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Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium
Mutation Research, 244 (1990) 209-214
209
Elsevier MUTLET 0364
Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium Qiyue Hu and Shoupeng Zhu Department of Radiotoxicology, Suzhou Medical College, Suzhou, Jiangsu 215007 (China) (Accepted 25 January 1990)
Keywords: Uranyl fluoride; Chromosomal aberration; Mouse germ cells
Summary Cytogenetic damage induced by a wide range of concentrations of uranyl fluoride injected into mouse testes was evaluated by determining the frequencies of chromosomal aberrations in spermatogonia and primary spermatocytes. Breaks, gaps and polyploids were observed in spermatogonia. The frequencies of the significant type of aberration, breaks, were induced according to the injected doses of uranyl fluoride. Primary spermatocytes were examined for fragments, univalents and multivalents. The multivalents observed in this study resulted either from chromatid interchanges or from reciprocal translocations. The reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes. For primary spermatocytes the incidence of aberrant ceils largely depended on the administered dose. Sampling time after treatment could affect the frequencies of chromosomal aberrations in male mouse germ cells.
Enriched uranium is one of the main nuclear fuels for nuclear power stations. Uranium hexafluoride is used to enrich uranium and easily forms uranyl fluoride when it leaks out into ambient air and meets with moisture. Once formed, uranyl fluoride is most likely to enter the worker's body on the spot. So far many studies have been done on uranium Correspondence: Dr. Qiyue Hu, Department of Radiotoxicology, Suzhou Medical College, Suzhou, Jiangsu 215007 (China).
toxicity (Boback, 1975; Durbin and Wrenn, 1975; Hursh and Spoor, 1973; Morrow et al., 1972; Sullivan, 1980). In blood uranium can combine with organic and inorganic acids and proteins to form different compounds. Some of the compounds are 'diffusible', that is, they can reach every part of the body via the circulation although they largely deposit in kidneys, skeleton, liver or lungs depending on the way they enter the body and their chemical valence (Morrow et al., 1982; Ballou et al., 1986). Of all uranyl compounds uranyl fluoride was found to be the most toxic in
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
210
acute lethality studies (Yuile, 1973). But there is little information on its genetic toxicity. In order to study the genetic effects of a trace amount of uranyl fluoride in male mouse germ cells without evident damage to other tissues, intratesticular (i.t.) injection was adopted for administration. It had been used to study factors influencing the mutagenicity of ethyl methanesulfonate in mice by Soares (1976) and to inject [3H]deoxythymidine into the mouse testis for labeling by Sega and Sotomayor (1982). We now report the results of an investigation of the effects of a trace amount of uranyl fluoride in testes on the induction of chromosomal aberrations in spermatogonia and primary spermatocytes o f male B A L B / c mice. Materials and methods
Uranyl fluoride containing enriched uranium, in which the uranium-235 isotope component is 18.9070, was obtained from a nuclear plant (China). Sexually mature male B A L B / c mice, 10-12 weeks old and weighing 23-27 g, were purchased from the Center of Experimental Animals of our college. They were acclimatized to our laboratory for 7 days after arrival and were housed in groups o f 5 in polypropylene cages with hardwood-chip bedding. Water and rodent chow were provided ad libitum throughout the period of animal holding and experimentation. The time from treatment to chromosome preparation was fixed at 1, 13, 36 and 60 days, respectively, to investigate the induction of chromosomal aberrations by uranyl fluoride at different stages of germ-cell development. There were 5 experimental groups, treated with single i.t. injections of different doses of uranyl fluoride ranging from 0.05 to 1.00 ~g/testis. The i.t. injection procedure was as described by Sega and Sotomayor (1982). A control group was treated with isotonic saline in the same way for each fixtation time. Each group included 5 males. The volume of uranyl fluoride solution or isotonic saline injected into each testis was 5 /zl. Colchicine, 4 m g / k g body weight, was injected intraperitoneally 6 h before
the mice were killed. Chromosome specimens were prepared from the testes according to the technique of Evans et al. (1964) with minor modifications, and stained with Giemsa in phosphate buffer (pH 6.8). 100 well-spread metaphase spermatogonia and, where possible, about the same number of primary spermatocytes per animal were analyzed under the light microscope. Gaps were defined as achromatic lesions smaller than the width of the chromatid and without dislocation of the distal segment. Breaks were scored when the achromatic region was accompanied by displacement of the distal segment. The form of multivalents in meiosis I depends on the number of chromosomes involved in pairing, the number and location of chiasmata, and the degree o f chiasma terminalization. In this study multivalents observed at metaphase I were the result of either a chromatid interchange induced in diplotene or preleptotene of meiosis or a reciprocal chromosome-type exchange induced in spermatogonia. The chromatid interchanges were mostly irregular and non-symmetrical configurations. The chromosome-type exchanges were reciprocal translocations in that they were symmetrical configurations. The variance test was calculated to check whether variance of the data was homogeneous. When heterogeneity of variance was found arcsine transformation was carried out before Dunnett's ttest was used to determine statistically significant differences between treatment and control groups. Results
The changes in chromosomal structure and number of spermatogonia obtained 1, 13, 36 and 60 days after treatment with uranyl fluoride are presented in Table 1. Uranyl fluoride did not significantly induce gaps although there was a slight increase, compared to the control, in gap number in treatment groups, especially at the 1-day fixation time. Also the number of polyploids in treated groups was higher than that of controls but this effect was not clearly dose-dependent and not statistically different from the control groups ex-
211 TABLE 1 CHANGES IN CHROMOSOMAL STRUCTURE AND NUMBER INDUCED IN SPERMATOGONIA BY URANYL FLUORIDE a Sampling Dose Gaps time after ~g/testis) (%) exposure (days)
Breaks (%)
Polyploids (%)
0.05 0.10 0.25 0.50 1.00 control
0.2±0.2 0.2±0.2 0.6±0.4 0.4±0.2 0.8±0.4 0.2±0.2
0.6+0.4 0.4±0.4 1.2 ±0.7 1.8±0.7" 2.2±0.6** 0.0±0.0
25.2+ 1.0 22.6±2.0 21.8± 1.5 24.8± 1.6 28.0±3.2 19.0±2.8
13
0.05 0.10 0.25 0.50 1.00 control
0.0±0.0 0.0±0.0 0.2±0.2 0.4±0.2 0.2±0.2 0.0±0.0
0.2±0.2 0.6±0.4 0.4±0.2 1.2±0.4" 1.2±0.5" 0.0±0.0
15.2± 1.4 15.6±0.5 16.8±2.1 24.8± 1.6"* 21.6±1.1"* 13.4±2.0
36
0.05 0.10 0.25 0.50 1.00 control
0.0±0.0 0.0± 0.0 0.4±0.2 0.2±0.2 0.2±0.2 0.0±0.0
0.4±0.4 0.2±0.2 0.2±0.2 1.0±0.4" 1.4±0.2"* 0.0± 0.0
14.6±2.0 16.8± 1.5 15.8± 1.6 15.2±1.9 17.4±2.2 13.8± 1.5
60
0.05 0.10 0.25 0.50 1.00 control
0.0+0.0 0.0±0.0 0.0+0.0 0.0±0.0 0.2+0.2 0.0± 0.0
0.2±0.2 0.2+0.2 0.6+0.4 0.6+0.2 0.8±0.2 0.2±0.2
14.7+2.0 16.0+0.8 17.4±2.5 1%6±0.6 16.8±1.6 14.6+_ 1.7
a Values are means + SE for 5 mice, 100 cells per mouse. *P<0.05, **P<0.01 compared to control.
cept for 1.00 and 0.50/zg/testis at the 13-day fixation. The significant difference between the 1.00 or 0.50 #g/testis dose group and the control was due to the relatively low concurrent control. Breaks were found to increase with increasing doses at 4 fixation times after uranyl fluoride injection and their frequency was statistically significant in highdose groups at 1, 13 and 36 days. The frequencies o f breaks are more meaningful in cytogenetic evaluation than the other criteria listed in Table 1 since either gaps or polyploids are no good in-
dicators of chromosome damage according to the general opinion (Ito et al., 1988; Kill±an et al., 1977; Watson et al., 1976). In treated groups more breaks were found at the 1-day fixation than at other fixation times. There is no other explanation but prolonged exposure to uranium deposited in the testes, for the significant increase in break yields at the 13- and 36-day fixation times. Table 2 summarizes the detailed results of chromosome aberrations in primary spermatocytes induced by uranyl fluoride. Uranyl fluoride induced fragments, univalents and multivalents. The XY or autosome univalent frequency did not significantly increase with dose. The frequency of fragments only increased at the 1-day fixation. And it was noted that all multivalents observed in this study were 'quadrivalents'. They were presented in the form o f either a ring or a chain. The frequency of chromatid interchanges significantly increased at the 13-day fixation. The reason for this may be that cells in preleptotene at the time of treatment were the most likely ones to show aberrations according to Preston et al. (1981). No significantly increased production of reciprocal translocations was observed compared to the control. In Table 2 cells with univalents were not included in the category of aberrant cells because rather high frequencies of univalents were found in control groups. Discussion
The aim o f the present study was to determine the clastogenic effects o f a trace amount of uranyl fluoride in mouse testes on chromosomes of germ cells. We chose the i.t. route to administer uranyl fluoride. In pilot experiments we had found that other ways, such as intraperitoneal and intravenous injections, resulted in damage to the kidneys and the liver of the animals and thus influenced the animals' survival for the experimental period of 2 months. In evaluating genetic damage in male germ cells by any treatment it is necessary to recognize that in the seminiferous tubules of the testis there is a wide range o f germ-cell types. Different germ-cell types
212 TABLE 2 C H R O M O S O M A L A B E R R A T I O N S I N D U C E D IN P R I M A R Y S P E R M A T O C Y T E S BY U R A N Y L F L U O R I D E a
Sampling
Dose ~ g /
Number of
Fragments
Univalents (%)
Multivalents (%)b
time after exposure (days)
testis)
cells analyzed
(%)
XY
Autosomes
c.i.
r.t.
Incidence c of aberrant cells (o70)
0.05 0.10 0.25 0.50 1.00 control
491 489 5~ 5~ 5~ 5~
0.8±0.4 0.6±0.3 0.6±0.4 1.4±0.2"* 1.6±0.2"* 0.0±0.0
8.8±1.4 7.8±1.2 8.8±0.8 8.8±1.3 9.8±1.5 4.6±0.9
4.6±1.2 4.3±0.8 4.0±1.0 4.8±1.0 6.6±0.8 4.2±1.0
0.0±0.0 0.0±0.0 0.0±0.0 0.2±0.2 0.0±0.0 0.0±0.0
0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.8±0.4 0.6±0.3 0.6±0.4 1.6±0.2"* 1.6±0.2"* 0.0±0.0
13
0.05 0.10 0.25 0.50 1.~ control
470 5~ 5~ 488 5~ 5~
0.6±0.4 0.4±0.2 0.4±0.2 0.8±0.4 0.8±0.2 0.0±0.0
7.0±1.0 6.4±1.0 7.0±1.3 8.8±1.4 10.0±0.6 5.4±0.5
5.6±0.9 4.4±0.9 4.2±0.8 5.6±1.0 7.8±1.4 4.4±1.0
0.0±0.0 0.2±0.2 0.6±0.5* 0.8±0.4** 1.2±0.4"* 0.0±0.0
0.0±0.0 0.0±0.0 0.0+0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.6±0.4 0.6±0.2 1.0±0.3" 1.4±0.2"* 2.0±0.3** 0.0±0.0
36
0.05 0.10 0.25 0.50 1.~ control
5~ 5~ 5~ 489 497 5~
0.0±0.0 0.4±0.4 0.4±0.2 0.4±0.2 0.8±0.4 0.2±0.2
5.8±1.4 6.8±0.5 8.4±0.5 8.0±0.9 8.0±1.1 5.6±0.7
5.0±1.1 4.2±0.8 4.2±0.6 5.9±0.3 4.8±0.6 3.8±0.6
0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.2±0.2 0.4±0.2 0.2±0.2 0.2±0.2 0.6±0.5 0.0±0.0
0.2±0.2 0.8±0.5 0.6±0.4 0.6±0.4 1.4±0.2 0.2±0.2
60
0.05 0.10 0.25 0.50 1.~ control
497 5~ 481 5~ 492 486
0.2±0.2 0.2±0.2 0.0±0.0 0.2±0.2 0.4±0.4 0.2±0.2
5.2±0.7 6.4±1.4 8.4±1.7 6.8±1.5 8.7±1.1 4.9±0.8
4.2±0.7 4.0±0.5 4.0±0.9 5.0±1.0 5.3±0.8 4.0±1.0
0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.2±0.2 0.2±0.2 0.2±0.2 0.2±0.2 0.6±0.9 0.0±0.0
0.4±0.2 0.4±0.2 0.2±0.2 0.4±0.2 0.6±0.2 0.2±0.2
a Values are means ± SE for 5 mice. b c.i., chromatid interchanges; r.t., reciprocal translocations. c Not including the cells with univalents. * P < 0 . 0 5 , * * P < 0 . 0 1 compared to control.
are differently sensitive to mutagens. The spermatogonial stem cell population is complex (Cattanach and Rasberry, 1987). It contains cells with different sensitivities to genetic damage. The spermatogonial cell renewal process has been described by Searle (1974). Results of the experiment (Table 1) show a general tendency for break production to increase with the dose of uranyl fluoride. At high dose levels the statistically significant difference of break frequencies between treated and control groups disappeared 60 days after treatment although more
breaks were found in treated groups than in the control group. This indicates that the effect of uranyl fluoride on chromosome aberration induction lasted at least 36 days after a single injection into the testis, which might result from uranium activity being incorporated in the testes. Unfortunately in this paper we could not provide exact information about its deposition and retention in the mouse testes. Data in Table 1 also indicate that spermatogonia were sensitive to a trace amount of uranyl fluoride. Spermatogonia are important target cells and an elevation of the aberrations in-
213
duced in spermatogonia is important for the estimation of genetic risk since spermatogonia will later give rise to mature germ cells. But many spermatogonia, carrying more than one aberration after treatment, may be eliminated before reaching the spermatocyte stage (Tates and Natarajan, 1976). For that reason it is genetically meaningful that the reciprocal translocations induced in spermatogonia are recorded in spermatocytes. The data in Table 2 show that on all days of sampling multivalents were found in treated groups at high dose levels but it should be made clear that multivalents observed on day 1 or 13 cannot be reciprocal translocations induced in spermatogonia but are chromatid interchanges induced in diplotene or preleptotene of meiosis. Their shape is different from reciprocal translocation multivalents although sometimes similar to them. A symmetrical chromatid interchange in a spermatocyte will result in 6/16 sperm with duplications/deficiencies, 9/16 normal sperm and 1/16 sperm with a balanced reciprocal translocation (Brewen et al., 1976). An asymmetrical interchange will lead to deletions in 50°70 of the sperm. Multivalents observed on day 36 or 60 are reciprocal translocations. These translocations could persist over mitotic cell divisions, usually just as well as the normal chromosome in the complement (Bender et al., 1988). By meiotic segregation a spermatocyte with a reciprocal translocation will give rise to 50% sperm with a duplication/deficiency, 25°7o normal sperm and 25070 sperm with a balanced reciprocal translocation. All chromosomally imbalanced sperm will cause embryonic death. The most common type of abnormality observed in spermatocytes was the presence of univalents. This may be the result o f precocious separation of the chromosomes from the bivalents (Tates and Natarajan, 1976). It should be noticed that X and Y were more often separated than autosomes. However, this observation was not only made in treated groups but also in controls. With regard to the incidence of aberrant cells, it appears that only the high doses, within 13 days of treatment, produced significantly more aberrant cells than con-
trois although a higher incidence o f aberrant cells was found in nearly all treated groups compared to the controls in this study. At the 36-day fixation the fact that there is no significant difference in incidence of aberrant cells between the 1.00 #g/testis dose group and the control is due to the relatively high concurrent control. From the results of the present study, it can be concluded that uranyl fluoride, which has both chemical toxicity and radiotoxicity, in the testes induced chromosome aberrations in spermatogonia and spermatocytes, yet it is not clear whether the aberrations were produced mainly because of chemical toxicity or radiotoxicity. This remains to be studied further. References Ballou, J.E., R.A. Gies, A.C. Case, D.L. Haggard, R.L. Buschbom and J.L. Ryan (1986) Deposition and early disposition of inhaled 233UO2(NO3)2 and 232UOE(NO3)2 in the rat, Health Phys., 51,755-771. Bender, M.A, A.A. Awa, A.L. Brooks, H.J. Evans, P.G. Groer, L.G. Littlefield, C. Pereira, R.J. Preston and B.W. Wacholz (1988) Current status of cytogenetic procedures to detect and quantify previous exposures to radiation, Mutation Res., 196, 103-159. Boback, M.W. (1975) A review of uranium excretion and clinical urinalysis data in accidental exposure cases, in: Report ERDA 93, Conference on Occupational Health Experience with Uranium, 28-30 April, pp. 226-231. Brewen, J.G., H.S. Payne and R.J. Preston (1976) X-rayinduced chromosome aberrations in mouse dictyate oocytes. I. Time and dose relationships, Mutation Res., 35, 111-120. Cattanach, B.M., and C. Rasberry (1987) Genetic effects of combined chemical-X-ray treatments in male mouse germ cells, Int. J. Radiat. Biol., 51,985-996. Durbin, P.W., and M.E. Wrenn (1975) Metabolism and effects of uranium in animals, in: Report ERDA 93, Conference on Occupational Health Experience with Uranium, 28-30 April, pp. 68-129. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3,289-294. Hursh, J.B., and N.L. Spoor (1973) Data on man, in: H.C. Hodge, J.N. Stannard and J.B. Hursh (Eds.), Uranium, Plutonium, Transplutonic Elements, Springer-Verlag, New York, pp. 197-239. Ito, Y., N. Ueda, S. Murao, T. Sugiyama, H. Lee and R.G. Harvey (1988) Induction of chromosomal aberrations in rat bone marrow cells and mutations in Salmonella typhimurium
214 by benz[a|anthracene derivatives, Mutation Res., 206, 55-63. Killian, J.D., F.E. Moreland, M.C. Benge, M.S. Legator and E.B. Whorton Jr. (1977) in: B.J. Kilbey, W.W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 243-260. Morrow, P.E., F.R. Gibb and H.D. Beiter (1972) Inhalation studies of uranium trioxide, Health Phys., 23,273-280. Morrow, P.E., R. Gelein, H. Beiter, J. Scott, J. Picano and C. Yuile (1982) Inhalation and intravenous studies of UFjUO2F2 in dogs, Health Phys., 43, 859-873. Preston, R.J., W. Au, M.A Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. Wolff and J.S. Wassom (1981) Mammalian in vivo and vitro cytogenetic assays: a report of the U.S. EPA's Gene-Tox Program, Mutation Res., 87, 143-188. Searle, A.G. (1974) Mutation induction in mice, Adv. Radiat. Biol., 4, 131-207. Sega, G.A., and R.E. Sotomayor (1982) Unscheduled DNA synthesis in mammalian germ cells - its potential use in mutagenicity testing, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. 7, Plenum Press, New York, pp. 421-445.
Soares, E.R. (1976) Studies of factors influencing the mutagenicity of EMS in mice, Mutation Res., 37, 245-252. Sullivan, M.F. (1980) Absorption of actinide elements from the gastrointestinal tract of rats, guinea pigs and dogs, Health Phys., 88, 159-171. Tates, A.B., and A.T. Natarajan (1976) A correlative study on the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice, Mutation Res., 37, 267-278. Watson, W.A.F., J.C. Petrie, D.B. Galloway, I. Bullock and J.C. Gilbert (1976) In vivo cytogenetic activity of sulphonylurea drugs in man, Mutation Res., 38, 71-80. Yuile, C.L. (1973) Animal experiments, in: H.C. Hodge, J.N. Stannard and J.B. Hursh (Eds.), Uranium, Plutonium, Transplutonic Elements, Springer-Verlag, New York, pp. 165-196. Communicated by F.H. Sobels
209
Elsevier MUTLET 0364
Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium Qiyue Hu and Shoupeng Zhu Department of Radiotoxicology, Suzhou Medical College, Suzhou, Jiangsu 215007 (China) (Accepted 25 January 1990)
Keywords: Uranyl fluoride; Chromosomal aberration; Mouse germ cells
Summary Cytogenetic damage induced by a wide range of concentrations of uranyl fluoride injected into mouse testes was evaluated by determining the frequencies of chromosomal aberrations in spermatogonia and primary spermatocytes. Breaks, gaps and polyploids were observed in spermatogonia. The frequencies of the significant type of aberration, breaks, were induced according to the injected doses of uranyl fluoride. Primary spermatocytes were examined for fragments, univalents and multivalents. The multivalents observed in this study resulted either from chromatid interchanges or from reciprocal translocations. The reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes. For primary spermatocytes the incidence of aberrant ceils largely depended on the administered dose. Sampling time after treatment could affect the frequencies of chromosomal aberrations in male mouse germ cells.
Enriched uranium is one of the main nuclear fuels for nuclear power stations. Uranium hexafluoride is used to enrich uranium and easily forms uranyl fluoride when it leaks out into ambient air and meets with moisture. Once formed, uranyl fluoride is most likely to enter the worker's body on the spot. So far many studies have been done on uranium Correspondence: Dr. Qiyue Hu, Department of Radiotoxicology, Suzhou Medical College, Suzhou, Jiangsu 215007 (China).
toxicity (Boback, 1975; Durbin and Wrenn, 1975; Hursh and Spoor, 1973; Morrow et al., 1972; Sullivan, 1980). In blood uranium can combine with organic and inorganic acids and proteins to form different compounds. Some of the compounds are 'diffusible', that is, they can reach every part of the body via the circulation although they largely deposit in kidneys, skeleton, liver or lungs depending on the way they enter the body and their chemical valence (Morrow et al., 1982; Ballou et al., 1986). Of all uranyl compounds uranyl fluoride was found to be the most toxic in
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
210
acute lethality studies (Yuile, 1973). But there is little information on its genetic toxicity. In order to study the genetic effects of a trace amount of uranyl fluoride in male mouse germ cells without evident damage to other tissues, intratesticular (i.t.) injection was adopted for administration. It had been used to study factors influencing the mutagenicity of ethyl methanesulfonate in mice by Soares (1976) and to inject [3H]deoxythymidine into the mouse testis for labeling by Sega and Sotomayor (1982). We now report the results of an investigation of the effects of a trace amount of uranyl fluoride in testes on the induction of chromosomal aberrations in spermatogonia and primary spermatocytes o f male B A L B / c mice. Materials and methods
Uranyl fluoride containing enriched uranium, in which the uranium-235 isotope component is 18.9070, was obtained from a nuclear plant (China). Sexually mature male B A L B / c mice, 10-12 weeks old and weighing 23-27 g, were purchased from the Center of Experimental Animals of our college. They were acclimatized to our laboratory for 7 days after arrival and were housed in groups o f 5 in polypropylene cages with hardwood-chip bedding. Water and rodent chow were provided ad libitum throughout the period of animal holding and experimentation. The time from treatment to chromosome preparation was fixed at 1, 13, 36 and 60 days, respectively, to investigate the induction of chromosomal aberrations by uranyl fluoride at different stages of germ-cell development. There were 5 experimental groups, treated with single i.t. injections of different doses of uranyl fluoride ranging from 0.05 to 1.00 ~g/testis. The i.t. injection procedure was as described by Sega and Sotomayor (1982). A control group was treated with isotonic saline in the same way for each fixtation time. Each group included 5 males. The volume of uranyl fluoride solution or isotonic saline injected into each testis was 5 /zl. Colchicine, 4 m g / k g body weight, was injected intraperitoneally 6 h before
the mice were killed. Chromosome specimens were prepared from the testes according to the technique of Evans et al. (1964) with minor modifications, and stained with Giemsa in phosphate buffer (pH 6.8). 100 well-spread metaphase spermatogonia and, where possible, about the same number of primary spermatocytes per animal were analyzed under the light microscope. Gaps were defined as achromatic lesions smaller than the width of the chromatid and without dislocation of the distal segment. Breaks were scored when the achromatic region was accompanied by displacement of the distal segment. The form of multivalents in meiosis I depends on the number of chromosomes involved in pairing, the number and location of chiasmata, and the degree o f chiasma terminalization. In this study multivalents observed at metaphase I were the result of either a chromatid interchange induced in diplotene or preleptotene of meiosis or a reciprocal chromosome-type exchange induced in spermatogonia. The chromatid interchanges were mostly irregular and non-symmetrical configurations. The chromosome-type exchanges were reciprocal translocations in that they were symmetrical configurations. The variance test was calculated to check whether variance of the data was homogeneous. When heterogeneity of variance was found arcsine transformation was carried out before Dunnett's ttest was used to determine statistically significant differences between treatment and control groups. Results
The changes in chromosomal structure and number of spermatogonia obtained 1, 13, 36 and 60 days after treatment with uranyl fluoride are presented in Table 1. Uranyl fluoride did not significantly induce gaps although there was a slight increase, compared to the control, in gap number in treatment groups, especially at the 1-day fixation time. Also the number of polyploids in treated groups was higher than that of controls but this effect was not clearly dose-dependent and not statistically different from the control groups ex-
211 TABLE 1 CHANGES IN CHROMOSOMAL STRUCTURE AND NUMBER INDUCED IN SPERMATOGONIA BY URANYL FLUORIDE a Sampling Dose Gaps time after ~g/testis) (%) exposure (days)
Breaks (%)
Polyploids (%)
0.05 0.10 0.25 0.50 1.00 control
0.2±0.2 0.2±0.2 0.6±0.4 0.4±0.2 0.8±0.4 0.2±0.2
0.6+0.4 0.4±0.4 1.2 ±0.7 1.8±0.7" 2.2±0.6** 0.0±0.0
25.2+ 1.0 22.6±2.0 21.8± 1.5 24.8± 1.6 28.0±3.2 19.0±2.8
13
0.05 0.10 0.25 0.50 1.00 control
0.0±0.0 0.0±0.0 0.2±0.2 0.4±0.2 0.2±0.2 0.0±0.0
0.2±0.2 0.6±0.4 0.4±0.2 1.2±0.4" 1.2±0.5" 0.0±0.0
15.2± 1.4 15.6±0.5 16.8±2.1 24.8± 1.6"* 21.6±1.1"* 13.4±2.0
36
0.05 0.10 0.25 0.50 1.00 control
0.0±0.0 0.0± 0.0 0.4±0.2 0.2±0.2 0.2±0.2 0.0±0.0
0.4±0.4 0.2±0.2 0.2±0.2 1.0±0.4" 1.4±0.2"* 0.0± 0.0
14.6±2.0 16.8± 1.5 15.8± 1.6 15.2±1.9 17.4±2.2 13.8± 1.5
60
0.05 0.10 0.25 0.50 1.00 control
0.0+0.0 0.0±0.0 0.0+0.0 0.0±0.0 0.2+0.2 0.0± 0.0
0.2±0.2 0.2+0.2 0.6+0.4 0.6+0.2 0.8±0.2 0.2±0.2
14.7+2.0 16.0+0.8 17.4±2.5 1%6±0.6 16.8±1.6 14.6+_ 1.7
a Values are means + SE for 5 mice, 100 cells per mouse. *P<0.05, **P<0.01 compared to control.
cept for 1.00 and 0.50/zg/testis at the 13-day fixation. The significant difference between the 1.00 or 0.50 #g/testis dose group and the control was due to the relatively low concurrent control. Breaks were found to increase with increasing doses at 4 fixation times after uranyl fluoride injection and their frequency was statistically significant in highdose groups at 1, 13 and 36 days. The frequencies o f breaks are more meaningful in cytogenetic evaluation than the other criteria listed in Table 1 since either gaps or polyploids are no good in-
dicators of chromosome damage according to the general opinion (Ito et al., 1988; Kill±an et al., 1977; Watson et al., 1976). In treated groups more breaks were found at the 1-day fixation than at other fixation times. There is no other explanation but prolonged exposure to uranium deposited in the testes, for the significant increase in break yields at the 13- and 36-day fixation times. Table 2 summarizes the detailed results of chromosome aberrations in primary spermatocytes induced by uranyl fluoride. Uranyl fluoride induced fragments, univalents and multivalents. The XY or autosome univalent frequency did not significantly increase with dose. The frequency of fragments only increased at the 1-day fixation. And it was noted that all multivalents observed in this study were 'quadrivalents'. They were presented in the form o f either a ring or a chain. The frequency of chromatid interchanges significantly increased at the 13-day fixation. The reason for this may be that cells in preleptotene at the time of treatment were the most likely ones to show aberrations according to Preston et al. (1981). No significantly increased production of reciprocal translocations was observed compared to the control. In Table 2 cells with univalents were not included in the category of aberrant cells because rather high frequencies of univalents were found in control groups. Discussion
The aim o f the present study was to determine the clastogenic effects o f a trace amount of uranyl fluoride in mouse testes on chromosomes of germ cells. We chose the i.t. route to administer uranyl fluoride. In pilot experiments we had found that other ways, such as intraperitoneal and intravenous injections, resulted in damage to the kidneys and the liver of the animals and thus influenced the animals' survival for the experimental period of 2 months. In evaluating genetic damage in male germ cells by any treatment it is necessary to recognize that in the seminiferous tubules of the testis there is a wide range o f germ-cell types. Different germ-cell types
212 TABLE 2 C H R O M O S O M A L A B E R R A T I O N S I N D U C E D IN P R I M A R Y S P E R M A T O C Y T E S BY U R A N Y L F L U O R I D E a
Sampling
Dose ~ g /
Number of
Fragments
Univalents (%)
Multivalents (%)b
time after exposure (days)
testis)
cells analyzed
(%)
XY
Autosomes
c.i.
r.t.
Incidence c of aberrant cells (o70)
0.05 0.10 0.25 0.50 1.00 control
491 489 5~ 5~ 5~ 5~
0.8±0.4 0.6±0.3 0.6±0.4 1.4±0.2"* 1.6±0.2"* 0.0±0.0
8.8±1.4 7.8±1.2 8.8±0.8 8.8±1.3 9.8±1.5 4.6±0.9
4.6±1.2 4.3±0.8 4.0±1.0 4.8±1.0 6.6±0.8 4.2±1.0
0.0±0.0 0.0±0.0 0.0±0.0 0.2±0.2 0.0±0.0 0.0±0.0
0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.8±0.4 0.6±0.3 0.6±0.4 1.6±0.2"* 1.6±0.2"* 0.0±0.0
13
0.05 0.10 0.25 0.50 1.~ control
470 5~ 5~ 488 5~ 5~
0.6±0.4 0.4±0.2 0.4±0.2 0.8±0.4 0.8±0.2 0.0±0.0
7.0±1.0 6.4±1.0 7.0±1.3 8.8±1.4 10.0±0.6 5.4±0.5
5.6±0.9 4.4±0.9 4.2±0.8 5.6±1.0 7.8±1.4 4.4±1.0
0.0±0.0 0.2±0.2 0.6±0.5* 0.8±0.4** 1.2±0.4"* 0.0±0.0
0.0±0.0 0.0±0.0 0.0+0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.6±0.4 0.6±0.2 1.0±0.3" 1.4±0.2"* 2.0±0.3** 0.0±0.0
36
0.05 0.10 0.25 0.50 1.~ control
5~ 5~ 5~ 489 497 5~
0.0±0.0 0.4±0.4 0.4±0.2 0.4±0.2 0.8±0.4 0.2±0.2
5.8±1.4 6.8±0.5 8.4±0.5 8.0±0.9 8.0±1.1 5.6±0.7
5.0±1.1 4.2±0.8 4.2±0.6 5.9±0.3 4.8±0.6 3.8±0.6
0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.2±0.2 0.4±0.2 0.2±0.2 0.2±0.2 0.6±0.5 0.0±0.0
0.2±0.2 0.8±0.5 0.6±0.4 0.6±0.4 1.4±0.2 0.2±0.2
60
0.05 0.10 0.25 0.50 1.~ control
497 5~ 481 5~ 492 486
0.2±0.2 0.2±0.2 0.0±0.0 0.2±0.2 0.4±0.4 0.2±0.2
5.2±0.7 6.4±1.4 8.4±1.7 6.8±1.5 8.7±1.1 4.9±0.8
4.2±0.7 4.0±0.5 4.0±0.9 5.0±1.0 5.3±0.8 4.0±1.0
0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
0.2±0.2 0.2±0.2 0.2±0.2 0.2±0.2 0.6±0.9 0.0±0.0
0.4±0.2 0.4±0.2 0.2±0.2 0.4±0.2 0.6±0.2 0.2±0.2
a Values are means ± SE for 5 mice. b c.i., chromatid interchanges; r.t., reciprocal translocations. c Not including the cells with univalents. * P < 0 . 0 5 , * * P < 0 . 0 1 compared to control.
are differently sensitive to mutagens. The spermatogonial stem cell population is complex (Cattanach and Rasberry, 1987). It contains cells with different sensitivities to genetic damage. The spermatogonial cell renewal process has been described by Searle (1974). Results of the experiment (Table 1) show a general tendency for break production to increase with the dose of uranyl fluoride. At high dose levels the statistically significant difference of break frequencies between treated and control groups disappeared 60 days after treatment although more
breaks were found in treated groups than in the control group. This indicates that the effect of uranyl fluoride on chromosome aberration induction lasted at least 36 days after a single injection into the testis, which might result from uranium activity being incorporated in the testes. Unfortunately in this paper we could not provide exact information about its deposition and retention in the mouse testes. Data in Table 1 also indicate that spermatogonia were sensitive to a trace amount of uranyl fluoride. Spermatogonia are important target cells and an elevation of the aberrations in-
213
duced in spermatogonia is important for the estimation of genetic risk since spermatogonia will later give rise to mature germ cells. But many spermatogonia, carrying more than one aberration after treatment, may be eliminated before reaching the spermatocyte stage (Tates and Natarajan, 1976). For that reason it is genetically meaningful that the reciprocal translocations induced in spermatogonia are recorded in spermatocytes. The data in Table 2 show that on all days of sampling multivalents were found in treated groups at high dose levels but it should be made clear that multivalents observed on day 1 or 13 cannot be reciprocal translocations induced in spermatogonia but are chromatid interchanges induced in diplotene or preleptotene of meiosis. Their shape is different from reciprocal translocation multivalents although sometimes similar to them. A symmetrical chromatid interchange in a spermatocyte will result in 6/16 sperm with duplications/deficiencies, 9/16 normal sperm and 1/16 sperm with a balanced reciprocal translocation (Brewen et al., 1976). An asymmetrical interchange will lead to deletions in 50°70 of the sperm. Multivalents observed on day 36 or 60 are reciprocal translocations. These translocations could persist over mitotic cell divisions, usually just as well as the normal chromosome in the complement (Bender et al., 1988). By meiotic segregation a spermatocyte with a reciprocal translocation will give rise to 50% sperm with a duplication/deficiency, 25°7o normal sperm and 25070 sperm with a balanced reciprocal translocation. All chromosomally imbalanced sperm will cause embryonic death. The most common type of abnormality observed in spermatocytes was the presence of univalents. This may be the result o f precocious separation of the chromosomes from the bivalents (Tates and Natarajan, 1976). It should be noticed that X and Y were more often separated than autosomes. However, this observation was not only made in treated groups but also in controls. With regard to the incidence of aberrant cells, it appears that only the high doses, within 13 days of treatment, produced significantly more aberrant cells than con-
trois although a higher incidence o f aberrant cells was found in nearly all treated groups compared to the controls in this study. At the 36-day fixation the fact that there is no significant difference in incidence of aberrant cells between the 1.00 #g/testis dose group and the control is due to the relatively high concurrent control. From the results of the present study, it can be concluded that uranyl fluoride, which has both chemical toxicity and radiotoxicity, in the testes induced chromosome aberrations in spermatogonia and spermatocytes, yet it is not clear whether the aberrations were produced mainly because of chemical toxicity or radiotoxicity. This remains to be studied further. References Ballou, J.E., R.A. Gies, A.C. Case, D.L. Haggard, R.L. Buschbom and J.L. Ryan (1986) Deposition and early disposition of inhaled 233UO2(NO3)2 and 232UOE(NO3)2 in the rat, Health Phys., 51,755-771. Bender, M.A, A.A. Awa, A.L. Brooks, H.J. Evans, P.G. Groer, L.G. Littlefield, C. Pereira, R.J. Preston and B.W. Wacholz (1988) Current status of cytogenetic procedures to detect and quantify previous exposures to radiation, Mutation Res., 196, 103-159. Boback, M.W. (1975) A review of uranium excretion and clinical urinalysis data in accidental exposure cases, in: Report ERDA 93, Conference on Occupational Health Experience with Uranium, 28-30 April, pp. 226-231. Brewen, J.G., H.S. Payne and R.J. Preston (1976) X-rayinduced chromosome aberrations in mouse dictyate oocytes. I. Time and dose relationships, Mutation Res., 35, 111-120. Cattanach, B.M., and C. Rasberry (1987) Genetic effects of combined chemical-X-ray treatments in male mouse germ cells, Int. J. Radiat. Biol., 51,985-996. Durbin, P.W., and M.E. Wrenn (1975) Metabolism and effects of uranium in animals, in: Report ERDA 93, Conference on Occupational Health Experience with Uranium, 28-30 April, pp. 68-129. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3,289-294. Hursh, J.B., and N.L. Spoor (1973) Data on man, in: H.C. Hodge, J.N. Stannard and J.B. Hursh (Eds.), Uranium, Plutonium, Transplutonic Elements, Springer-Verlag, New York, pp. 197-239. Ito, Y., N. Ueda, S. Murao, T. Sugiyama, H. Lee and R.G. Harvey (1988) Induction of chromosomal aberrations in rat bone marrow cells and mutations in Salmonella typhimurium
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