Mutation Research, 229 (1990) 129-134 Elsevier
129
MUT 02705
Reversibility of the effects of chronic paternal exposure to cyclophosphamide on pregnancy outcome in rats B a r b a r a F. H a l e s
a
and Bernard Robaire b
Departments of Pharmacology and Therapeutics a,b and of Obstetrics and Gynecology b, Centre for the Study of Reproduction a.b McGill University, Montreal, Que. H3G 1 Y6 (Canada) (Received 6 August 1988) (Accepted 30 January 1989)
Keywords: Cyclophosphamide; Male-mediated toxicity; Pre-implantation loss; Post-implantation loss; Reversal; Pregnancy outcome in rats
Summary Low-dose chronic treatment of the male rat with the antitumor drug cyclophospharnide causes a timeand dose-dependent increase in pre- and post-implantation loss in the untreated females to which he is mated. The objective of the present study was to determine whether such effects are reversed, and if so at what time after cessation of drug treatment. Adult male Sprague-Dawley rats were gavage fed daffy, 6 times per week for 9 weeks, with saline (control) or with 1 of 3 doses of cyclophosphamide, 1.4, 3.4 or 5.1 mg/kg/day. After the 9 weeks of treatment and at 2-week intervals thereafter, each male was matdd with 2 females in proestrus. The females were caesarian sectioned 20 days later and pregnancy outcome assessed. After 9 weeks of drug treatment, pre-implantation loss increased more than 3-fold from 6% in the control group to 21% in the 5.1 m g / k g / d a y cyclophosphamide treatment group. Post-implantation loss increased in a dose dependent fashion from 5% in the control group to 74% in the 5.1 m g / k g / d a y cyclophosphamide treatment group. Pre-implantation loss rapidly decreased upon cessation of treatment with cyclophosphamide: within 2 weeks it had returned to within the control range. Within just 2 weeks after termination of drug treatment in the 5.1 m g / k g / d a y cyclophosphamide treatment group, post-implantation loss decreased by half to 44%; it had decreased to 11% by 4 weeks and then was maintained at 4-6% thereafter. In the 3.4 m g / k g / d a y cyclophosphamide treatment group, post-implantation loss returned to the control range by 4 weeks. Thus, the effects of paternally administered cyclophosphamide on progeny outcome are reversible. The timing of reversal suggests that the effects on pre-implantation loss are due to a drug effect on spermatozoa either in the epididymis or near the time of spermiation while those on post-implantation loss are due to an additional effect on spermatids in the seminiferous tubules.
Cyclophosphamide is extensively used as an anticancer agent. This alkylating agent has clearly been demonstrated to damage DNA; this results
Correspondence: Dr. Barbara F. Hales, Department of Pharmacology and Therapeutics, McCrill University, 3655 Drummond Street, Montreal, Que. H3G 1Y6 (Canada).
in base substitution mutations and chromosomal aberrations (Au et al., 1980; Hales, 1982; Mohn and Ellenberger, 1976). In addition, cyclophosphamide is a teratogen capable of causing various malformations, depending on the stage of development when the mother a n d / o r conceptus is exposed (Gibson and Becket, 1968; Hales, 1981; Schardein, 1976; Spielmann et al., 1977). The
0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
130 long-term administration of cyclophosphamide in man has been associated with gonadal dysfunction resulting in azoo- or oligozoo-spermia, and with an increase in follicle-stimulating hormone and luteir~iring hormone concentrations in response to stimulation with luteinizing hormone releasing hormone (Watson et al., 1985). It has been established in several laboratories that exposure of male rodents to cyclophosphamide can result in dominant lethality and in progeny whose behaviour is altered (Adams et al., 1981; Auroux and Dulioust, 1985; Auroux et al., 1986; Moreland et al., 1981). The post-meiotic germ cell, especially the spermatid, is most sensitive to the dominant lethal effect of an alkylating agent like cyclophosphamide. However, at least some of the effects of cyclophosphamide have been associated with the exposure of pre-meiotic germ cells to drug. For example, behavioural changes are found in the F 1 progeny produced from cyclophosphamide treated male rats 60 or 100 days post-treatment (Auroux and Dulioust, 1985; Auroux et al., 1986). The incidence of congenital malformations in progeny is also increased after continuous administration of cyclophosphamide to the male rat for 7 - 9 weeks (Trasler et al., 1985, 1986). Previous studies from our laboratory have shown that chronic low dose exposure of male rats to cyclophosphamide will result in dose- and time-dependent increases in both pre- and postimplantation loss and in congenital malformations in the females mated to treated males (Trasler et al., 1985, 1986, 1987). Pre-implantation loss was maximal 5 - 6 weeks after the initiation of drug treatment of the father, i.e. when the male germ cells were first exposed as spermatocytes or early spermatids, while post-implantation loss was increased after 2 - 4 weeks of drug treatment, i.e. when the germ cells were first exposed as spermatozoa or late spermatids. Unlike in the human situation, these effects are not associated with major alterations in the quantity of sperm produced, the reproductive-endocrine profile of the treated males or their sexual behaviour (Trasler et al., 1985). Such treatments did cause, expectedly, a decrease in leukocyte numbers (Trasler et al., 1987). In addition, the treatment of male rats with this drug did cause several significant but subtle
changes in the morphological and biochemical features of their epididymides (Trasler et al., 1988; Trasler and Robaire, 1988). The reversibility of any adverse effect on progeny which results from treatment of the father with an anticancer drug like cyclophosphamide is an important question. It is apparent that at least with respect to some parameters (e.g. learning ability of the progeny) a long-term risk exists. The objective of the present study was to determine if, an if so when, some of the deleterious effects of chronic treatment of males with cyclophosphamide on their progeny would be reversed after termination of drug treatment. Our results indicate a rapid and complete reversal of the dramatic effects of cyclophosphamide treatment of adult male rats on pre- and post-implantation loss. Materials and methods Animal treatments and mating schedule Adult male (300-350 g) and female (200-225 g) Sprague-Dawley rats from Charles River Canada Ltd. (St. Constant, Quebec) were maintained in the Mclntyre Animal Centre on a 14-h light:10-h dark cycle and provided with food and water ad libitum. One week after arrival the males were randomly divided into 4 treatment groups (n = 6/group). The control group was gavage fed saline (5 m l / k g / d a y ) 6 days a week for 9 weeks; the cyclophosphamide (Procytox R, Frank Homer, Montreal, Quebec) treatment groups received a dose of 1.4, 3.4 or 5.1 m g / k g / d a y . On the 7th day of the 9th week of treatment each male in each treatment group was mated overnight with 2 females in proestrus. Each male was also mated to 2 females in proestrus at 2-week intervals up to 12 weeks after the cessation of treatment. Hence 336 females were used in this study. Assessment of pregnancy outcome The vaginas of the females exposed to the male rats were flushed with saline the morning after exposure (day 0) to assess the presence of spermatozoa. On day 20 the spermatozoa positive females were killed. The ovaries were removed and the number of corpora lutea were counted. The number of implantations, resorptions and live
131
fetuses per pregnant female was determined. Preimplantation loss was assessed by calculating the difference between the number of implantations and the number of corpora lutea (representing the number of eggs ovulated) for each female. Thus, this loss may represent ova that were not fertilized or embryos that died prior to implantation. Postimplantation loss represents the number of resorption sites or dead fetuses per female. Live fetuses were blotted dry, weighed and examined for external malformations.
Data analysis The data were analyzed on a per litter basis using one way analysis of variance (ANOVA) with the new Duncan's multiple range test (Dunnet, 1967). P < 0.05 was the significance level used throughout. Results
After 9 weeks of treatment with cyclophosphamide, a significant increase in pre-implantation loss was noted in females mated to males treated with the highest dose of cyclophosphamide (5.1 mg/kg/day) used (Fig. 1). The pre-implantation loss in this group was 3.6 + 0.6 per litter. Expressed as a percentile (i.e. the difference between the implantations and the corpora lutea per corpora lutea), this loss was 21%. The two lower
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129
MUT 02705
Reversibility of the effects of chronic paternal exposure to cyclophosphamide on pregnancy outcome in rats B a r b a r a F. H a l e s
a
and Bernard Robaire b
Departments of Pharmacology and Therapeutics a,b and of Obstetrics and Gynecology b, Centre for the Study of Reproduction a.b McGill University, Montreal, Que. H3G 1 Y6 (Canada) (Received 6 August 1988) (Accepted 30 January 1989)
Keywords: Cyclophosphamide; Male-mediated toxicity; Pre-implantation loss; Post-implantation loss; Reversal; Pregnancy outcome in rats
Summary Low-dose chronic treatment of the male rat with the antitumor drug cyclophospharnide causes a timeand dose-dependent increase in pre- and post-implantation loss in the untreated females to which he is mated. The objective of the present study was to determine whether such effects are reversed, and if so at what time after cessation of drug treatment. Adult male Sprague-Dawley rats were gavage fed daffy, 6 times per week for 9 weeks, with saline (control) or with 1 of 3 doses of cyclophosphamide, 1.4, 3.4 or 5.1 mg/kg/day. After the 9 weeks of treatment and at 2-week intervals thereafter, each male was matdd with 2 females in proestrus. The females were caesarian sectioned 20 days later and pregnancy outcome assessed. After 9 weeks of drug treatment, pre-implantation loss increased more than 3-fold from 6% in the control group to 21% in the 5.1 m g / k g / d a y cyclophosphamide treatment group. Post-implantation loss increased in a dose dependent fashion from 5% in the control group to 74% in the 5.1 m g / k g / d a y cyclophosphamide treatment group. Pre-implantation loss rapidly decreased upon cessation of treatment with cyclophosphamide: within 2 weeks it had returned to within the control range. Within just 2 weeks after termination of drug treatment in the 5.1 m g / k g / d a y cyclophosphamide treatment group, post-implantation loss decreased by half to 44%; it had decreased to 11% by 4 weeks and then was maintained at 4-6% thereafter. In the 3.4 m g / k g / d a y cyclophosphamide treatment group, post-implantation loss returned to the control range by 4 weeks. Thus, the effects of paternally administered cyclophosphamide on progeny outcome are reversible. The timing of reversal suggests that the effects on pre-implantation loss are due to a drug effect on spermatozoa either in the epididymis or near the time of spermiation while those on post-implantation loss are due to an additional effect on spermatids in the seminiferous tubules.
Cyclophosphamide is extensively used as an anticancer agent. This alkylating agent has clearly been demonstrated to damage DNA; this results
Correspondence: Dr. Barbara F. Hales, Department of Pharmacology and Therapeutics, McCrill University, 3655 Drummond Street, Montreal, Que. H3G 1Y6 (Canada).
in base substitution mutations and chromosomal aberrations (Au et al., 1980; Hales, 1982; Mohn and Ellenberger, 1976). In addition, cyclophosphamide is a teratogen capable of causing various malformations, depending on the stage of development when the mother a n d / o r conceptus is exposed (Gibson and Becket, 1968; Hales, 1981; Schardein, 1976; Spielmann et al., 1977). The
0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
130 long-term administration of cyclophosphamide in man has been associated with gonadal dysfunction resulting in azoo- or oligozoo-spermia, and with an increase in follicle-stimulating hormone and luteir~iring hormone concentrations in response to stimulation with luteinizing hormone releasing hormone (Watson et al., 1985). It has been established in several laboratories that exposure of male rodents to cyclophosphamide can result in dominant lethality and in progeny whose behaviour is altered (Adams et al., 1981; Auroux and Dulioust, 1985; Auroux et al., 1986; Moreland et al., 1981). The post-meiotic germ cell, especially the spermatid, is most sensitive to the dominant lethal effect of an alkylating agent like cyclophosphamide. However, at least some of the effects of cyclophosphamide have been associated with the exposure of pre-meiotic germ cells to drug. For example, behavioural changes are found in the F 1 progeny produced from cyclophosphamide treated male rats 60 or 100 days post-treatment (Auroux and Dulioust, 1985; Auroux et al., 1986). The incidence of congenital malformations in progeny is also increased after continuous administration of cyclophosphamide to the male rat for 7 - 9 weeks (Trasler et al., 1985, 1986). Previous studies from our laboratory have shown that chronic low dose exposure of male rats to cyclophosphamide will result in dose- and time-dependent increases in both pre- and postimplantation loss and in congenital malformations in the females mated to treated males (Trasler et al., 1985, 1986, 1987). Pre-implantation loss was maximal 5 - 6 weeks after the initiation of drug treatment of the father, i.e. when the male germ cells were first exposed as spermatocytes or early spermatids, while post-implantation loss was increased after 2 - 4 weeks of drug treatment, i.e. when the germ cells were first exposed as spermatozoa or late spermatids. Unlike in the human situation, these effects are not associated with major alterations in the quantity of sperm produced, the reproductive-endocrine profile of the treated males or their sexual behaviour (Trasler et al., 1985). Such treatments did cause, expectedly, a decrease in leukocyte numbers (Trasler et al., 1987). In addition, the treatment of male rats with this drug did cause several significant but subtle
changes in the morphological and biochemical features of their epididymides (Trasler et al., 1988; Trasler and Robaire, 1988). The reversibility of any adverse effect on progeny which results from treatment of the father with an anticancer drug like cyclophosphamide is an important question. It is apparent that at least with respect to some parameters (e.g. learning ability of the progeny) a long-term risk exists. The objective of the present study was to determine if, an if so when, some of the deleterious effects of chronic treatment of males with cyclophosphamide on their progeny would be reversed after termination of drug treatment. Our results indicate a rapid and complete reversal of the dramatic effects of cyclophosphamide treatment of adult male rats on pre- and post-implantation loss. Materials and methods Animal treatments and mating schedule Adult male (300-350 g) and female (200-225 g) Sprague-Dawley rats from Charles River Canada Ltd. (St. Constant, Quebec) were maintained in the Mclntyre Animal Centre on a 14-h light:10-h dark cycle and provided with food and water ad libitum. One week after arrival the males were randomly divided into 4 treatment groups (n = 6/group). The control group was gavage fed saline (5 m l / k g / d a y ) 6 days a week for 9 weeks; the cyclophosphamide (Procytox R, Frank Homer, Montreal, Quebec) treatment groups received a dose of 1.4, 3.4 or 5.1 m g / k g / d a y . On the 7th day of the 9th week of treatment each male in each treatment group was mated overnight with 2 females in proestrus. Each male was also mated to 2 females in proestrus at 2-week intervals up to 12 weeks after the cessation of treatment. Hence 336 females were used in this study. Assessment of pregnancy outcome The vaginas of the females exposed to the male rats were flushed with saline the morning after exposure (day 0) to assess the presence of spermatozoa. On day 20 the spermatozoa positive females were killed. The ovaries were removed and the number of corpora lutea were counted. The number of implantations, resorptions and live
131
fetuses per pregnant female was determined. Preimplantation loss was assessed by calculating the difference between the number of implantations and the number of corpora lutea (representing the number of eggs ovulated) for each female. Thus, this loss may represent ova that were not fertilized or embryos that died prior to implantation. Postimplantation loss represents the number of resorption sites or dead fetuses per female. Live fetuses were blotted dry, weighed and examined for external malformations.
Data analysis The data were analyzed on a per litter basis using one way analysis of variance (ANOVA) with the new Duncan's multiple range test (Dunnet, 1967). P < 0.05 was the significance level used throughout. Results
After 9 weeks of treatment with cyclophosphamide, a significant increase in pre-implantation loss was noted in females mated to males treated with the highest dose of cyclophosphamide (5.1 mg/kg/day) used (Fig. 1). The pre-implantation loss in this group was 3.6 + 0.6 per litter. Expressed as a percentile (i.e. the difference between the implantations and the corpora lutea per corpora lutea), this loss was 21%. The two lower
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