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Effects of paternal ethylene dibromide exposure on F1 generation behavior in the rat
Mutation Research, 139 (1984) 133-138
133
Elsevier
MRLett 0533
Effects of paternal ethylene dibromide exposure o n F1 generation behavior in the rat* Donatella
F a n i n i a, M a r v i n
S. L e g a t o r b a n d P e r r i e M . A d a m s c
Department o f Pharmacology, Libero Istituto Universitario di Medicina e Chirurgia, L "Aquila (Italy), b Department o f Preventive Medicine and Community Health, and c Departments o f Psychiatry and Behavioral Sciences, Pharmacology and Toxicology, The University o f Texas Medical Branch, Galveston, T X (U.S.A.) (Accepted 14 December 1983)
Summary The effects of ethylene dibromide (EDB) exposure to the male rat were studied through behavioral assessments of their F1 progeny. Exposed males were bred with untreated female rats at 4 or 9 w e e ~ after 5 daily EDB treatments. Behavioral assessment of motor reflexes and motor coordination were examined up to 21 days of age. Significant differences in the development of motor coordination and motor activity were observed in the F~ progeny of EDB-exposed males. These results support the evidence of EDB genotoxicity and further demonstrates the utility of behavioral end-points of the offspring as a sensitive means of assessing paternal reproductive risk.
Ethylene dibromide (EDB) was first produced on a commercial scale in the beginning of the 1920's. Production has increased rapidly since about 1960. The major use for EDB is as a gasoline additive in combination with ethylene dichloride. EDB is also used as a soil, grain and fruit fumigant, as a chemical intermediate in the synthesis of dyes and pharmaceuticals, and as a solvent for resins, gums and waxes. The adverse effects of EDB described in humans [22], were of acute toxicity resulting from inhalation, skin absorption and ingestion. Main observed
* Send correspondence to: Perrie M. Adams, Ph.D., Department of Psychiatry and Behavioral Sciences, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.). 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
target organs were liver, kidneys and testes. Also upper respiratory irritation and extensive degeneration of the heart as well as painful skin inflammation have been reported [26, 35, 24]. EDB produced squamous cell carcinoma of the stomach in rats and mice [23, 27], damaged hepatic DNA in rats [20, 21], and increased the tumor incidence in mammary gland, spleen, adrenal, liver, kidney and subcutaneous tissue in rats [37, 18]. Recent studies have shown that EDB may produce genetic and reproductive toxicity. EDB was shown to be mutagenic in bacteria and Drosophila melanogaster [10, 34, 28], affected spermatogenesis in rats, bulls and rams [12, 3-8, 11, 13], and impaired the permeability of hen-egg vitelline membrane to macromolecules [15]. However, there was no evidence of dominant
134
lethality in male rats that inhaled 19 or 39 ppm of EDB, while inhalation of 89 ppm of EDB produced infertility [29-31, 19]. The anti-fertility effects o f EDB in male Wistar rats were investigated by Edwards et al. [12]. Their results showed subfertility during the 3rd week after treatment, and that the sterility had resulted from damage to spermatids. A number of chemicals and drugs have been reported to have adverse behavioral effects on the offspring of exposed male parents including alcohol, lead, caffeine, morphine, methadone, marijuana and cyclophosphamide [17, 25, 9, 16, 1-2, 14]. A recent study of Smith and Goldman [32] on the behavioral effects of prenatal exposure to EDB showed significant abnormalities compared to the control litters. However, they suggested that the observed behavioral effects on the offspring may be mediated by a stress reaction in the medium and high dosage EDB exposed mothers. The present work is part of a project designed to demonstrate the sensitivity of behavioral assessments of the developing offspring from exposed male parents as a possible measure of EDB genotoxicity.
Materials and methods
Animals Adult Fisher 344 strain male rats (90-120 days old) derived from the breeding colony in our laboratory (1 lth generation) were used. An inbred strain was chosen in order to reduce genetic heterogeneity.
Treatment EDB was obtained from Aldrich Chemical Company, Milwaukee, WI, with a purity of 99O7o. The male rats were treated with a daily dosage of EDB (1.25, 2.5, 5.0, 10.0 mg/kg) on a subacute i.p. schedule (for 5 successive days). Corn oil was used as a vehicle and the solutions were prepared daily. The amount of fluid administered was 0.1 ml/100 g of body weight. Groups of males that received a
5-day i.p. dose of corn oil (0.1 ml/100 g) served as a vehicle control. An additional saline control group was used to control for the possible effects of corn oil alone. During the treatment, the rats were kept under a class A hood in order to reduce the exposure of laboratory workers to the exhaled EDB.
Breeding Beginning 4 weeks after the last injection, the EDB-treated males were crossed with untreated virgin females (2:1 female:male ratio). The breeding continued for 7 days. Successful mating was indicated by the presence o f a vaginal sperm plug (day 0 of pregnancy). A second breeding with untreated females occurred 9 weeks after the last EDB treatment. This breeding schedule was followed in order to compare a mid-stage (spermatocyte) with an early germ cell stage (i.e. spermatogonia).
Preweaning behavioral assessment On postnatal day 3 the pups were weighed and the sex determined. Subsequent measurement of body weight was made on days 7, 10, 14 and 21. The assessments of the behavioral development were made on all the pups using an extensive testing battery. The battery included assessment of simple reflexes (e.g. surface righting, cliff avoidance and negative geotaxis), motor coordination (e.g. swimming), and locomotor activity. These behaviors develop quite rapidly in the rat and reach essentially an adult level at an early stage. This allows for early detection of a genotoxic effect resulting from paternal exposure. All of the pups received the same amount of testing regardless of their previous performance in order to control for handling and testing effects.
Surface righting The ability of the young rat to right itself was determined on 3 daily trials beginning on postnatal day 3. The pups were placed on their back on a smooth wooden surface and the time required to right themselves to a position where all four touch the surface was recorded. A criterion of successful
135
righting within two seconds was used. The pups were tested daily on postnatal days 3-6.
Cliff avoidance
Negative geotaxis Negative geotaxis was assessed by recording the time required for the neonatal rat to re-orient from a head down to a head up position on a 25 ° inclined plane. The plane was made of plywood and each test rat was given one trial on day 10 and 15. Pups sliding off the plane were tested again. The time required to complete a 180 ° turn was the dependent measure.
Beginning on postnatal day 4 each pup was placed on a wooden platform elevated 20 cm above a table top. The forepaws and snout of the animal were positioned so that the edge of the platform passed just behind an imaginary line drawn between the eye orbits. The latency required for a retraction of the head completely behind the edge of the platform was recorded daily. A criterion of a complete head retraction within 20.0 sec was used. If the animal was not successful within 60 sec the trial was terminated. If the animal fell it was given a second attempt and if it fell again a latency o f 60 sec was recorded. All pups were tested daily on postnatal days 4 - 1 0 regardless of whether they had reached criterion.
The data were statistically analyzed using 2-way analysis of variance methods (ANOVA) [36]. The mean of the litter for the behavioral assessment was used as the unit of measurement in most of analyses. For cliff avoidance and swimming the percent of the litter reaching the criterion for that behavior was used. The level for statistical significance was selected to be a p < 0 . 0 5 .
Swimming
Results
The ability of the neonatal rat to swim was assessed in a 30 × 30 cm plastic tank with the water temperature maintained at 24°C. On postnatal days 6, 8, 10, 12, 14 and 16 each animal was placed into the tank and swimming behavior rated for direction (straight = 3, circling = 2, floating = 1) and head angle (ears out of the water = 4, ears half out of the water = 3, nose and top of the head out of the water = 2, and unable to hold head up = 1). Limb movement was rated as either 1 = all four limbs used, or 2 = back limbs only used. Latency to begin limb movement after being placed in the water was also recorded. The animals were given a maximum of 15 sec in the water and were then removed to a warm towel to dry.
Open field activity On days 14 and 21 open field activity was measured. The pups were placed in the center of a 45-cm Plexiglas circle for three 1-min periods separated by 30 sec between each period. The circle was divided into 3 concentric circles with each quarter divided into 6 areas. The number of entries made during each 1-min period was measured.
Statistical methods
A total of 19 litters composed of 172 animals (84 males and 88 females) were obtained from breeding the exposed males with untreated females. There were no significant differences in litter size between the control and EDB treatment at any of the dosage-breeding time combinations.
Body weight The results on a repeated measures A N O V A on body weight at postnatal days 3, 7, 10, 14 and 21 revealed no significant differences for the offspring of either the 4- or 9-week breeding times regardless of treatment group.
Surface righting and negative geotaxis Paternal EDB exposure did not significantly alter the development of surface righting ability in the 3 - 6 - d a y old neonates. Similarly, the ability of day 10 and day 15 neonates to demonstrate negative geotaxis was not affected by paternal EDB exposure.
Cliff avoidance The results of the A N O V A on the acquisition of
136
cliff avoidance in the F1 progeny of EDB exposed males when bred at 9 weeks were not significant. The results of the ANOVA on the F~ progeny of males exposed to EDB and bred at 4 weeks were significant (F 12.3, p <0.01, 4, 70 df). An examination of the EDB dosage differences revealed that these ANOVA results were due to the suppressed performance of the 25 mg/kg EDB exposure offspring on postnatal days 4 and 5. These animals were not significantly different from the control offspring after day 5.
Swimming performance (a) Direction. The result of the ANOVA on swimming direction for postnatal days 6-12 indicated no significant impairment in the offspring of the 9-week breeding but a significantly smaller percentage of the offspring swimming straight following EDB exposure in the 4-week breeding litters (F 3.1, p < 0.05, 4, 32 df). This difference was accounted for by the greater percentage of offspring swimming in circles on postnatal day 6 and 8 (Fig. 1). (b)Head angle. The development of the neonatal rat to raise its head higher with age when forced to swim was found to be significantly slower (F7.22,p <0.01, 3, 32 df) for the offspring of EDB exposed males bred 9 weeks following exposure. The offspring of the 4-week post-EDB ex-
posure were not significantly different than the controls. (c) Limb movement. The rat typically swims with the hindlimbs and keeps the forelimbs stationary by postnatal day 16. The offspring of the EDB exposed males bred at 4 weeks were found to have significantly fewer animals swimming in this way on day 16 (F 61.8, p <0.01, 4, 10 df). These analyses indicate that swimming development is significantly impaired in the F1 offspring by paternal EDB exposure and that the manifestations of the impairment is dependent upon the time of breeding following exposure and the particular component of swimming behavior analyzed.
Open field activity The amount of ambulation in the open-field was significantly suppressed in the F1 offspring of EDB males regardless of breeding time (Fig. 2). In the offspring of the 4-week breeding there was a significant decrease in activity on both postnatal day 14 and 21 (F 7.62, p <0.01, 4, 24 df; F 5 . 1 , p < 0.01, 4, 24 df, respectively). Similarly, the offspring of the 9-week breeding were significantly less active on both day 14 (F 10.2, p <0.01, 3, 24 df) and day 21 (F 6.5, p <0.01, 3, 24 df). Day 14
10.08,06.0-
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SALINE
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~--~ EDB 25.0-4
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~ <
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20-
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• EDB 250 4 • EDB 250-9 [] EDB 50 0-9
12.0 80 4.0. 12
A g e (Days)
Fig. 1. The effect of paternal EDB exposure on the development o f swimming behavior in the F] progeny.
1
2
3
Minutes
Fig. 2. The effect of paternal EDB exposure on the FL progeny open-field activity on postnatal days 14 and 21.
137
Discussion The results presented here extend a previous report [32] on the sensitivity of behavioral endpoints to in utero EDB exposures by demonstrating the utility of behavioral end-points to assess the effects of paternal exposure. The present findings demonstrate the sensitivity of specific behavioral end-points (e.g. swimming and open field activity) to detect possible genotoxicity from paternal exposure to a chemical by assessing F1 offspring early in their development. Further, the sensitivity of these behavioral end-points was found to be greater than that reported with other genotoxic end-points such as dominant lethality or sperm morphology. Dominant lethality has not been demonstrated to be sensitive to EDB genotoxicity in the rat [33]. It was found to be insensitive to detect genotoxic effects at low EDB dosages and caused infertility at high dosages. Unpublished data using the sperm morphology assay indicated that the dosage o f EDB needed to produce abnormal sperm morphology was very close to the LD50 (80 m g / k g × 5). We were able to detect consistent behavioral abnormalities in the offspring of EDB-treated males at dosages as low as 6.25 (1.25 m g / k g / d a y × 5 days) in the 4-week breeding treatment. This dosage is considerably lower than the ones demonstrating dominant lethality, infertility or sperm morphology changes. The behavioral abnormalities were significant in the progeny of the 4th and 9th week breeding period and are consistent with an interpretation that extensive spermatogenic damage may occur with EDB which is not repaired. It should be pointed out that while we did observe significant behavioral effects on the FI offspring of EDB exposed male rats these effects did not always follow a linear dose-response function. This observation is frequently encountered in behavioral teratology when in utero drug exposure effects are being examined in the developing offspring. Similar difficulties are expected in studies where genetic toxicity is being evaluated in the offspring of exposed male parents.
These data suggest that premeiotic stages of rat spermatogenesis are sensitive to the genotoxic effects of EDB and demonstrate quite clearly that behavioral assessments of the developing Ft offspring from an EDB-exposed male are capable of detecting such an effect induced during spermatogenesis and transmitted through the sperm. Further studies will determine whether postmeiotic exposure to EDB in the male will produce behavioral differences in the F1 offspring and whether these first generation differences are transmitted to subsequent generations.
Acknowledgements This research was supported by N I O S H grant O H 01245. The authors would like to thank Ms. Carolyn J. Holt for her excellent assistance in preparation of this manuscript, Ms. Sing-Ling Luh, Mr. Peter Mancillas and Mr. Bruce Baily for technical assistance. Parts of this paper were presented at the annual meeting of the Behavioral Teratology Society at Atlantic City in June 1983.
References 1 Adams, P.M., J.D. Fabricant and M.S. Legator, Cyclophosphamide induced spermatogeniceffects detected in the F1 generation by behavioral testing, Science, 211 (1981) 80-83. 2 Adams, P.M., J.D. Fabricant and M.S. Legator, Active avoidance behavior in the Fl progeny of male rats exposed to cyclophosphamide prior to fertilization, Neurobehav. Toxicol. Teratog., 4 (1982) 531-534. 3 Amir, D., and R. Volcani, Effect of dietary ethylene dibromide on bull semen, Nature (London), 206 (1965) 99-100. 4 Amir, D., and R. Volcani, Effect of dietary ethylene dibromide on the testes of the bulls, Fertfl. Steril., 18 (1967) 144-148. 5 Amir, D., The sites of spermicidal action of ethylene dibromide in bulls, J. Reprod. Fertil., 35 (1973) 519-525. 6 Amir, D., Individual and age differences in the spermicidal effects of ethylene dibromide in bulls, J. Reprod. Fertil., 51 (1977) 453-456. 7 Amir, D., C. Esnault, J.C. Nicolle and M. Courot, DNA and protein changes in the spermatozoa of bulls treated oral-
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8
9
10
11
12
13
14
15
16 17
18
19
20
21 22
ly with ethylene dibromide, J. Reprod. Fertil., 51 (1977) 453-456. Amir, D., J.C. Nicolle and M. Courot, Changes induced in bull spermatids and testicular spermatozoa by a single peritesticular injection of ethylene dibromide, Int. J. Androl., 2(2) (1979) 162-170. Brady, K., Y. Herrera and H. Zenick, Influence of paternal lead exposure on subsequent learning ability of offspring, Pharm. Biochem. Behav., 3 (1975) 561-565. Brem, H., A.B. Stein and H.S. Rosenkranz, The mutagenicity and DNA-modifying effect of haloalkanes, Cancer Res., 34 (1974) 2576-2579. Courtens, J.L., D. Amir and J. Duranc, Abnormal spermiogenesis in bulls treated with ethylene dibromide: An ultrastructural and ultracytochemical study, J. Ultrastruct. Res., 71 (1980) 103-115. Edwards, K., H. Jackson and A.R. Jones, Studies with alkylating esters, II. A chemical interpretation through metabolic studies of the antifertility effects of ethylene dimethanesulphonate and ethylene dibromide, a commonly used pesticide, EMS Newslett., 3 (1970) 36-37. Eljack, A.H., and F. Hrudka, Pattern and dynamics of teratospermia induced in rams by paternal treatment with ethylene dibromide, J. Ultrastruct. Res., 67 (1979) 124-134. Fabricant, J.D., M.S. Legator and P.M. Adams, Postmeiotic cell mediation of behavior in progeny of male rats treated with cyclophosphamide, Mutation Res., 119 (1983) 185-190. Harduf, Z., and E. Alumot, Impaired permeability of the vitelline membrane to macromolecules in small eggs of hens poisoned with ethylene dibromide, Gen. Pharmacol., II (1979) 181-184. Joffe, J., Influence of drug exposure of the father on perinatal outcome, Clin. Perinatal., 6 (1979) 21. Klassen, R.W., and T. Persaud, Experimental studies on the influence of male alcoholism on pregnancy and progeny, Exp. Pathol., 12 (1976) 38. Kluwe, M., R. McNish and J.B. Hook, Acute nephrotoxicities and hepatotoxicities of 2-dibromo-3-chloropropane and 1,2-dibromoethane in male and female F344 rats, Toxicol. Lett., 8 (1981) 317-321. Minor, J.L., R.D. Short, J. Seifter and C.C. Lee, Effects of ethylene dibromide inhaled by rats and mice during gestation, Toxicol. Appl. Pharmacol., 45 (1978) 347. Nachtomi, E., and D.S.R. Sarma, Repair of rat liver DNA in vivo damaged by ethylene dibromide, Biochem. Pharmacol., 26 (1977) 1941-1945. Nachtomi, E., and E. Farber, Ethylene dibromide as a mitogen for liver, Lab. Invest., 38 (1978) 279-283. Olmstead, E.V., Pathological changes in ethylene dibromide poisoning, AMA Arch. Ind. Hyg. Occup. Med., 21 (1960) 45-49.
23 Olson, W.A., R.T. Haberman, E.K. Weisburger, J.M. Ward and J.H. Weisburger, Induction of stomach cancer in rats and mice by halogenated aliphatic fumigants, J. Natl. Cancer Inst., 51 (1973) 1993-1995. 24 Ott, M.G., H.C. Scharnweber and R.R. Langner, Mortality experience of 161 employees exposed to ethylene dibromide in two production units, Br. J. Ind. Med., 37 (1980) 163-168. 25 Pfeifer, W.D., J.R. Mackinnon and R.L. Seiser, Adverse effects of paternal alcohol consumption on offspring in the rat, Bull. Psychonom. Soc., 10 (1977) 246. 26 Pflesser, G., Skin damaging effect of ethylene dibromide, A constituent of liquid from remote water gauges (German), Arch. Gewerbepathol. Gewerbehyg., 8 (1928) 591-600. 27 Powers, M.B., R.W. Voelker, N.P. Page, E.K. Weisburger and H.F. Kraybill, Carcinogenicity of ethylene dibromide (EDB) and 1,2-dibromo-3-cyclopropane (DBCP) after oral administration in rats and mice, Toxicol. Appl. Pharmacol., 33 (1975) 171-172. 28 Rannug, U., Genotoxic effects of 1,2-dibromoethane and 1,2-dichloroethane, Mutation Res., 76 (1980) 269-295. 29 Short, R.D., J.L. Minor, B. Ferguson, T. Unger and C.C. Lee, Toxicity studies of selected chemicals, 1. Developmental toxicity of ethylene dibromide inhaled by rats and mice during organogenesis, USNTIS PB Report (PB-256 659): II (1976) 30 Short, R.D., J.L. Minor, J.M. Winston, J. Seifer and C.C. Lee, Inhalation of ethylene dibromide during gestation by rats and mice, Toxicol. Appl. Pharmacol., 46 (1978) 173-182. 31 Short, R.D., J.M. Winston, C.B. Hong, J.L. Minor, C.C. Lee and J. Seifer, Effects of ethylene dibromide on reproduction in male and female rats, Toxicol. Appl. Pharmacol., 49 (1979) 97-105. 32 Smith, R.F., and L. Goldman, Behavioral effects of prenatal exposure to ethylene dibromide, in press. 33 Teramoto, S., R. Saito, H. Aoyama and Y. Shirasu, Dominant lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane, Mutation Res., 77 (1980) 71-78. 34 Vogel, E., and J.L.R. Chandler, Mutagenicity testing of cyclamate and some pesticides in Drosophila melanogaster, Experientia, 30 (1974) 621-623. 35 Von Oettingen, The halogenated hydrocarbons: Toxicity and potential dangers, Publ. Hlth. Serv. Rep. No. 414 (1958) 152. 36 Winer, B.J., Statistical Principles in Experimental Design (1962), McGraw-Hill, New York. 37 Wong, C.K., J.M. Winston, C.B. Hong and H. Plotnick, Carcinogenicity and toxicity of 1,2-dibromoethane in the rat, Toxicot. Appl. Pharmacol., 63 (1982) 155-165.
133
Elsevier
MRLett 0533
Effects of paternal ethylene dibromide exposure o n F1 generation behavior in the rat* Donatella
F a n i n i a, M a r v i n
S. L e g a t o r b a n d P e r r i e M . A d a m s c
Department o f Pharmacology, Libero Istituto Universitario di Medicina e Chirurgia, L "Aquila (Italy), b Department o f Preventive Medicine and Community Health, and c Departments o f Psychiatry and Behavioral Sciences, Pharmacology and Toxicology, The University o f Texas Medical Branch, Galveston, T X (U.S.A.) (Accepted 14 December 1983)
Summary The effects of ethylene dibromide (EDB) exposure to the male rat were studied through behavioral assessments of their F1 progeny. Exposed males were bred with untreated female rats at 4 or 9 w e e ~ after 5 daily EDB treatments. Behavioral assessment of motor reflexes and motor coordination were examined up to 21 days of age. Significant differences in the development of motor coordination and motor activity were observed in the F~ progeny of EDB-exposed males. These results support the evidence of EDB genotoxicity and further demonstrates the utility of behavioral end-points of the offspring as a sensitive means of assessing paternal reproductive risk.
Ethylene dibromide (EDB) was first produced on a commercial scale in the beginning of the 1920's. Production has increased rapidly since about 1960. The major use for EDB is as a gasoline additive in combination with ethylene dichloride. EDB is also used as a soil, grain and fruit fumigant, as a chemical intermediate in the synthesis of dyes and pharmaceuticals, and as a solvent for resins, gums and waxes. The adverse effects of EDB described in humans [22], were of acute toxicity resulting from inhalation, skin absorption and ingestion. Main observed
* Send correspondence to: Perrie M. Adams, Ph.D., Department of Psychiatry and Behavioral Sciences, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.). 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
target organs were liver, kidneys and testes. Also upper respiratory irritation and extensive degeneration of the heart as well as painful skin inflammation have been reported [26, 35, 24]. EDB produced squamous cell carcinoma of the stomach in rats and mice [23, 27], damaged hepatic DNA in rats [20, 21], and increased the tumor incidence in mammary gland, spleen, adrenal, liver, kidney and subcutaneous tissue in rats [37, 18]. Recent studies have shown that EDB may produce genetic and reproductive toxicity. EDB was shown to be mutagenic in bacteria and Drosophila melanogaster [10, 34, 28], affected spermatogenesis in rats, bulls and rams [12, 3-8, 11, 13], and impaired the permeability of hen-egg vitelline membrane to macromolecules [15]. However, there was no evidence of dominant
134
lethality in male rats that inhaled 19 or 39 ppm of EDB, while inhalation of 89 ppm of EDB produced infertility [29-31, 19]. The anti-fertility effects o f EDB in male Wistar rats were investigated by Edwards et al. [12]. Their results showed subfertility during the 3rd week after treatment, and that the sterility had resulted from damage to spermatids. A number of chemicals and drugs have been reported to have adverse behavioral effects on the offspring of exposed male parents including alcohol, lead, caffeine, morphine, methadone, marijuana and cyclophosphamide [17, 25, 9, 16, 1-2, 14]. A recent study of Smith and Goldman [32] on the behavioral effects of prenatal exposure to EDB showed significant abnormalities compared to the control litters. However, they suggested that the observed behavioral effects on the offspring may be mediated by a stress reaction in the medium and high dosage EDB exposed mothers. The present work is part of a project designed to demonstrate the sensitivity of behavioral assessments of the developing offspring from exposed male parents as a possible measure of EDB genotoxicity.
Materials and methods
Animals Adult Fisher 344 strain male rats (90-120 days old) derived from the breeding colony in our laboratory (1 lth generation) were used. An inbred strain was chosen in order to reduce genetic heterogeneity.
Treatment EDB was obtained from Aldrich Chemical Company, Milwaukee, WI, with a purity of 99O7o. The male rats were treated with a daily dosage of EDB (1.25, 2.5, 5.0, 10.0 mg/kg) on a subacute i.p. schedule (for 5 successive days). Corn oil was used as a vehicle and the solutions were prepared daily. The amount of fluid administered was 0.1 ml/100 g of body weight. Groups of males that received a
5-day i.p. dose of corn oil (0.1 ml/100 g) served as a vehicle control. An additional saline control group was used to control for the possible effects of corn oil alone. During the treatment, the rats were kept under a class A hood in order to reduce the exposure of laboratory workers to the exhaled EDB.
Breeding Beginning 4 weeks after the last injection, the EDB-treated males were crossed with untreated virgin females (2:1 female:male ratio). The breeding continued for 7 days. Successful mating was indicated by the presence o f a vaginal sperm plug (day 0 of pregnancy). A second breeding with untreated females occurred 9 weeks after the last EDB treatment. This breeding schedule was followed in order to compare a mid-stage (spermatocyte) with an early germ cell stage (i.e. spermatogonia).
Preweaning behavioral assessment On postnatal day 3 the pups were weighed and the sex determined. Subsequent measurement of body weight was made on days 7, 10, 14 and 21. The assessments of the behavioral development were made on all the pups using an extensive testing battery. The battery included assessment of simple reflexes (e.g. surface righting, cliff avoidance and negative geotaxis), motor coordination (e.g. swimming), and locomotor activity. These behaviors develop quite rapidly in the rat and reach essentially an adult level at an early stage. This allows for early detection of a genotoxic effect resulting from paternal exposure. All of the pups received the same amount of testing regardless of their previous performance in order to control for handling and testing effects.
Surface righting The ability of the young rat to right itself was determined on 3 daily trials beginning on postnatal day 3. The pups were placed on their back on a smooth wooden surface and the time required to right themselves to a position where all four touch the surface was recorded. A criterion of successful
135
righting within two seconds was used. The pups were tested daily on postnatal days 3-6.
Cliff avoidance
Negative geotaxis Negative geotaxis was assessed by recording the time required for the neonatal rat to re-orient from a head down to a head up position on a 25 ° inclined plane. The plane was made of plywood and each test rat was given one trial on day 10 and 15. Pups sliding off the plane were tested again. The time required to complete a 180 ° turn was the dependent measure.
Beginning on postnatal day 4 each pup was placed on a wooden platform elevated 20 cm above a table top. The forepaws and snout of the animal were positioned so that the edge of the platform passed just behind an imaginary line drawn between the eye orbits. The latency required for a retraction of the head completely behind the edge of the platform was recorded daily. A criterion of a complete head retraction within 20.0 sec was used. If the animal was not successful within 60 sec the trial was terminated. If the animal fell it was given a second attempt and if it fell again a latency o f 60 sec was recorded. All pups were tested daily on postnatal days 4 - 1 0 regardless of whether they had reached criterion.
The data were statistically analyzed using 2-way analysis of variance methods (ANOVA) [36]. The mean of the litter for the behavioral assessment was used as the unit of measurement in most of analyses. For cliff avoidance and swimming the percent of the litter reaching the criterion for that behavior was used. The level for statistical significance was selected to be a p < 0 . 0 5 .
Swimming
Results
The ability of the neonatal rat to swim was assessed in a 30 × 30 cm plastic tank with the water temperature maintained at 24°C. On postnatal days 6, 8, 10, 12, 14 and 16 each animal was placed into the tank and swimming behavior rated for direction (straight = 3, circling = 2, floating = 1) and head angle (ears out of the water = 4, ears half out of the water = 3, nose and top of the head out of the water = 2, and unable to hold head up = 1). Limb movement was rated as either 1 = all four limbs used, or 2 = back limbs only used. Latency to begin limb movement after being placed in the water was also recorded. The animals were given a maximum of 15 sec in the water and were then removed to a warm towel to dry.
Open field activity On days 14 and 21 open field activity was measured. The pups were placed in the center of a 45-cm Plexiglas circle for three 1-min periods separated by 30 sec between each period. The circle was divided into 3 concentric circles with each quarter divided into 6 areas. The number of entries made during each 1-min period was measured.
Statistical methods
A total of 19 litters composed of 172 animals (84 males and 88 females) were obtained from breeding the exposed males with untreated females. There were no significant differences in litter size between the control and EDB treatment at any of the dosage-breeding time combinations.
Body weight The results on a repeated measures A N O V A on body weight at postnatal days 3, 7, 10, 14 and 21 revealed no significant differences for the offspring of either the 4- or 9-week breeding times regardless of treatment group.
Surface righting and negative geotaxis Paternal EDB exposure did not significantly alter the development of surface righting ability in the 3 - 6 - d a y old neonates. Similarly, the ability of day 10 and day 15 neonates to demonstrate negative geotaxis was not affected by paternal EDB exposure.
Cliff avoidance The results of the A N O V A on the acquisition of
136
cliff avoidance in the F1 progeny of EDB exposed males when bred at 9 weeks were not significant. The results of the ANOVA on the F~ progeny of males exposed to EDB and bred at 4 weeks were significant (F 12.3, p <0.01, 4, 70 df). An examination of the EDB dosage differences revealed that these ANOVA results were due to the suppressed performance of the 25 mg/kg EDB exposure offspring on postnatal days 4 and 5. These animals were not significantly different from the control offspring after day 5.
Swimming performance (a) Direction. The result of the ANOVA on swimming direction for postnatal days 6-12 indicated no significant impairment in the offspring of the 9-week breeding but a significantly smaller percentage of the offspring swimming straight following EDB exposure in the 4-week breeding litters (F 3.1, p < 0.05, 4, 32 df). This difference was accounted for by the greater percentage of offspring swimming in circles on postnatal day 6 and 8 (Fig. 1). (b)Head angle. The development of the neonatal rat to raise its head higher with age when forced to swim was found to be significantly slower (F7.22,p <0.01, 3, 32 df) for the offspring of EDB exposed males bred 9 weeks following exposure. The offspring of the 4-week post-EDB ex-
posure were not significantly different than the controls. (c) Limb movement. The rat typically swims with the hindlimbs and keeps the forelimbs stationary by postnatal day 16. The offspring of the EDB exposed males bred at 4 weeks were found to have significantly fewer animals swimming in this way on day 16 (F 61.8, p <0.01, 4, 10 df). These analyses indicate that swimming development is significantly impaired in the F1 offspring by paternal EDB exposure and that the manifestations of the impairment is dependent upon the time of breeding following exposure and the particular component of swimming behavior analyzed.
Open field activity The amount of ambulation in the open-field was significantly suppressed in the F1 offspring of EDB males regardless of breeding time (Fig. 2). In the offspring of the 4-week breeding there was a significant decrease in activity on both postnatal day 14 and 21 (F 7.62, p <0.01, 4, 24 df; F 5 . 1 , p < 0.01, 4, 24 df, respectively). Similarly, the offspring of the 9-week breeding were significantly less active on both day 14 (F 10.2, p <0.01, 3, 24 df) and day 21 (F 6.5, p <0.01, 3, 24 df). Day 14
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Fig. 1. The effect of paternal EDB exposure on the development o f swimming behavior in the F] progeny.
1
2
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Fig. 2. The effect of paternal EDB exposure on the FL progeny open-field activity on postnatal days 14 and 21.
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Discussion The results presented here extend a previous report [32] on the sensitivity of behavioral endpoints to in utero EDB exposures by demonstrating the utility of behavioral end-points to assess the effects of paternal exposure. The present findings demonstrate the sensitivity of specific behavioral end-points (e.g. swimming and open field activity) to detect possible genotoxicity from paternal exposure to a chemical by assessing F1 offspring early in their development. Further, the sensitivity of these behavioral end-points was found to be greater than that reported with other genotoxic end-points such as dominant lethality or sperm morphology. Dominant lethality has not been demonstrated to be sensitive to EDB genotoxicity in the rat [33]. It was found to be insensitive to detect genotoxic effects at low EDB dosages and caused infertility at high dosages. Unpublished data using the sperm morphology assay indicated that the dosage o f EDB needed to produce abnormal sperm morphology was very close to the LD50 (80 m g / k g × 5). We were able to detect consistent behavioral abnormalities in the offspring of EDB-treated males at dosages as low as 6.25 (1.25 m g / k g / d a y × 5 days) in the 4-week breeding treatment. This dosage is considerably lower than the ones demonstrating dominant lethality, infertility or sperm morphology changes. The behavioral abnormalities were significant in the progeny of the 4th and 9th week breeding period and are consistent with an interpretation that extensive spermatogenic damage may occur with EDB which is not repaired. It should be pointed out that while we did observe significant behavioral effects on the FI offspring of EDB exposed male rats these effects did not always follow a linear dose-response function. This observation is frequently encountered in behavioral teratology when in utero drug exposure effects are being examined in the developing offspring. Similar difficulties are expected in studies where genetic toxicity is being evaluated in the offspring of exposed male parents.
These data suggest that premeiotic stages of rat spermatogenesis are sensitive to the genotoxic effects of EDB and demonstrate quite clearly that behavioral assessments of the developing Ft offspring from an EDB-exposed male are capable of detecting such an effect induced during spermatogenesis and transmitted through the sperm. Further studies will determine whether postmeiotic exposure to EDB in the male will produce behavioral differences in the F1 offspring and whether these first generation differences are transmitted to subsequent generations.
Acknowledgements This research was supported by N I O S H grant O H 01245. The authors would like to thank Ms. Carolyn J. Holt for her excellent assistance in preparation of this manuscript, Ms. Sing-Ling Luh, Mr. Peter Mancillas and Mr. Bruce Baily for technical assistance. Parts of this paper were presented at the annual meeting of the Behavioral Teratology Society at Atlantic City in June 1983.
References 1 Adams, P.M., J.D. Fabricant and M.S. Legator, Cyclophosphamide induced spermatogeniceffects detected in the F1 generation by behavioral testing, Science, 211 (1981) 80-83. 2 Adams, P.M., J.D. Fabricant and M.S. Legator, Active avoidance behavior in the Fl progeny of male rats exposed to cyclophosphamide prior to fertilization, Neurobehav. Toxicol. Teratog., 4 (1982) 531-534. 3 Amir, D., and R. Volcani, Effect of dietary ethylene dibromide on bull semen, Nature (London), 206 (1965) 99-100. 4 Amir, D., and R. Volcani, Effect of dietary ethylene dibromide on the testes of the bulls, Fertfl. Steril., 18 (1967) 144-148. 5 Amir, D., The sites of spermicidal action of ethylene dibromide in bulls, J. Reprod. Fertil., 35 (1973) 519-525. 6 Amir, D., Individual and age differences in the spermicidal effects of ethylene dibromide in bulls, J. Reprod. Fertil., 51 (1977) 453-456. 7 Amir, D., C. Esnault, J.C. Nicolle and M. Courot, DNA and protein changes in the spermatozoa of bulls treated oral-
138
8
9
10
11
12
13
14
15
16 17
18
19
20
21 22
ly with ethylene dibromide, J. Reprod. Fertil., 51 (1977) 453-456. Amir, D., J.C. Nicolle and M. Courot, Changes induced in bull spermatids and testicular spermatozoa by a single peritesticular injection of ethylene dibromide, Int. J. Androl., 2(2) (1979) 162-170. Brady, K., Y. Herrera and H. Zenick, Influence of paternal lead exposure on subsequent learning ability of offspring, Pharm. Biochem. Behav., 3 (1975) 561-565. Brem, H., A.B. Stein and H.S. Rosenkranz, The mutagenicity and DNA-modifying effect of haloalkanes, Cancer Res., 34 (1974) 2576-2579. Courtens, J.L., D. Amir and J. Duranc, Abnormal spermiogenesis in bulls treated with ethylene dibromide: An ultrastructural and ultracytochemical study, J. Ultrastruct. Res., 71 (1980) 103-115. Edwards, K., H. Jackson and A.R. Jones, Studies with alkylating esters, II. A chemical interpretation through metabolic studies of the antifertility effects of ethylene dimethanesulphonate and ethylene dibromide, a commonly used pesticide, EMS Newslett., 3 (1970) 36-37. Eljack, A.H., and F. Hrudka, Pattern and dynamics of teratospermia induced in rams by paternal treatment with ethylene dibromide, J. Ultrastruct. Res., 67 (1979) 124-134. Fabricant, J.D., M.S. Legator and P.M. Adams, Postmeiotic cell mediation of behavior in progeny of male rats treated with cyclophosphamide, Mutation Res., 119 (1983) 185-190. Harduf, Z., and E. Alumot, Impaired permeability of the vitelline membrane to macromolecules in small eggs of hens poisoned with ethylene dibromide, Gen. Pharmacol., II (1979) 181-184. Joffe, J., Influence of drug exposure of the father on perinatal outcome, Clin. Perinatal., 6 (1979) 21. Klassen, R.W., and T. Persaud, Experimental studies on the influence of male alcoholism on pregnancy and progeny, Exp. Pathol., 12 (1976) 38. Kluwe, M., R. McNish and J.B. Hook, Acute nephrotoxicities and hepatotoxicities of 2-dibromo-3-chloropropane and 1,2-dibromoethane in male and female F344 rats, Toxicol. Lett., 8 (1981) 317-321. Minor, J.L., R.D. Short, J. Seifter and C.C. Lee, Effects of ethylene dibromide inhaled by rats and mice during gestation, Toxicol. Appl. Pharmacol., 45 (1978) 347. Nachtomi, E., and D.S.R. Sarma, Repair of rat liver DNA in vivo damaged by ethylene dibromide, Biochem. Pharmacol., 26 (1977) 1941-1945. Nachtomi, E., and E. Farber, Ethylene dibromide as a mitogen for liver, Lab. Invest., 38 (1978) 279-283. Olmstead, E.V., Pathological changes in ethylene dibromide poisoning, AMA Arch. Ind. Hyg. Occup. Med., 21 (1960) 45-49.
23 Olson, W.A., R.T. Haberman, E.K. Weisburger, J.M. Ward and J.H. Weisburger, Induction of stomach cancer in rats and mice by halogenated aliphatic fumigants, J. Natl. Cancer Inst., 51 (1973) 1993-1995. 24 Ott, M.G., H.C. Scharnweber and R.R. Langner, Mortality experience of 161 employees exposed to ethylene dibromide in two production units, Br. J. Ind. Med., 37 (1980) 163-168. 25 Pfeifer, W.D., J.R. Mackinnon and R.L. Seiser, Adverse effects of paternal alcohol consumption on offspring in the rat, Bull. Psychonom. Soc., 10 (1977) 246. 26 Pflesser, G., Skin damaging effect of ethylene dibromide, A constituent of liquid from remote water gauges (German), Arch. Gewerbepathol. Gewerbehyg., 8 (1928) 591-600. 27 Powers, M.B., R.W. Voelker, N.P. Page, E.K. Weisburger and H.F. Kraybill, Carcinogenicity of ethylene dibromide (EDB) and 1,2-dibromo-3-cyclopropane (DBCP) after oral administration in rats and mice, Toxicol. Appl. Pharmacol., 33 (1975) 171-172. 28 Rannug, U., Genotoxic effects of 1,2-dibromoethane and 1,2-dichloroethane, Mutation Res., 76 (1980) 269-295. 29 Short, R.D., J.L. Minor, B. Ferguson, T. Unger and C.C. Lee, Toxicity studies of selected chemicals, 1. Developmental toxicity of ethylene dibromide inhaled by rats and mice during organogenesis, USNTIS PB Report (PB-256 659): II (1976) 30 Short, R.D., J.L. Minor, J.M. Winston, J. Seifer and C.C. Lee, Inhalation of ethylene dibromide during gestation by rats and mice, Toxicol. Appl. Pharmacol., 46 (1978) 173-182. 31 Short, R.D., J.M. Winston, C.B. Hong, J.L. Minor, C.C. Lee and J. Seifer, Effects of ethylene dibromide on reproduction in male and female rats, Toxicol. Appl. Pharmacol., 49 (1979) 97-105. 32 Smith, R.F., and L. Goldman, Behavioral effects of prenatal exposure to ethylene dibromide, in press. 33 Teramoto, S., R. Saito, H. Aoyama and Y. Shirasu, Dominant lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane, Mutation Res., 77 (1980) 71-78. 34 Vogel, E., and J.L.R. Chandler, Mutagenicity testing of cyclamate and some pesticides in Drosophila melanogaster, Experientia, 30 (1974) 621-623. 35 Von Oettingen, The halogenated hydrocarbons: Toxicity and potential dangers, Publ. Hlth. Serv. Rep. No. 414 (1958) 152. 36 Winer, B.J., Statistical Principles in Experimental Design (1962), McGraw-Hill, New York. 37 Wong, C.K., J.M. Winston, C.B. Hong and H. Plotnick, Carcinogenicity and toxicity of 1,2-dibromoethane in the rat, Toxicot. Appl. Pharmacol., 63 (1982) 155-165.