Faguet et el. 10
B
C
D
g vX
C
8 w @
1
(J
@ •~
/
/ /
"O
10
0 P
@
E e-
o ' '
~-7o o'
'
..7o ~'
'
~-~o ~'
'
~-7o
Observation time in days
Figure 4 Incidence of chromatid-type abnormalities (chromatid gaps/breaks, [~: isochromatid gaps/breaks, A; total, O} in nonirradiated cells unexposed (series A) and exposed to serum from irradiated rats (series B) compared with that seen in cells from irradiated rats preincubated (series D) or not (series C) with nonirradiated rat serum. Cells and sera were obtained at the times indicated following 400 R irradiation. irradiation} was clastogenic to nonirradiated cells {8-11), has drawn little attention as judged by the scarcity of reports generated (12-19). However, renewed interest in diffusable CF parallels the recognition of clastogenic activity in the plasma of patients with certain congenital {22-24) or acquired diseases (25-28) collectively known as "chromosomal breakage syndromes" and the suggestion that CF might play a significant role in the pathogenesis of certain diseases and their complications (27). Our data show that whole body irradiation of rats consistently induced the emergence of humoral factors that are clastogenic to nonirradiated cells. Induced cytogenetic abnormalities included chromatid and isochromatid gaps and breaks, all previously associated with CF, and hyperdiploidy, not previously recognized. While the incidence of the changes we detected is highly significant, the apparent influence of culture media on the activity of CF (29) suggests that our in vitro system may have underestimated the real potential of radiation-induced CF. The clastogenic activity of the plasma was detectable immediately after irradiation and persisted in circulation for the duration of the study, in agreement with published reports (16, 18). The failure of nonirradiated serum to abrogate the clastogenic activity of irradiated serum and the lack of clastogenic activity in rat serum irradiated in vitro suggests that the clastogenic effect of irradiated rat plasma does not result from radiation-induced depletion of protective factors normally found in plasma, nor does it reflect radiation-induced physical or chemical changes of normal plasma components, as suggested by two earlier studies (13, 14). Our data pro-
Clastogenic Plasma Factors
81
vides additional evidence in support of the existence of radiation-dependent CF and suggest their origin in cellular elements as a result of radiation-dependent effect, activation, or cell damage. Similar conclusions were r~ached by several investigators who viewed radiation-dependent or independent CF as products of normal (23) or cancerous cells (11). Further support for the cellular origin of the CF comes from the observation that cocultivation of lymphocytes from patients with lupus erythematosus (harboring CF) with normal lymphocytes increased the frequency of chromosomal abnormalities in normal cells (27). In addition, lymphocyte extracts from these patients also induce chromosomal breaks in normal lymphocytes exposed in vitro (27). The exact nature of CF is unknown. However, endogenous viruses, inhibitors of enzymatic systems responsible for DNA repair, or increased production of free radicals have all been implicated (30). It has been postulated that endogenous viruses can be activated by environmental factors, such as hormonal changes or irradiation, and become mutagenic (31). Indeed, most virus- or radiation-induced experimental tumors have been shown to contain radiation leukemia virus-specific cell surface antigens (32). Thus, activated retroviruses released into circulation might be responsible for the clastogenic activity of the plasma. However, this is most unlikely by virtue of the fact that the clastogenic activity of plasma from NZB-HB mice and from patients with lupus erythematosus, scleroderma and Bloom's syndrome, was found in the ultrafiltrate fraction containing substances with a molecular weight of less than 10,000 daltons (24, 25, 27, 30). The mechanism of action of CF appears to be mediated by free radicals. This is suggested by the demonstration of CF-induced increased production of O; by PHA-stimulated lymphocytes (27), the observation that CF, like photochemicallyor enzymatically-induced free radicals induce only single-strand DNA abnormalities (33), and the fact that free radical scavengers, such as superoxide dismutase, penicillamine, and cysteine, strongly reduce or abolish the activity of CF (24, 27, 28, 33). The clinical significance of CF may be far-reaching. They may play a significant role in the expression and severity of certain diseases and may be a risk factor in the development of late neoplasias resulting from therapeutic or accidental irradiation. The observation that superoxide dismutase administered to one patient with lupus erythematosus reduced both the incidence of chromosomal breaks and the titers of DNA antibodies in one study, lead the authors to suggest that certain symptoms in this disease could be secondary to DNA damage followed by release into circulation and subsequent antibody formation (27). Similarly, the clastogenic activity of plasma from patients with Crohn's disease, in some instances, has been shown to correlate with the extent and activity of intestinal lesions (28). Both manifestations of the disease were improved in three patients treated with penicillamine (28). It is conceivable that a common denominator to the NZB mice, certain patients with the chromosome breakage syndrome and individuals exposed to therapeutic or accidental irradiation with respect to their increased incidence of malignancy, might relate to the repetitive or sustained effects of their long-lived clastogenic plasma. While the correlations mentioned are speculative, the carcinogenicity of radiation-induced clastogenic plasma has been confirmed experimentally (8). In these experiments, nonirradiated rats infused with plasma from irradiated rats or with plasma ultrafiltrate from irradiated sheep developed a significantly increased incidence of tumors compared with control rats infused with nonirradiated rat plasma, nonirradiated sheep plasma ultrafiltrate, or normal saline. Elucidation of the origin, nature, and mechanism of action of CF is of considerable importance to radiation biology. Such knowledge should accrue the database needed to maximize utilization of irradiation as a diagnostic and therapeutic tool, while minimizing its
82
G, B, Faguet et al.
d e l e t e r i o u s effects. T h e d e m o n s t r a t i o n that C F - i n d u c e d c h r o m o s o m a l d a m a g e can be abrogated by certain substances, i n c l u d i n g r a d i o p r o t e c t o r s k n o w n to d e c r e a s e the i n c i d e n c e and s e v e r i t y of c o m p l i c a t i o n s of irradiation i n c l u d i n g d e a t h (34), suggests that the i m m e d i a t e and l o n g - t e r m effects of r a d i a t i o n - i n d u c e d CF m i g h t be p r e v e n t able. The authors thank Nancy Bailey and Gloria Logan for technical assistance and June Schulte and Mary Anne Jones for typing this manuscript.
REFERENCES 1. Arseneau JD, Sponzo RW, Levin DL, Schnipper LE, Bonner H, Young RC, Cannellos GP, Johnson RE, DeVita VT, Jr: (1972): Non-lymphomatous malignant tumors complicating Hodgkins's disease. N Engl J Med 287:1119-1122. 2. Storer JB (1975): A comprehensive treatise. Etiology: Chemical and Physical carcinogenesis, Vol. I. New York, Plenum Press. 3. Bloom AD, Neriishi S, Kamada N, Iseki T, Keehn RJ (1966): Cytogenetic investigation of survivors of the atomic bombings of Hiroshima and Nagasaki. Lancet Ih672-674. 4. Bender MA (1969}: Human radiation cytogenetics. In Advances in Radiation Biology, Vol. III. New York, Academic Press, pp. 215-275. 5. MacMahon B (1962): Prenatal x-ray exposure and childhood cancer. J Natl Cancer Inst 28:1173-1191. 6. Miller JA (1970): Carcinogenesis by chemicals: An overview. GHA Clowes Memorial Lecture. Cancer Res 30:559-576. 7. Jankovic-Stejin VD, Kanazir DT (1978): Late effects of X-irradiation in rats. I. Lasting chromosome abnormalities in marrow cells. Jap J Genet 53:227-240. 8. Souto J (1962): Tumor development in the rat induce by blood of irradiated animals. Nature 195:1317-1318. 9. Hollowell JG Jr, Littlefield LG (1967): Chromosome aberrations induced by plasma from irradiated patients. A brief report. J South Carolina Med Assoc 63:437-442. 10. Hollowell JG Jr, Littlefield LG (1968): Chromosome damage induced by plasma x-rayed patients. An indirect effect of x-ray. Proc Soc Exper Biol Med 129:240-244. 11. Littlefield LG, Hollowell JG Jr, Pool WH Jr, (1969): Chromosomal aberrations induced by plasma from irradiated patients: An indirect effect of X radiation. Radiol 93:878-886. 12. Goh KO, Sumner H (1968): Breaks in normal human chromosomes: Are they induced by a transferable substance in the plasma of persons exposed to total-body irradiation? Radiation Res 35:171-181. 13. Scott D (1969): The effect of irradiated plasma on normal human chromosomes and its relevance to the long-lived lymphocyte hypothesis. Cell Tissue Kinet 2:295-305. 14. Liniecki J, Bajerska A (1970): Irradiation of culture-medium and chromosomal aberrations in cultured non-irradiated lymphocytes. Bull L'Acad Pol Sci 18:293-296. 15. Goyanes Villa-Escusa V (1971): Chromosomal abnormalities in lymphocytes of children and baby rabbits born from mothers treated by X-irradiation before pregnancy. A transplacentary plasmatic chromosome damage factor? Blur 22:93-96. 16. Goh K (1975): Total-body irradiation and human chromosomes. V. Additional evidence of a transferable substance in the plasma of irradiated persons to induce chromosomal breakages. J Med 6:51-6o. 17. Kamada N, Oguma N (1977): Leukemogenic factors and chromosome aberrations. Jap J Clin Hematol 18:765-770. 18. Pant GS, Kamada N (1977): Chromosome aberrations in normal leukocytes induced by the plasma of exposed individuals. Hiroshima J Med Sci 26:149-154. 19. Demoise CF, Conard RA (1972): Effects of age and radiation exposure on chromosomes in a Marshall island population. J Gerontol 27:197-201. 20. Evans HJ, O'Riordan ML (1975): Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Murat Res 31:135-148.
Clastogenic P l a s m a Factors
83
21. Bruning L, Kintz BL (eds.) (1977): Computational Handbook of Statistics, 2nd ed. Glenview, IL, Scott, Foresman & Co. 22. Tice R, Windler G, Rary JM (1978): Effect of cocultivation on sister chromatid exchange frequencies in Bloom syndrome and normal fibroblast cells. Nature (London) 273:538540. 23. Shaham M, Becket Y, Cohen MM (1980): A diffusable clastogenic factor in ataxia telangiectasia. Cytogenet Cell Genet 27:155-161. 24. Emerit I, Cerutti P (1981): Clastogenic activity from Bloom syndrome fibroblast cultures. Proc Natl Acad Sci 78:1868-1872. 25. Emerit I, Levy A, Housset E (1973): Scl~rodermie g~n~ralis~e et cassures chromosomiques. Mise en 6vidence d'un facteur cassant dans le s6rum des malades. Ann Genet (Paris) 16:135-138. 26. Emerit [, Levy A, Housset E (1974): Breakage factor in systemic sclerosis and protector effect of L-cysteine. Hum Genet 25:221-226. 27. Emerit I, Michelson AN, Levy A, Camus JP, Emerit J (1980): Chromosome breakup agent of low molecular weight in human systemic lupus erythematosus. Hum Genet 55:341344. 28. Emerit I, Emerit J, Levy A, Keck M (1979): Chromosomal breakage in Crohn's disease: Anticlastogenic effect of D-pencillamine and L-cysteine. Hum Genet 50:51-57. 29. Keck M, Emerit I (1979): The influence of culture medium composition on the incidence of chromosomal breakage. Hum Genet 50:277-283. 30. Emerit I, Levy A, de Vaux Saint-Cyr C (1980): Chromosome damaging agent of low molecular weight in the serum of New Zealand black mice. Cytogent Cell Genet 26:41-48. 31. Kaplan HS (1967): On the natural history of the murine leukemias: Presidential address. Cancer Res 27:1325-1340. 32. Ferrer JF, Gibbs FA Jr (1969): Concomitant loss of specific cell-surface antigen and demonstrable type-C virus particles in lymphomas induced by radiation leukemia virus in rats. J Natl Cancer Inst 43:1317-1330. 33. Emerit I, Michelson AM (1979): Chromosomal breakage, free radicals and superoxide dismutase in collagen disease In: Proc Sixth Int Congr Radiat Res, Tokyo. 34. Gebhart E (1974]: Antimutagens. Data and problems. Humangenetik 24:1-32. 35. Welter DA, Henry S, Hodge LD (1982): Preparation of prenuclear structures from HeLa $3 cells for visualization. Sem J Cell Biol 95:307A.
ABSTRACT: Ionizing irradiation induces chromosomal aberrations in directly exposed cells and is known to have mutagenic and carcinogenic potential for the exposed host. Under controlled conditions, we examined whether such clastogenic effects of irradiation might be due in part to radiation-induced plasma factors. Irradiated cells and sera from CF-Nelson rats were used at 15 min, and 1, 7, 14, and 56-70 days after total body irradiation (250 R, n = 67 or 400 R, n = 39). Control rats (n = 44) served as donors of nonirradiated sera and cells. In addition, sara from six rats were irradiated (250 R or 400 R) in vitro. On the average, 298 metaphases from six rats were studied at each time-point. Cy~ogenetic abnormalities observed included chromatid- and chromosome-type lesions and hyperdiploidy. The frequency of abnormalities was comparable at both radiation doses. Nonirradiated cells exposed in vitro to irradiated serum (15 rain postirradiation) exhibited a 36- to 48-fold increment in hyperdiploidy (p = 0.0001) and a 2.- to 2.2-fold rise in chromatid gaps and breaks (p < 0.01), but none of the chromosome-type aberrations seen in cells exposed to radiation. The clastogenic activity of irradiated plasma persisted in circulation for the lO-wk duration of the study and was not abrogated by dilution with nonirradiated serum. Serum irradiated in vitro was not clastogenic. This study shows that irradiation of rats results in the prompt appearance of clastogenic activity in their plasma. This activity is not due to radiation-induced depletion of protective factors nor to chemical-physical changes of normal plasma components, but results from circulating factors released by irradiated cells. INTRODUCTION Chromosomal aberrations resulting from ionizing radiation are well documented. While irreparable chromosome damage results in cell death but has no long-term consequences to the exposed host, persistence of nonlethal alterations to the genome is of greater concern by virtue of its mutagenic and carcinogenic potential [15]. The exact m e c h a n i s m by which radiation and chemicals induce cancer is unknown. At present, carcinogenesis is viewed as the result of somatic mutations reflecting alterations in the genetic material of exposed cells. This hypothesis finds experimental support from data demonstrating b i n d i n g of chemical carcinogens to DNA [6], and from the associated high frequency of chromosomal aberrations and increased incidence of malignancies in animals [7] and in h u m a n s [1] exposed to
From the Departments of Medicine and Cellular and Molecular biology (G.B.F.), Department of Radiology (S.M.R.], and Department of Anatomy (D.A.W.), Medical College of Georgia; and the Medical Research Service (G.B.F.), VA Medical Center, Augusta, GA.
Address requests for reprints to Guy B. Faguet, M.D., Medical Research Service, VA Medical Center, Augusta, GA 309]0. Received May 6, 1983; accepted June 29, 1983.
73 © 1984 by Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 12, 73-83 (1984) 0165-4608/84/$03.00
74
G.B. Faguet et al. radiation. While a large body of evidence supports a direct effect of ionizing radiation on the chromosomes of exposed cells, preliminary data has shown that plasma from irradiated animals and from patients exposed to accidental or therapeutic radiation contain factors capable of inducing chromosomal aberrations in unexposed cells [6-19]. In this communication, we report that whole body irradiation of rats results in the prompt emergence of clastogenic factors (CF), which persist in circulation for at least 10 wk postirradiation. Our data show that the CF do not reflect radiation-induced depletion of protective factors nor radiation-induced physical or chemical changes of normal plasma components, but rather represent products secreted or excreted by cellular elements as a result of radiation effect, activation, or cell damage.
METHODS
Animals Young adult CF-Nelson female rats weighing 200-250 gm {Harlan-Sprague Dawley Co., Indianapolis, IN) were used in this study.
Cytogenetic Preparations Ether anesthetized rats were exsanguinated by aortic puncture, performed under direct visualization, using sterile techniques. Blood was drawn in 10-15 U/ml of preservative-free heparin (Connaught Laboratories, Toronto) immediately and on days 1, 7, 14, and 56-70 postirradiation. Nonirradiated blood was obtained at similar intervals from control rats. Twelve drops (approximately 0.6 ml) of whole blood was cultured in Gibco chromosome medium 1A (Grand Island Biological, Grand Island, NY) maintained at 38.5°C at a 30 ° angle and rotated every 12 hr. To maximize detection of unstable chromatid aberrations in first generation cells and to minimize chromosomal aberrations resulting from duplication of aberrations that were initially chromatid-type, culture time was limited to 40-42 hr [20]. Two hours prior to harvesting, mitoses were blocked in metaphase by exposure to colchicine (l~.g/ml culture medium) for 2 hr. After hypotonic shock (75 mM KC1) for 10 min, cells were fixed in a 3:1 ratio of absolute methanol:glacial acetic acid, spread on the glass slide, heat-dried, and stained with 1:50 Giemsa in Sorenson's buffer.
Cell-Serum Combinations Six cell-plasma (or serum) combinations were evaluated: Combination A, whole blood from nonirradiated animals; combination B, nonirradiated cells preincubated for 2 hr in serum from irradiated animals prior to addition to the culture medium; combination C, whole blood from irradiated animals; combination D, cells from irradiated animals preincubated in serum from nonirradiated animals; combination E, nonirradiated cells preincubated in a 1:1 ratio of irradiated:nonirradiated sera; and combination F, nonirradiated cells preincubated in serum irradiated in vitro. For cell-serum combination experiments B, D, E, and F, the cell and serum fractions were separated from whole blood by centrifugation at 300 x g for 15 min and reconstituted to generate the desired cell-serum combination, while preserving the original cell density. After incubating the cells in their new serum environment for 2 hr at 37°C, 0.6 ml of the reconstituted whole blood was processed as described herein for chromosome analysis.
Clastogenic Plasma Factors
75
Radiation Delivery Whole body irradiation was administered by h therapeutic x-ray unit operated at 200kVp and 15 mA with a filter of 0.5 mmCu and 1 mmAl at dose rates of 40-100 R/min. The half-value is equivalent to 0.9 mmCu. The dose delivered to the center of the body/area in R/min in air is measured by a Victoreen dosimeter. One hundred-six irradiated rats (250 R, n = 67 or 400 R, n = 39) were used as donors of irradiated cells and irradiated sera 15 min following irradiation and on postirradiation days 1, 7, 14, and 56-70. Nonirradiated animals (n = 44) donated nonirradiated sera and nonirradiated cells. In addition, sera from six rats were irradiated in vitro (250 R, n = 3, or 400 R, n = 3) using the same apparatus and technique described herein.
Data Collection and analysis Data at each time-point were derived from duplicate cultures from each of an average of six rats (range, 3-11). Triplicate slides were made from each culture and coded. An average of 298 metaphases per time-point (range, 35-800) were examined under the microscope by one of us (DAW) without previous knowledge of code or type of experiment. Chromatid-type damage recorded included chromatid and isochromatid gaps and breaks (Fig. 1). Chromosome-type aberrations observed included dicentrics, ring chromosomes, acentric fragments, and translocations. Because chromosome-type aberrations were not induced by exposing nonirradiated cells to irradiated plasma, however, these changes are tangential to our study and are not included in the analysis. Hyperdiploidy (>2n but <4n) noticed in initial cultures was subsequently monitored and recorded. Most gains were of one or a few chromosomes (Fig. 1). Abnormalities observed per slide were recorded and appropriately pooled to generate each time-point of each experimental series. Results are reported as a percentage of the corresponding total number of metaphases examined. Significance of difference between two proportions was determined by standard two-tailed tests [21]. Because the numbers of specific lesions are small, meaningful analysis were derived by comparing chromatid-type abnormalities. RESULTS
Assessment of Spontaneous Cytogenetic Aberrations in Nonirradiated Rat Cells As shown in Figures 2, 3, and 4, series A, hyperdiploidy and chromatid-type changes (single and isochromatid gaps and breaks) were seen, on the average, in 0.16% (range, 0%-0.52%) and 4.6% (range, 3.7%-4.9%), respectively, of cells from 44 nonirradiated control rats studied over a period of 10 wk. No chromosome-type aberrations were observed.
Assessment of Cytogenetic Aberrations in Cells from Irradiated Rats Because of the well known radiation-induced lymphocytopenia and the inhibitory effects of radiation on lymphocyte transformation and mitosis, experiments performed immediately following irradiation yielded a low number of analyzable metaphases. Experiments that yielded fewer than 10 analyzable metaphases per culture or 35 metaphases per time-point were not considered to be reliable and were excluded from data analysis. As shown in Figures 2, 3, and 4, when compared to nonirradiated blood (series A), blood cells from irradiated animals (series C, 400 R
76
G.B. Faguet et al.
A
• t
6
I Q
I
O V
c
4
Figure 1A Representative metaphases showing: (a) hyperdiploidy (43 chromosomes), (b) single chromatid break, and (c) single chromatid gap.
and 250 R) showed a marked increase in hyperdiploidy (p .< 0.0001) and chromatid-type lesions (p -< 0.01). In addition, chromosome-type aberrations (acentrics, dicentrics, rings, and translocations) were observed in these experiments, whereas they were not seen in nonirradiated cells. The radiation-induced increased incidence of hyperdiploidy and chromatid abnormalities seemed to abate 2-10 wk post-250 R irradiation, although hyperdiploidy remained significantly above base-
Clastogenic Plasma Factors
77
Figure IB Scanning electron micrograph (35) of the large submetacentric chromosome demonstrating a single chromatid gap. Marker bar represents 0.5 p.. line values (p ~ 0.0001). Whether this reflects gradual elimination of unstable types of aberrant chromosomes resulting from in vivo cell division of lymphocytes harboring abnormal chromosomes or whether it reflects experimental variation cannot be ascertained from our data. Assessment of the Clastogenic Effect of Irradiated Sera on Nonirradiated Cells Incubation of nonirradiated cells in sera from irradiated rats prior to culturing markedly increased the baseline frequency of hyperdiploidy (Fig. 2, series B) and chromatid-type lesions (figs. 3 and 4, series B) but did not induce chromosome-type aberrations. As shown, the clastogenic activity of irradiated sere on nonirradiated cells was demonstrable immediately postirradiation and persisted throughout the lO-wk study. The effect of sera from rats irradiated with either 250 R or 400 R on the frequency of hyperdiploidy of nonirradiated cells was highly significant (P ~ 0.0001) throughout the study-period (Fig. 2, series A versus B). This effect was comparable to that observed in irradiated cells (series B versus C, p : NS) at each time-point (except 14 days post-400 R irradiation, p ~ 0.001). The increased incidence of chromatid-type abnormalities induced by irradiated rat serum was less pronounced although highly significant (Fig. 3, series A versus B, p -~ 0.01) for all
78
G.B. Faguet et al. time-points except 1 and 56-70 days following 250 R irradiation (p = NS). The type and i n c i d e n c e of these changes were comparable to those seen in cells from irradiated animals at each time-point and for both radiation doses (Figs. 3 and 4, series B versus C, p = NS). Figures 3 and 4 suggest that the clastogenic effect of rat serum irradiated with 250 R d i m i n i s h e s with time, as e v i d e n c e d by its decreased capacity to i n d u c e h y p e r d i p l o i d y and c h r o m a t i d changes at 56-70 days postirradiation. Whether or not a similar reduction in the clastogenic activity of sera from animals postirradiated with 400 R occurs was not ascertained.
Assessment of the Clastogenic Plasma Factors The clastogenic activity in the p l a s m a of irradiated rats could be due to the generation of circulating clastogenic factors, d e p l e t i o n of c h r o m o s o m a l protective factors present in n o n i r r a d i a t e d plasma, or to r a d i a t i o n - i n d u c e d physical or chemical changes of normal p l a s m a components. If cytogenetic abnormalities found in cells from irradiated animals were due to d e p l e t i o n of protective serum factors, it w o u l d be expected that (a) reconstitution of the cell's normal serum e n v i r o n m e n t immediately following irradiation w o u l d preclude the d e v e l o p m e n t of cytogenetic abnormalities; (b) normal plasma should reconstitute " d e p l e t e d " (irradiated) p l a s m a and, thus, preclude its clastogenic activity; (c) depletion of putative c h r o m o s o m a l protective plasma factors by irradiation should be achievable by irradiation of plasma in vitro as well as in vivo. To evaluate these possibilities, the following studies were conducted: (a) cells from irradiated animals were p r e i n c u b a t e d in nonirradiated serum prior to culturing. As shown in Figs. 2, 3, and 4 (series D), nonirra-
Figure 2 Incidence of hyperdiploidy observed in nonirradiated cells unexposed (series A) and exposed to serum from irradiated rats [series B) compared with that seen in cells from radiated rats preincubated (series D) or not (series C) with nonirradiated rat serum. Cells and sera were obtained at the times indicated following irradiation (O = 250 R, A = 400 R).
A
B
C
0
10-
"0 °-0 ¢k
0
56-70 0
~'76
0 r '
~-76
Observation t i m e in days
0 ' r
G6-70
79
Clastogenic Plasma Factors 10] A
X
4 C I
0
ID
•~
C
p,,
o
B
1
O
l
't 10-
0 ¢
"O o~
e
J Observation time in days
Figure 3 Incidence of chromatid-type abnormalities (chromatid gaps/breaks, []; isochromatid gaps/breaks, A; total, 0) in nonirradiated cells unexposed (series A) and exposed to serum from irradiated rats (series B) compared with that seen in cells from irradiated rats preincubated (series D) or not (series C) with nonirradiated rat serum. Cells and sera were obtained at the times indicated following 250 R irradiation. diated rat serum had no protective effect on r a d i a t i o n - i n d u c e d cytogenetic abnormalities. The incidence of chromatid-type aberrations in irradiated cells obtained at various times postirradiation were not altered by a d d i t i o n of nonirradiated serum (series C versus D, p -- NS). The same is true for the incidence of h y p e r d i p l o i d y , except for cells obtained 14 days post-400 R irradiation (p < 0.01); (b) cells from nonirradiated rats were incubated with a 1:1 mixture of nonirradiated and irradiated rat sera for 2 hr prior to culturing. The incidence of h y p e r d i p l o i d y (5.9%) and c h r o m a t i d - t y p e abnormalities C8.7%) observed in these experiments (not shown) were comparable (p = NS) to that i n d u c e d by u n d i l u t e d irradiated serum on nonirradiated cells (Figs. 3 and 4, series B); (c) finally, cells from n o n i r r a d i a t e d animals were p r e i n c u b a t e d for 2 hr in rat serum irradiated in vitro (250 R or 400 R) prior to culturing. Irradiation of serum in vitro failed to generate clastogenic activity. Indeed, p r e i n c u b a t i o n of n o n i r r a d i a t e d cells in serum irradiated in vitro had no effect (p -- NS) on the i n c i d e n c e of h y p e r d i p l o i d y (0% at both radiation doses), or chromatid-type changes (5.0% at 250 R and 4.0% at 400 R), w h e n c o m p a r e d to baseline values (series A). DISCUSSION W h i l e the deleterious effects of ionizing radiation on cells e x p o s e d directly have been long recognized, the initial observation that p l a s m a obtained from irradiated animals (and, subsequently, from i n d i v i d u a l s exposed to accidental or therapeutic
80
G.B. Faguet et el. 10
B
C
D
g vX
C
8 w @
1
(J
@ •~
/
/ /
"O
10
0 P
@
E e-
o ' '
~-7o o'
'
..7o ~'
'
~-~o ~'
'
~-7o
Observation time in days
Figure 4 Incidence of chromatid-type abnormalities (chromatid gaps/breaks, [~: isochromatid gaps/breaks, A; total, O} in nonirradiated cells unexposed (series A) and exposed to serum from irradiated rats (series B) compared with that seen in cells from irradiated rats preincubated (series D) or not (series C) with nonirradiated rat serum. Cells and sera were obtained at the times indicated following 400 R irradiation. irradiation} was clastogenic to nonirradiated cells {8-11), has drawn little attention as judged by the scarcity of reports generated (12-19). However, renewed interest in diffusable CF parallels the recognition of clastogenic activity in the plasma of patients with certain congenital {22-24) or acquired diseases (25-28) collectively known as "chromosomal breakage syndromes" and the suggestion that CF might play a significant role in the pathogenesis of certain diseases and their complications (27). Our data show that whole body irradiation of rats consistently induced the emergence of humoral factors that are clastogenic to nonirradiated cells. Induced cytogenetic abnormalities included chromatid and isochromatid gaps and breaks, all previously associated with CF, and hyperdiploidy, not previously recognized. While the incidence of the changes we detected is highly significant, the apparent influence of culture media on the activity of CF (29) suggests that our in vitro system may have underestimated the real potential of radiation-induced CF. The clastogenic activity of the plasma was detectable immediately after irradiation and persisted in circulation for the duration of the study, in agreement with published reports (16, 18). The failure of nonirradiated serum to abrogate the clastogenic activity of irradiated serum and the lack of clastogenic activity in rat serum irradiated in vitro suggests that the clastogenic effect of irradiated rat plasma does not result from radiation-induced depletion of protective factors normally found in plasma, nor does it reflect radiation-induced physical or chemical changes of normal plasma components, as suggested by two earlier studies (13, 14). Our data pro-
Clastogenic Plasma Factors
81
vides additional evidence in support of the existence of radiation-dependent CF and suggest their origin in cellular elements as a result of radiation-dependent effect, activation, or cell damage. Similar conclusions were r~ached by several investigators who viewed radiation-dependent or independent CF as products of normal (23) or cancerous cells (11). Further support for the cellular origin of the CF comes from the observation that cocultivation of lymphocytes from patients with lupus erythematosus (harboring CF) with normal lymphocytes increased the frequency of chromosomal abnormalities in normal cells (27). In addition, lymphocyte extracts from these patients also induce chromosomal breaks in normal lymphocytes exposed in vitro (27). The exact nature of CF is unknown. However, endogenous viruses, inhibitors of enzymatic systems responsible for DNA repair, or increased production of free radicals have all been implicated (30). It has been postulated that endogenous viruses can be activated by environmental factors, such as hormonal changes or irradiation, and become mutagenic (31). Indeed, most virus- or radiation-induced experimental tumors have been shown to contain radiation leukemia virus-specific cell surface antigens (32). Thus, activated retroviruses released into circulation might be responsible for the clastogenic activity of the plasma. However, this is most unlikely by virtue of the fact that the clastogenic activity of plasma from NZB-HB mice and from patients with lupus erythematosus, scleroderma and Bloom's syndrome, was found in the ultrafiltrate fraction containing substances with a molecular weight of less than 10,000 daltons (24, 25, 27, 30). The mechanism of action of CF appears to be mediated by free radicals. This is suggested by the demonstration of CF-induced increased production of O; by PHA-stimulated lymphocytes (27), the observation that CF, like photochemicallyor enzymatically-induced free radicals induce only single-strand DNA abnormalities (33), and the fact that free radical scavengers, such as superoxide dismutase, penicillamine, and cysteine, strongly reduce or abolish the activity of CF (24, 27, 28, 33). The clinical significance of CF may be far-reaching. They may play a significant role in the expression and severity of certain diseases and may be a risk factor in the development of late neoplasias resulting from therapeutic or accidental irradiation. The observation that superoxide dismutase administered to one patient with lupus erythematosus reduced both the incidence of chromosomal breaks and the titers of DNA antibodies in one study, lead the authors to suggest that certain symptoms in this disease could be secondary to DNA damage followed by release into circulation and subsequent antibody formation (27). Similarly, the clastogenic activity of plasma from patients with Crohn's disease, in some instances, has been shown to correlate with the extent and activity of intestinal lesions (28). Both manifestations of the disease were improved in three patients treated with penicillamine (28). It is conceivable that a common denominator to the NZB mice, certain patients with the chromosome breakage syndrome and individuals exposed to therapeutic or accidental irradiation with respect to their increased incidence of malignancy, might relate to the repetitive or sustained effects of their long-lived clastogenic plasma. While the correlations mentioned are speculative, the carcinogenicity of radiation-induced clastogenic plasma has been confirmed experimentally (8). In these experiments, nonirradiated rats infused with plasma from irradiated rats or with plasma ultrafiltrate from irradiated sheep developed a significantly increased incidence of tumors compared with control rats infused with nonirradiated rat plasma, nonirradiated sheep plasma ultrafiltrate, or normal saline. Elucidation of the origin, nature, and mechanism of action of CF is of considerable importance to radiation biology. Such knowledge should accrue the database needed to maximize utilization of irradiation as a diagnostic and therapeutic tool, while minimizing its
82
G, B, Faguet et al.
d e l e t e r i o u s effects. T h e d e m o n s t r a t i o n that C F - i n d u c e d c h r o m o s o m a l d a m a g e can be abrogated by certain substances, i n c l u d i n g r a d i o p r o t e c t o r s k n o w n to d e c r e a s e the i n c i d e n c e and s e v e r i t y of c o m p l i c a t i o n s of irradiation i n c l u d i n g d e a t h (34), suggests that the i m m e d i a t e and l o n g - t e r m effects of r a d i a t i o n - i n d u c e d CF m i g h t be p r e v e n t able. The authors thank Nancy Bailey and Gloria Logan for technical assistance and June Schulte and Mary Anne Jones for typing this manuscript.
REFERENCES 1. Arseneau JD, Sponzo RW, Levin DL, Schnipper LE, Bonner H, Young RC, Cannellos GP, Johnson RE, DeVita VT, Jr: (1972): Non-lymphomatous malignant tumors complicating Hodgkins's disease. N Engl J Med 287:1119-1122. 2. Storer JB (1975): A comprehensive treatise. Etiology: Chemical and Physical carcinogenesis, Vol. I. New York, Plenum Press. 3. Bloom AD, Neriishi S, Kamada N, Iseki T, Keehn RJ (1966): Cytogenetic investigation of survivors of the atomic bombings of Hiroshima and Nagasaki. Lancet Ih672-674. 4. Bender MA (1969}: Human radiation cytogenetics. In Advances in Radiation Biology, Vol. III. New York, Academic Press, pp. 215-275. 5. MacMahon B (1962): Prenatal x-ray exposure and childhood cancer. J Natl Cancer Inst 28:1173-1191. 6. Miller JA (1970): Carcinogenesis by chemicals: An overview. GHA Clowes Memorial Lecture. Cancer Res 30:559-576. 7. Jankovic-Stejin VD, Kanazir DT (1978): Late effects of X-irradiation in rats. I. Lasting chromosome abnormalities in marrow cells. Jap J Genet 53:227-240. 8. Souto J (1962): Tumor development in the rat induce by blood of irradiated animals. Nature 195:1317-1318. 9. Hollowell JG Jr, Littlefield LG (1967): Chromosome aberrations induced by plasma from irradiated patients. A brief report. J South Carolina Med Assoc 63:437-442. 10. Hollowell JG Jr, Littlefield LG (1968): Chromosome damage induced by plasma x-rayed patients. An indirect effect of x-ray. Proc Soc Exper Biol Med 129:240-244. 11. Littlefield LG, Hollowell JG Jr, Pool WH Jr, (1969): Chromosomal aberrations induced by plasma from irradiated patients: An indirect effect of X radiation. Radiol 93:878-886. 12. Goh KO, Sumner H (1968): Breaks in normal human chromosomes: Are they induced by a transferable substance in the plasma of persons exposed to total-body irradiation? Radiation Res 35:171-181. 13. Scott D (1969): The effect of irradiated plasma on normal human chromosomes and its relevance to the long-lived lymphocyte hypothesis. Cell Tissue Kinet 2:295-305. 14. Liniecki J, Bajerska A (1970): Irradiation of culture-medium and chromosomal aberrations in cultured non-irradiated lymphocytes. Bull L'Acad Pol Sci 18:293-296. 15. Goyanes Villa-Escusa V (1971): Chromosomal abnormalities in lymphocytes of children and baby rabbits born from mothers treated by X-irradiation before pregnancy. A transplacentary plasmatic chromosome damage factor? Blur 22:93-96. 16. Goh K (1975): Total-body irradiation and human chromosomes. V. Additional evidence of a transferable substance in the plasma of irradiated persons to induce chromosomal breakages. J Med 6:51-6o. 17. Kamada N, Oguma N (1977): Leukemogenic factors and chromosome aberrations. Jap J Clin Hematol 18:765-770. 18. Pant GS, Kamada N (1977): Chromosome aberrations in normal leukocytes induced by the plasma of exposed individuals. Hiroshima J Med Sci 26:149-154. 19. Demoise CF, Conard RA (1972): Effects of age and radiation exposure on chromosomes in a Marshall island population. J Gerontol 27:197-201. 20. Evans HJ, O'Riordan ML (1975): Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Murat Res 31:135-148.
Clastogenic P l a s m a Factors
83
21. Bruning L, Kintz BL (eds.) (1977): Computational Handbook of Statistics, 2nd ed. Glenview, IL, Scott, Foresman & Co. 22. Tice R, Windler G, Rary JM (1978): Effect of cocultivation on sister chromatid exchange frequencies in Bloom syndrome and normal fibroblast cells. Nature (London) 273:538540. 23. Shaham M, Becket Y, Cohen MM (1980): A diffusable clastogenic factor in ataxia telangiectasia. Cytogenet Cell Genet 27:155-161. 24. Emerit I, Cerutti P (1981): Clastogenic activity from Bloom syndrome fibroblast cultures. Proc Natl Acad Sci 78:1868-1872. 25. Emerit I, Levy A, Housset E (1973): Scl~rodermie g~n~ralis~e et cassures chromosomiques. Mise en 6vidence d'un facteur cassant dans le s6rum des malades. Ann Genet (Paris) 16:135-138. 26. Emerit [, Levy A, Housset E (1974): Breakage factor in systemic sclerosis and protector effect of L-cysteine. Hum Genet 25:221-226. 27. Emerit I, Michelson AN, Levy A, Camus JP, Emerit J (1980): Chromosome breakup agent of low molecular weight in human systemic lupus erythematosus. Hum Genet 55:341344. 28. Emerit I, Emerit J, Levy A, Keck M (1979): Chromosomal breakage in Crohn's disease: Anticlastogenic effect of D-pencillamine and L-cysteine. Hum Genet 50:51-57. 29. Keck M, Emerit I (1979): The influence of culture medium composition on the incidence of chromosomal breakage. Hum Genet 50:277-283. 30. Emerit I, Levy A, de Vaux Saint-Cyr C (1980): Chromosome damaging agent of low molecular weight in the serum of New Zealand black mice. Cytogent Cell Genet 26:41-48. 31. Kaplan HS (1967): On the natural history of the murine leukemias: Presidential address. Cancer Res 27:1325-1340. 32. Ferrer JF, Gibbs FA Jr (1969): Concomitant loss of specific cell-surface antigen and demonstrable type-C virus particles in lymphomas induced by radiation leukemia virus in rats. J Natl Cancer Inst 43:1317-1330. 33. Emerit I, Michelson AM (1979): Chromosomal breakage, free radicals and superoxide dismutase in collagen disease In: Proc Sixth Int Congr Radiat Res, Tokyo. 34. Gebhart E (1974]: Antimutagens. Data and problems. Humangenetik 24:1-32. 35. Welter DA, Henry S, Hodge LD (1982): Preparation of prenuclear structures from HeLa $3 cells for visualization. Sem J Cell Biol 95:307A.