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Association of cyclophosphamide-induced male-mediated, foetal abnormalities with reduced paternal germ-cell apoptosis
Mutation Research 447 Ž2000. 149–154 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
Association of cyclophosphamide-induced male-mediated, foetal abnormalities with reduced paternal germ-cell apoptosis Martin H. Brinkworth, Eberhard Nieschlag ) Institute of ReproductiÕe Medicine of the UniÕersity, Domagkstraße 11, D-48129 Munster, Germany ¨ Received 1 June 1999; received in revised form 20 September 1999; accepted 20 September 1999
Abstract To investigate the mechanism by which malformed offspring can result from the exposure of males to mutagens, we treated adult male rats with 0, 1.4, 3.4 or 5.1 mgrkg cyclophosphamide, 6 days per week for 9 weeks, a treatment regimen known to induce heritable abnormalities. Testis samples from some of the animals were then collected for fixation in Carnoy’s fluid and subsequent analysis of germ-cell apoptosis and proliferation. The remainder were mated, resulting in a greater than 11-fold increase in the proportion of abnormal offspring produced in the 5.1 mgrkg group. The number of apoptotic cells per stage XIIrXIII tubular cross-section decreased with increasing dose, significantly so at 5.1 mgrkg Ž P - 0.05.. No statistically significant effect was found on spermatocyte numbers at this dose, indicating that a reduction in the amount of cells available to undergo apoptosis cannot explain the decrease. The inappropriate survival of damaged germ-cells caused by a lowering of the incidence of apoptosis may, therefore, account for the rise in the proportion of foetal malformations. q 2000 Elsevier Science B.V. All rights reserved. Keywords: ApoptosisrFoetal abnormality; Cyclophosphamide; Rat; Male germ-cell
1. Introduction The majority of foetal malformations have long been believed to be caused in utero. However, increased attention has been focused recently on the possibility that mutations causing effects in the offspring may also arise in the male germ line. This has been suggested to occur in humans, most notably by Gardner et al. w1x to explain a cluster of childhood
)
Corresponding author. Tel.: q49-251-8356097; fax: q49251-8356093. E-mail address: [email protected] ŽE. Nieschlag..
leukemias at Sellafield in the UK. The mechanism was subsequently criticised w2x and after exhaustive investigations it appears that paternal radiation exposure is unlikely to be responsible for excesses of childhood leukaemia or non-Hodgkin lymphoma in Britain w3x. Nonetheless, radiation exposure following the explosion at Chernobyl has been confirmed to have induced male germ-line mutations in minisatellites that are heritable w4x. Furthermore, significant associations have been found between paternal smoking and the incidence of childhood cancer, especially leukaemia w5–7x. This indicates that mutagens are indeed capable of producing changes in the human male germ-line, some of which may be capable of affecting the next generation.
0027-5107r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 1 8 9 - X
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M.H. Brinkworth, E. Nieschlagr Mutation Research 447 (2000) 149–154
Paternally administered X-rays can cause skeletal defects in mice w8x and preconceptional, sub-chronic exposure of male mice to 239 plutonium can potentiate the carcinogenic effects of subsequent methyl nitrosourea administration to the offspring w9x. Similarly, repeated administration of a low dose Že.g., 5.1 mgrkg 6 days a week for 7–30 weeks, equivalent to a therapeutic maintenance regimen. of the anticancer drug cyclophosphamide ŽCP. to male rats results in an elevated number of foetal malformations among their offspring w10–12x. The requirement for prolonged exposure to low doses makes this model particularly relevant to humans because it mimics most human exposure to mutagens w13x. One of the defenses against the induction of germ-line mutations is apoptosis, which can be induced in spermatogenic cells following genotoxin exposure. A single dose of 70 mgrkg bodyweight CP to rats can, for example, result in apoptosis among certain spermatocytes and spermatogonia, that is apparent 12–24 h after administration w14x. However, such a dose of CP does not cause mutations leading to effects in the offspring and it is not known whether apoptosis has a protective role against heritable genetic damage. We therefore performed the following experiment using sub-chronic administration of CP to induce foetal abnormalities and examining its effect on apoptosis.
2. Materials and methods 2.1. Animal treatment and mating Groups of 15 male Sprague–Dawley rats were treated orally with 0.9% saline or 1.4, 3.4 or 5.1 mgrkg CP ŽCAS number: 6055-19-2. in saline, 6 days per week for 9 weeks. One hour after the final dose 4–5 animals per group were anaesthetised by ether ŽAsid Bonz, Boblingen, Germany. inhalation ¨ and the right testis was removed and fixed in Bouin’s fluid for future gene expression studies. All rats recovered within a few minutes of the procedure and exhibited no signs of discomfort. They were sacrificed 1 h later and the left testis was weighed, then fixed in Carnoy’s fluid Ž60% vrv chloroform ŽCAS number: 67-66-6., Merck, Darmstadt, Germany, 30% vrv absolute ethanol ŽCAS
number: 64-17-5., J.T. Baker, Deventer, Holland, 10% vrv glacial acetic acid ŽCAS number: 64-19-7., Merck, Darmstadt, Germany. and embedded in paraffin. Sections of 2 mm-thickness were cut from the blocks and mounted on slides for studies of apoptosis and cell numbers. The remaining animals Ž10 per group. were mated to two virgin females each, which were sacrificed on the day before parturition for evaluation of the uterine contents for dominant lethality and the proportion of abnormal, live foetuses. All procedures were conducted in accordance with the German Federal Law on the Care and Use of Laboratory Animals. 2.2. Apoptosis Apoptotic cells were quantified by counting labelled cells on 2 mm thick, Carnoy-fixed testis sections following end-labelling with biotin-labelled, deoxyuridine triphosphate ŽBoehringer Mannheim, Mannheim, Germany. using 0.3 Urml terminal deoxynucleotide transferase ŽPromega, Madison, WI, USA. and visualisation with Extravidin–peroxidase complex and diamino-benzidine Žboth from SigmaAldrich Chemie, Deisenhof, Germany. ŽTUNEL labelling. as previously described w15 16x. Cells were scored from 50 rounded, tubule cross-sections per animal that were fully contained within the microscope-field of view at a total of 250 = magnification. Stages XIIrXIII were combined since they are difficult to distinguish reliably from each other by conventional light microscopy w17x. These stages were chosen because they exhibit a relatively high rate of spontaneous germ cell apoptosis and are known to show increased germ-cell apoptosis in response to a high, acute dose of CP w14x. Furthermore, in comparison with other stages they occur relatively frequently throughout a cross-section of a rat testis and can be reliably identified. The mean number of labelled Ži.e., apoptotic. cells per tubule cross-section was calculated for each animal and used to derive group means. In order to control for treatment-related alterations in germ-cell proliferation that could influence the number of apoptotic cells found, zygotene spermatocytes were scored from 20 tubule cross-sections per animal in the 0.0 mgrkg and 5.1 mgrkg CP dose
M.H. Brinkworth, E. Nieschlagr Mutation Research 447 (2000) 149–154
groups. These data were then used to derive group means. Expression of the apoptosis and zygotene spermatocyte data as cells per tubule cross-section is only valid if there is no change in the cross-sectional dimensions of the tubules. Therefore, the shortest diameter of 20, virtually circular, tubule cross-sections was measured per animal for all groups and used to derive group means. 2.3. Procedure for cell scoring Subsequent methods required quantification of cells on histological sections and the following approach was followed for each. Slides were randomised and coded by a third party and scored blind. The search for appropriate tubules commenced at the top left of the section Žas it appeared through the microscope. and proceeded across the section; the section was then moved downwards by the diameter of the field of view and searching recommenced from right to left. This was repeated until either the whole section had been covered Žin which case a further section was examined. or the requisite number of tubules had been scored. The Vernier coordinates of each tubule were recorded to prevent accidental rescoring. Fifty tubule cross-sections were analysed for apoptotic cells and 20 for zygotene spermatocyte numbers and tubule diameter measurements. The adequacy of these numbers was checked by statistical analysis of the first half of each data set. In all cases the results were qualitatively very similar and statistically identical to those obtained by analysis of the full data set Ždata not shown..
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3. Results 3.1. Mating studies The absolute testes weights and bodyweights were both lower in the treated groups compared with the controls but the differences were not statistically significant ŽTable 1.. A pronounced dose-dependent effect was observed as expected on dominant lethality, expressed as the percentage of the total implants that resulted in early embryonic deaths Ždefined as those decidua where foetal tissue was either not visible or was necrotic. per female per group. The control value was 11.3% and for 1.4, 3.4 and 5.1 mgrkg CP was 13.5%, 29.5% and 52.2% respectively, significantly different by Kruskal–Wallis oneway ANOVA Ž P - 0.001.. Foetuses that had externally visible abnormalities or weighed less than 75% of the weight of their normal litter mates w8x were scored as abnormal. The proportion of abnormal offspring as a percentage of live offspring produced per female per group was increased from 0.45% in the controls to 5.25% in the 5.1 mgrkg dose group ŽFig. 1a., which is a similar magnitude of increase as observed previously w10–12x As it is well-known that abnormalities are only induced by 5.1 mgrkgrday w10–12x we were only interested to confirm that this dose had indeed induced an effect. Accordingly, it was tested by Student’s t-test on arc-sine transformed data, using a one-tailed test since only an increase was expected, and is significantly different from the controls Ž P 0.05.. Abnormalities encountered included growth retardation, presumed spina bifida with limb defects, and apparent anaemia.
Table 1 The effect of treatment with 1.4, 3.4 or 5.1 mgrkg cyclophosphamide, 6 days per week for 9 weeks on bodyweight, testes weight and seminiferous tubule diameter in rats Group
0.0 mgrkg CP
1.4 mgrkg CP
3.4 mgrkg CP
5.1 mgrkg CP
Bodyweight 389.3 g " 31.61 g 379.3 g " 27.77 g 367.5 g " 21.19 g 353.0 g " 21.61 g Testes weight 3.82 g " 0.274 g 3.42 g " 0.508 g 3.32 g " 0.302 g 3.35 g " 0.295 g Seminiferous tubule diameter 263.625 mm " 8.85 mm 274.50 mm " 8.025 mm 259.125 mm " 13.65 mm 259.50 mm " 13.125 mm Data for bodyweight and testes weight are means" standard deviations and means" standard errors for seminiferous tubule diameters. Data were analysed by one-way analysis of variance.
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M.H. Brinkworth, E. Nieschlagr Mutation Research 447 (2000) 149–154
3.2. Apoptosis The microscopic appearance of the tubules was normal with no obvious abnormalities of spermatogenesis. Germ cells seen undergoing apoptosis in all groups are illustrated in Fig. 2 and were predominantly A-spermatogonia and zygotene spermatocytes; rarely late pachytene or diplotene spermatocytes were also seen to be labelled. Degenerating spermatid-heads at the base of the germinal epithelium were also occasionally encountered but were not included in the analysis since it is not known whether such TUNEL-labelling results from apoptosis or Sertoli cell-lysosome-mediated necrosis. Surprisingly, there was a dose-related decrease in the mean number of apoptotic cells per stage XIIrXIII tubule cross-section, the top dose group showing a reduction of nearly 2.5-fold Žwith all animals similarly affected.. This was significantly different from the controls by one-way ANOVA
Fig. 2. The detection of apoptotic, testicular germ-cells. The arrows indicate apoptotic cells: on the basis of their position in the germinal epithelium and spatial relationship to other cell types, they can be identified as 2 A-spermatogonia and 2 zygotene spermatocytes. The scale bar represents 20 mm.
followed by Student–Newman–Keul’s test Ž P 0.05. ŽFig. 1b.. ŽIn view of this finding, testicular DNA ladder data would be of very limited value since control levels of germ cell apoptosis in the rat testis are so low in relation to total cellular content w15x that a dose-related decrease would be virtually impossible to confirm by this approach.. 3.3. Controls for cell proliferation and tissue shrinkage The mean number Ž"standard error. of zygotene spermatocytes per stage XIIrXIII tubule cross-section was 63.5 Ž"8.12. in the controls and 65.3 Ž"9.01. in the 5.1 mgrkg CP treatment group. The data for mean tubule diameters are presented in Table 1. There was no significant difference between any of the groups Žone-way ANOVA..
4. Discussion
Fig. 1. Ža. Induction of foetal abnormalities by paternal administration of CP: data points represent the mean percentages of abnormal foetuses as a proportion of total live pups per litter; error bars are standard errors. The asterisk denotes a mean significantly different from that of the control group: P - 0.05. Žb. The incidence of apoptotic germ cells in the testes of rats treated with CP. Data points represent means per tubule cross-section per animal per group; error bars are standard errors. The asterisk denotes a mean significantly different from that of the control group: P - 0.05.
The most striking aspect of this study was the finding that sub-chronic treatment with CP induced a dose-related decrease in germ-cell apoptosis. Since there was no significant effect on seminiferous tubule diameters, that can be excluded as a source of bias. The germ cells present in rat stage XIIrXIII tubules are Žin ascending order of maturity. A-spermatogonia, zygotene spermatocytes, pachytenerdiplotene spermatocytes and step 12r13 spermatids Želongating.. Virtually all the apoptotic cells found belonged to the first two categories. It is conceivable that a treatment-related alteration in cellular prolifer-
M.H. Brinkworth, E. Nieschlagr Mutation Research 447 (2000) 149–154
ation could have altered the spermatogonia or spermatocyte population sizes, leading to apparent changes in the proportion of apoptotic cells. However, the lack of any difference between the mean number of zygotene spermatocytes per tubule crosssection effectively excludes this possibility. This is, therefore, to the best of our knowledge, the first report to demonstrate a toxin-induced reduction in the incidence of testicular germ-cell apoptosis. It is particularly interesting that this reduction is mediated by exposure to low doses of a compound that at higher doses can elevate the incidence. Furthermore, the dosing regimen of CP that leads to the decreased incidence of apoptosis also leads to foetal abnormalities. The significance of the findings thus extends to the understanding both of apoptotic processes in the testis and of the heritability of mutations. The principal cell types in which dominant lethal mutations are induced by prolonged dosing with low doses of CP are elongating spermatids and sperm undergoing epididymal maturation because the effect disappears within 4 weeks of cessation of treatment w18x. ŽThat these are the cell types affected by acute doses has also been directly shown by experiments involving ligation of the efferent ducts w19x.. Foetal abnormalities, however, are only seen after prolonged treatment w10–12x and the present work. Together, these data suggest that CP may induce a different spectrum of DNA damage in different cell types ie: exposure of pre-meiotic cells Že.g., spermatogonia and zygotene spermatocytes. may cause damage that can lead to foetal abnormalities whereas exposure of elongating spermatids and maturing sperm results in dominant lethal mutations. High doses Že.g., 70 mgrkg CP or above. increase apoptosis in pre-meiotic cells, which may serve to prevent the transmission of heritable abnormalities w14x. This accords with the present data, which provide the first experimental evidence showing that abnormalities occur when apoptosis is reduced. Lower doses may not induce sufficient damage to be a mutational hazard unless repeated, in which case the induction of apoptosis among these pre-meiotic cells Žand presumably also DNA repair. will compensate. However, the negative effect of low doses of CP on the induction of apoptosis evidently overwhelms these protective mechanisms,
153
so that a significant proportion of the sperm surviving to participate in fertilization and yield viable offspring carry heritable mutations. It is interesting that the level of apoptosis in the 3.4 mgrkg CP group was also much lower than that of the controls, whereas there was no evidence of an induction of abnormalities in this group, in agreement with previous studies w10,11x. The induction of genetic damage by CP does not show a similar threshold in the dose range tested as regards dominant lethality. Therefore, it may be more likely that the threshold for abnormalities stems not from the induction of damage but from its persistence. Although other factors such as DNA repair are likely to be involved, it may be that the lower amount of damage induced at 3.4 mgrkg, together with the higher rate of apoptosis, is sufficient to reduce the number of damaged cells surviving to become mature sperm to below the level at which they can be detected in a mating assay. How CP may act to reduce apoptosis presumably depends on being administered long-term in low doses. As a powerful alkylating agent, CP will alkylate a range of cellular macromolecules. These may include the mRNA and proteins that mediate apoptotic responses, thus poisoning the machinery of apoptosis in susceptible germ cells. Alternatively, CP may damage the DNA of genes required for germ cell apoptosis. This would have highly important implications for the transmission of abnormalities to the F1 generation, since it implies that inactivating mutations in such genes, by allowing the survival of damaged cells that would otherwise have died by apoptosis, will be more likely to be passed on to the F1 generation than other debilitating mutations. In F1 animals such mutations are likely to pre-dispose towards cancer or developmental abnormalities by impairing the ability of cells to die when instructed to, especially if ‘hit’ by a second inactivating event in the maternal allele. This, then, could provide a mechanism to explain the increase in cases of cancer among the children of men who smoke compared with non-smoking men w5–7x. Likewise, it may account for the induction of mutations pre-disposing to cancer in the male germ-cells of mice dosed with 239 plutonium w9x and for the occurrence of foetal abnormalities in the offspring of CP-treated rats w10– 12x wand the present workx.
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Acknowledgements We are grateful to M. Heuerman, M. Niemeier J. Esselmann and G. Stelke, for technical assistance and to Drs. T.G. Cooper, J. Gromoll, A. Lerchl, S. Schlatt and G.F. Weinbauer for helpful comments and suggestions during the preparation of the manuscript. The work was supported by the Bundesministerium fur ¨ Bildung, Wissenschaft, Forschung und Technologie of the Federal Republic of Germany, project: 07GTX02: ‘Umweltbedingte Veranderungen der vaterlichen Erbsubstanz’. ¨ ¨
w8x
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w10x
w11x
References w12x w1x M.J. Gardner, M.P. Snee, A.J. Hall, C.A. Powell, S. Downes, J.D. Terrell, Results of case-control study of leukaemia and lymphoma among young people near Sellafield nuclear plant in West Cumbria, Br. Med. J. 300 Ž1990. 423–429. w2x R. Doll, H.J. Evans, S.C. Darby, Paternal exposure not to blame, Nature 367 Ž1994. 678–680. w3x G.J. Draper, M.P. Little, T. Sorahan, L.J. Kinlen, K.J. Bunch, A.J. Conquest, G.M. Kendall, G.W. Kneale, R.J. Lancashire, C.R. Muirhead, C.M. O’Connor, T.J. Vincent, Cancer in the offspring of radiation workers: a record linkage study, Br. Med. J. 315 Ž1997. 1181–1188. w4x Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, R. Neumann, D.L. Neil, A.J. Jeffreys, Human minisatellite mutation rate after the Chernobyl accident, Nature 380 Ž1996. 683–686. w5x T. Sorahan, R.J. Lancashire, M.A. Hulten, I. Peck, A.M. Stewart, Childhood cancer and parental use of tobacco: deaths from 1953 to 1955, Br. J. Cancer 75 Ž1997. 134–138. w6x B.T. Ji, X.O. Shu, M.S. Linet, W. Zheng, S. Wacholder, Y.T. Gao, D.M. Ying, F. Jin, Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers, J. Natl. Cancer Inst. 89 Ž1997. 238–244. w7x T. Sorahan, P. Prior, R.J. Lancashire, S.P. Faux, M.A. Hulten, I.M. Peck, A.M. Stewart, Childhood cancer and
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parental use of tobacco: deaths from 1971 to 1976, Br. J. Cancer 76 Ž1997. 1525–1531. K.M. Kirk, M.F. Lyon, Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages, Mutat. Res. 125 Ž1984. 75–85. B.I. Lord, L.B. Woolford, L. Wang, V.A. Stones, D. McDonald, S.A. Lorimore, D. Papworth, E.G. Wright, D. Scott, Tumour induction by methyl-nitroso-urea following preconceptional paternal contamination with plutonium-239, Br. J. Cancer 78 Ž1998. 301–311. J.M. Trasler, B.F. Hales, B. Robaire, Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility, Nature 316 Ž1985. 144–146. J.M. Trasler, B.F. Hales, B. Robaire, Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny, Biol. Reprod. 34 Ž1986. 275–283. P.C. Jenkinson, D. Anderson, Malformed foetuses and karyotype abnormalities in the offspring of cyclophosphamide and allyl alcohol-treated male rats, Mutat. Res. 229 Ž1990. 173–184. P.H. Abelson, Risk assessments of low-level exposures, Science 265 Ž1994. 1507. L. Cai, B.F. Hales, B. Robaire, Induction of apoptosis in the germ cells of adult male rats after exposure to cyclophosphamide, Biol. Reprod. 56 Ž1997. 1490–1497. M.H. Brinkworth, G.F. Weinbauer, S. Schlatt, E. Nieschlag, Identification of male germ cells undergoing apoptosis in adult rats, J. Reprod. Fertil. 105 Ž1995. 25–33. M.H. Brinkworth, G.F. Weinbauer, M. Bergmann, E. Nieschlag, Apoptosis as a mechanism of germ cell loss in elderly men, Int. J. Androl. 20 Ž1997. 222–228. L.D. Russell, R.A. Ettlin, A.P. Sinha Hikim, in: Histological and histopathological evaluation of the testis. Staging for the rat, Cache River Press, Clearwater, 1990, pp. 63–118. B.F. Hales, B. Robaire, Reversibility of the effects of chronic paternal exposure to cyclophosphamide on pregnancy outcome in rats, Mutat. Res. 229 Ž1990. 129–134. J. Qiu, B.F. Hales, B. Robaire, Adverse effects of cyclophosphamide on progeny outcome can be mediated through posttesticular mechanisms in the rat, Biol. Reprod. 46 Ž1992. 926–931.
Association of cyclophosphamide-induced male-mediated, foetal abnormalities with reduced paternal germ-cell apoptosis Martin H. Brinkworth, Eberhard Nieschlag ) Institute of ReproductiÕe Medicine of the UniÕersity, Domagkstraße 11, D-48129 Munster, Germany ¨ Received 1 June 1999; received in revised form 20 September 1999; accepted 20 September 1999
Abstract To investigate the mechanism by which malformed offspring can result from the exposure of males to mutagens, we treated adult male rats with 0, 1.4, 3.4 or 5.1 mgrkg cyclophosphamide, 6 days per week for 9 weeks, a treatment regimen known to induce heritable abnormalities. Testis samples from some of the animals were then collected for fixation in Carnoy’s fluid and subsequent analysis of germ-cell apoptosis and proliferation. The remainder were mated, resulting in a greater than 11-fold increase in the proportion of abnormal offspring produced in the 5.1 mgrkg group. The number of apoptotic cells per stage XIIrXIII tubular cross-section decreased with increasing dose, significantly so at 5.1 mgrkg Ž P - 0.05.. No statistically significant effect was found on spermatocyte numbers at this dose, indicating that a reduction in the amount of cells available to undergo apoptosis cannot explain the decrease. The inappropriate survival of damaged germ-cells caused by a lowering of the incidence of apoptosis may, therefore, account for the rise in the proportion of foetal malformations. q 2000 Elsevier Science B.V. All rights reserved. Keywords: ApoptosisrFoetal abnormality; Cyclophosphamide; Rat; Male germ-cell
1. Introduction The majority of foetal malformations have long been believed to be caused in utero. However, increased attention has been focused recently on the possibility that mutations causing effects in the offspring may also arise in the male germ line. This has been suggested to occur in humans, most notably by Gardner et al. w1x to explain a cluster of childhood
)
Corresponding author. Tel.: q49-251-8356097; fax: q49251-8356093. E-mail address: [email protected] ŽE. Nieschlag..
leukemias at Sellafield in the UK. The mechanism was subsequently criticised w2x and after exhaustive investigations it appears that paternal radiation exposure is unlikely to be responsible for excesses of childhood leukaemia or non-Hodgkin lymphoma in Britain w3x. Nonetheless, radiation exposure following the explosion at Chernobyl has been confirmed to have induced male germ-line mutations in minisatellites that are heritable w4x. Furthermore, significant associations have been found between paternal smoking and the incidence of childhood cancer, especially leukaemia w5–7x. This indicates that mutagens are indeed capable of producing changes in the human male germ-line, some of which may be capable of affecting the next generation.
0027-5107r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 1 8 9 - X
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M.H. Brinkworth, E. Nieschlagr Mutation Research 447 (2000) 149–154
Paternally administered X-rays can cause skeletal defects in mice w8x and preconceptional, sub-chronic exposure of male mice to 239 plutonium can potentiate the carcinogenic effects of subsequent methyl nitrosourea administration to the offspring w9x. Similarly, repeated administration of a low dose Že.g., 5.1 mgrkg 6 days a week for 7–30 weeks, equivalent to a therapeutic maintenance regimen. of the anticancer drug cyclophosphamide ŽCP. to male rats results in an elevated number of foetal malformations among their offspring w10–12x. The requirement for prolonged exposure to low doses makes this model particularly relevant to humans because it mimics most human exposure to mutagens w13x. One of the defenses against the induction of germ-line mutations is apoptosis, which can be induced in spermatogenic cells following genotoxin exposure. A single dose of 70 mgrkg bodyweight CP to rats can, for example, result in apoptosis among certain spermatocytes and spermatogonia, that is apparent 12–24 h after administration w14x. However, such a dose of CP does not cause mutations leading to effects in the offspring and it is not known whether apoptosis has a protective role against heritable genetic damage. We therefore performed the following experiment using sub-chronic administration of CP to induce foetal abnormalities and examining its effect on apoptosis.
2. Materials and methods 2.1. Animal treatment and mating Groups of 15 male Sprague–Dawley rats were treated orally with 0.9% saline or 1.4, 3.4 or 5.1 mgrkg CP ŽCAS number: 6055-19-2. in saline, 6 days per week for 9 weeks. One hour after the final dose 4–5 animals per group were anaesthetised by ether ŽAsid Bonz, Boblingen, Germany. inhalation ¨ and the right testis was removed and fixed in Bouin’s fluid for future gene expression studies. All rats recovered within a few minutes of the procedure and exhibited no signs of discomfort. They were sacrificed 1 h later and the left testis was weighed, then fixed in Carnoy’s fluid Ž60% vrv chloroform ŽCAS number: 67-66-6., Merck, Darmstadt, Germany, 30% vrv absolute ethanol ŽCAS
number: 64-17-5., J.T. Baker, Deventer, Holland, 10% vrv glacial acetic acid ŽCAS number: 64-19-7., Merck, Darmstadt, Germany. and embedded in paraffin. Sections of 2 mm-thickness were cut from the blocks and mounted on slides for studies of apoptosis and cell numbers. The remaining animals Ž10 per group. were mated to two virgin females each, which were sacrificed on the day before parturition for evaluation of the uterine contents for dominant lethality and the proportion of abnormal, live foetuses. All procedures were conducted in accordance with the German Federal Law on the Care and Use of Laboratory Animals. 2.2. Apoptosis Apoptotic cells were quantified by counting labelled cells on 2 mm thick, Carnoy-fixed testis sections following end-labelling with biotin-labelled, deoxyuridine triphosphate ŽBoehringer Mannheim, Mannheim, Germany. using 0.3 Urml terminal deoxynucleotide transferase ŽPromega, Madison, WI, USA. and visualisation with Extravidin–peroxidase complex and diamino-benzidine Žboth from SigmaAldrich Chemie, Deisenhof, Germany. ŽTUNEL labelling. as previously described w15 16x. Cells were scored from 50 rounded, tubule cross-sections per animal that were fully contained within the microscope-field of view at a total of 250 = magnification. Stages XIIrXIII were combined since they are difficult to distinguish reliably from each other by conventional light microscopy w17x. These stages were chosen because they exhibit a relatively high rate of spontaneous germ cell apoptosis and are known to show increased germ-cell apoptosis in response to a high, acute dose of CP w14x. Furthermore, in comparison with other stages they occur relatively frequently throughout a cross-section of a rat testis and can be reliably identified. The mean number of labelled Ži.e., apoptotic. cells per tubule cross-section was calculated for each animal and used to derive group means. In order to control for treatment-related alterations in germ-cell proliferation that could influence the number of apoptotic cells found, zygotene spermatocytes were scored from 20 tubule cross-sections per animal in the 0.0 mgrkg and 5.1 mgrkg CP dose
M.H. Brinkworth, E. Nieschlagr Mutation Research 447 (2000) 149–154
groups. These data were then used to derive group means. Expression of the apoptosis and zygotene spermatocyte data as cells per tubule cross-section is only valid if there is no change in the cross-sectional dimensions of the tubules. Therefore, the shortest diameter of 20, virtually circular, tubule cross-sections was measured per animal for all groups and used to derive group means. 2.3. Procedure for cell scoring Subsequent methods required quantification of cells on histological sections and the following approach was followed for each. Slides were randomised and coded by a third party and scored blind. The search for appropriate tubules commenced at the top left of the section Žas it appeared through the microscope. and proceeded across the section; the section was then moved downwards by the diameter of the field of view and searching recommenced from right to left. This was repeated until either the whole section had been covered Žin which case a further section was examined. or the requisite number of tubules had been scored. The Vernier coordinates of each tubule were recorded to prevent accidental rescoring. Fifty tubule cross-sections were analysed for apoptotic cells and 20 for zygotene spermatocyte numbers and tubule diameter measurements. The adequacy of these numbers was checked by statistical analysis of the first half of each data set. In all cases the results were qualitatively very similar and statistically identical to those obtained by analysis of the full data set Ždata not shown..
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3. Results 3.1. Mating studies The absolute testes weights and bodyweights were both lower in the treated groups compared with the controls but the differences were not statistically significant ŽTable 1.. A pronounced dose-dependent effect was observed as expected on dominant lethality, expressed as the percentage of the total implants that resulted in early embryonic deaths Ždefined as those decidua where foetal tissue was either not visible or was necrotic. per female per group. The control value was 11.3% and for 1.4, 3.4 and 5.1 mgrkg CP was 13.5%, 29.5% and 52.2% respectively, significantly different by Kruskal–Wallis oneway ANOVA Ž P - 0.001.. Foetuses that had externally visible abnormalities or weighed less than 75% of the weight of their normal litter mates w8x were scored as abnormal. The proportion of abnormal offspring as a percentage of live offspring produced per female per group was increased from 0.45% in the controls to 5.25% in the 5.1 mgrkg dose group ŽFig. 1a., which is a similar magnitude of increase as observed previously w10–12x As it is well-known that abnormalities are only induced by 5.1 mgrkgrday w10–12x we were only interested to confirm that this dose had indeed induced an effect. Accordingly, it was tested by Student’s t-test on arc-sine transformed data, using a one-tailed test since only an increase was expected, and is significantly different from the controls Ž P 0.05.. Abnormalities encountered included growth retardation, presumed spina bifida with limb defects, and apparent anaemia.
Table 1 The effect of treatment with 1.4, 3.4 or 5.1 mgrkg cyclophosphamide, 6 days per week for 9 weeks on bodyweight, testes weight and seminiferous tubule diameter in rats Group
0.0 mgrkg CP
1.4 mgrkg CP
3.4 mgrkg CP
5.1 mgrkg CP
Bodyweight 389.3 g " 31.61 g 379.3 g " 27.77 g 367.5 g " 21.19 g 353.0 g " 21.61 g Testes weight 3.82 g " 0.274 g 3.42 g " 0.508 g 3.32 g " 0.302 g 3.35 g " 0.295 g Seminiferous tubule diameter 263.625 mm " 8.85 mm 274.50 mm " 8.025 mm 259.125 mm " 13.65 mm 259.50 mm " 13.125 mm Data for bodyweight and testes weight are means" standard deviations and means" standard errors for seminiferous tubule diameters. Data were analysed by one-way analysis of variance.
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3.2. Apoptosis The microscopic appearance of the tubules was normal with no obvious abnormalities of spermatogenesis. Germ cells seen undergoing apoptosis in all groups are illustrated in Fig. 2 and were predominantly A-spermatogonia and zygotene spermatocytes; rarely late pachytene or diplotene spermatocytes were also seen to be labelled. Degenerating spermatid-heads at the base of the germinal epithelium were also occasionally encountered but were not included in the analysis since it is not known whether such TUNEL-labelling results from apoptosis or Sertoli cell-lysosome-mediated necrosis. Surprisingly, there was a dose-related decrease in the mean number of apoptotic cells per stage XIIrXIII tubule cross-section, the top dose group showing a reduction of nearly 2.5-fold Žwith all animals similarly affected.. This was significantly different from the controls by one-way ANOVA
Fig. 2. The detection of apoptotic, testicular germ-cells. The arrows indicate apoptotic cells: on the basis of their position in the germinal epithelium and spatial relationship to other cell types, they can be identified as 2 A-spermatogonia and 2 zygotene spermatocytes. The scale bar represents 20 mm.
followed by Student–Newman–Keul’s test Ž P 0.05. ŽFig. 1b.. ŽIn view of this finding, testicular DNA ladder data would be of very limited value since control levels of germ cell apoptosis in the rat testis are so low in relation to total cellular content w15x that a dose-related decrease would be virtually impossible to confirm by this approach.. 3.3. Controls for cell proliferation and tissue shrinkage The mean number Ž"standard error. of zygotene spermatocytes per stage XIIrXIII tubule cross-section was 63.5 Ž"8.12. in the controls and 65.3 Ž"9.01. in the 5.1 mgrkg CP treatment group. The data for mean tubule diameters are presented in Table 1. There was no significant difference between any of the groups Žone-way ANOVA..
4. Discussion
Fig. 1. Ža. Induction of foetal abnormalities by paternal administration of CP: data points represent the mean percentages of abnormal foetuses as a proportion of total live pups per litter; error bars are standard errors. The asterisk denotes a mean significantly different from that of the control group: P - 0.05. Žb. The incidence of apoptotic germ cells in the testes of rats treated with CP. Data points represent means per tubule cross-section per animal per group; error bars are standard errors. The asterisk denotes a mean significantly different from that of the control group: P - 0.05.
The most striking aspect of this study was the finding that sub-chronic treatment with CP induced a dose-related decrease in germ-cell apoptosis. Since there was no significant effect on seminiferous tubule diameters, that can be excluded as a source of bias. The germ cells present in rat stage XIIrXIII tubules are Žin ascending order of maturity. A-spermatogonia, zygotene spermatocytes, pachytenerdiplotene spermatocytes and step 12r13 spermatids Želongating.. Virtually all the apoptotic cells found belonged to the first two categories. It is conceivable that a treatment-related alteration in cellular prolifer-
M.H. Brinkworth, E. Nieschlagr Mutation Research 447 (2000) 149–154
ation could have altered the spermatogonia or spermatocyte population sizes, leading to apparent changes in the proportion of apoptotic cells. However, the lack of any difference between the mean number of zygotene spermatocytes per tubule crosssection effectively excludes this possibility. This is, therefore, to the best of our knowledge, the first report to demonstrate a toxin-induced reduction in the incidence of testicular germ-cell apoptosis. It is particularly interesting that this reduction is mediated by exposure to low doses of a compound that at higher doses can elevate the incidence. Furthermore, the dosing regimen of CP that leads to the decreased incidence of apoptosis also leads to foetal abnormalities. The significance of the findings thus extends to the understanding both of apoptotic processes in the testis and of the heritability of mutations. The principal cell types in which dominant lethal mutations are induced by prolonged dosing with low doses of CP are elongating spermatids and sperm undergoing epididymal maturation because the effect disappears within 4 weeks of cessation of treatment w18x. ŽThat these are the cell types affected by acute doses has also been directly shown by experiments involving ligation of the efferent ducts w19x.. Foetal abnormalities, however, are only seen after prolonged treatment w10–12x and the present work. Together, these data suggest that CP may induce a different spectrum of DNA damage in different cell types ie: exposure of pre-meiotic cells Že.g., spermatogonia and zygotene spermatocytes. may cause damage that can lead to foetal abnormalities whereas exposure of elongating spermatids and maturing sperm results in dominant lethal mutations. High doses Že.g., 70 mgrkg CP or above. increase apoptosis in pre-meiotic cells, which may serve to prevent the transmission of heritable abnormalities w14x. This accords with the present data, which provide the first experimental evidence showing that abnormalities occur when apoptosis is reduced. Lower doses may not induce sufficient damage to be a mutational hazard unless repeated, in which case the induction of apoptosis among these pre-meiotic cells Žand presumably also DNA repair. will compensate. However, the negative effect of low doses of CP on the induction of apoptosis evidently overwhelms these protective mechanisms,
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so that a significant proportion of the sperm surviving to participate in fertilization and yield viable offspring carry heritable mutations. It is interesting that the level of apoptosis in the 3.4 mgrkg CP group was also much lower than that of the controls, whereas there was no evidence of an induction of abnormalities in this group, in agreement with previous studies w10,11x. The induction of genetic damage by CP does not show a similar threshold in the dose range tested as regards dominant lethality. Therefore, it may be more likely that the threshold for abnormalities stems not from the induction of damage but from its persistence. Although other factors such as DNA repair are likely to be involved, it may be that the lower amount of damage induced at 3.4 mgrkg, together with the higher rate of apoptosis, is sufficient to reduce the number of damaged cells surviving to become mature sperm to below the level at which they can be detected in a mating assay. How CP may act to reduce apoptosis presumably depends on being administered long-term in low doses. As a powerful alkylating agent, CP will alkylate a range of cellular macromolecules. These may include the mRNA and proteins that mediate apoptotic responses, thus poisoning the machinery of apoptosis in susceptible germ cells. Alternatively, CP may damage the DNA of genes required for germ cell apoptosis. This would have highly important implications for the transmission of abnormalities to the F1 generation, since it implies that inactivating mutations in such genes, by allowing the survival of damaged cells that would otherwise have died by apoptosis, will be more likely to be passed on to the F1 generation than other debilitating mutations. In F1 animals such mutations are likely to pre-dispose towards cancer or developmental abnormalities by impairing the ability of cells to die when instructed to, especially if ‘hit’ by a second inactivating event in the maternal allele. This, then, could provide a mechanism to explain the increase in cases of cancer among the children of men who smoke compared with non-smoking men w5–7x. Likewise, it may account for the induction of mutations pre-disposing to cancer in the male germ-cells of mice dosed with 239 plutonium w9x and for the occurrence of foetal abnormalities in the offspring of CP-treated rats w10– 12x wand the present workx.
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Acknowledgements We are grateful to M. Heuerman, M. Niemeier J. Esselmann and G. Stelke, for technical assistance and to Drs. T.G. Cooper, J. Gromoll, A. Lerchl, S. Schlatt and G.F. Weinbauer for helpful comments and suggestions during the preparation of the manuscript. The work was supported by the Bundesministerium fur ¨ Bildung, Wissenschaft, Forschung und Technologie of the Federal Republic of Germany, project: 07GTX02: ‘Umweltbedingte Veranderungen der vaterlichen Erbsubstanz’. ¨ ¨
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