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Prenatal versus postnatal effects on offspring weight gain of rats exposed to diphenhydramine: A critical evaluation of fostering procedures in rats
Comp. Biochem. Physiol. Vol. 99A, No. 1/2, pp. 219-221, 1991 Printed in Great Britain
0300-9629/91 $3.00 + 0.00 © 1991 Pergamon Press plc
PRENATAL VERSUS POSTNATAL EFFECTS ON OFFSPRING WEIGHT GAIN OF RATS EXPOSED TO DIPHENHYDRAMINE: A CRITICAL EVALUATION OF FOSTERING PROCEDURES IN RATS SILVANA CHIAVEGATTOand MARIA MARTHA BERNARDI*~" Departamento de Farmacologia, Instituto de Ci~ncias Biomrdicas, Universidade de S~o Paulo, 05508 Sho Paulo, SP, Brazil; *Departamento de Patologia, Faculdade de Medicina Veterin~ria e Zootecnia, Universidade de S~o Paulo, 05340 S~o Paulo, SP, Brazil. Telephone: 210-2122/438 (Received 10 August 1990) Abstract--1. In order to evaluate the relative contribution of fostering procedures in the analysis of the development of rats prenatally exposed to diphenhydramine (20 mg/kg/day, sc) the weight gain of litters fostered or not by their biological mothers were examined from 2-21 days of age. 2. Maternal behavior and milk production were also assessed. 3. The results showed a decreased weight gain only in offspring fostered by mothers from different prenatal treatments and a lack of effects on maternal behavior and milk produetion. 4. It was concluded that the reduced weight of cross-fostered litters was mediated through an interference with postnatal mother-offspring interaction, which was not herein identified.
INTRODUCTION
Longitudinal research into behavioral teratology is fraught with potentially confounding variables that may obscure or contribute to real effects. Litter size, changes in the mother's milk and maternal behavior due to prenatal drug administration, postnatal transmission of drugs administered prenatally, etc, might directly or indirectly affect the subsequent maturation and development of neonate (Spyker, 1975a). One of the most subtle of such influences is that o f postnatal effects induced by the mother. In mammals, any maternal treatment producing prenatal effects must also be considered capable of affecting offspring postnatally. In order to separate prenatal influences from subsequent maturation and development, fostering (exchanging offspring with similarly treated mothers) and crossfostering (exchanging treated progeny with control mothers and vice-versa) procedures should be employed prior to nursing. Additionally, control and experimental offspring should be raised by the biological mother to control the fostering variable itself (Spyker, 1975b). The present experiment was undertaken to evaluate the relative contribution of fostering procedures in the analysis of the development of rats prenatally exposed to diphenhydramine (D) an histamine-H~ antagonist. MATERIALS AND METHODS
Fifteen pregnant Wistar rats from our colony were housed in temperature-controlled (22°C) and artificially tCorrespondence should be addressed to: Dr M. M. Bernardi, Departamento de Patologia, Faculdade de Medicina Veterin~iria e Zootecnia, USP Av. Corifeu de Azevedo Marques, 2720, 05340 S~o Paulo, SP, Brazil. 219
lighted (12 hr light and 12hr dark, lights on at 6:00 a.m.) rooms with free access to food and water. Animals were treated with D (20 mg/kg) or the same volume of NaCl 0.9% solution (S), sc, daily, during the entire pregnancy. Following delivery, eight pups were left with each dam. Therefore, the progeny of two-three mothers were exchanged within the same (SSe and DDe groups) or different treatments (SD and DS groups) where the first letter indicates the mother treatment and the second the progeny prenatal treatment. The remaining litters (2-3) were raised by their own biological mothers (SS and DD groups). The body weight of all pups was measured at 2 and 21 days after birth and the weight gain was calculated by the body weight differences. The maternal behavior of the dams in their individual cages was evaluated by the scoring system of Sodersten and Eneroth (1984), which is based on the following observations: (0) absence of nest, (1) presence of a nest constructed from a sheet of paper towel available in each cage, (2) presence of all pups in the nest, (3) all pups warm, and (4) dam positioned over all pups in a nursing posture. These observations were performed once a day at 9:00 a.m., for every 21 days after delivery. Thirteen pregnant female rats, treated, as D and S groups above described with six and seven dams, respectively, were used to evaluate the milk production (Morag, 1970). Briefly, on the fourth day after the delivery, the litter of each dam was discarded at 9:00 hr. After 7 hr, the dam was anesthetised (pentobarbital, 30 mg/kg, ip), injected (sc) with oxytocin (0.5 IU) and placed on its flank in a cage. Three healthy control pups for each dam, 9-14 days old, and starved for 8 hr, were weighed and allowed to suckle for 40 min. A pup not sucking was discarded. After the first 20 min, an additional 0.5 IU of oxytocin was administered and the dam was turned on its other flank. The milk production during the 7 hr secretory period was taken as the difference between initial and final body weight of the pups. The milk production was analysed by the Student t-test (Snedecor, 1946) and the maternal behavior and weight gain by the Kruskal-Wallis analysis of variance followed by multiple comparison test (Hayes, 1989) since Bartlet's test (Johnson and Leone, 1964) showed that the data were respectively, homocedastic (P < 0.05) or not (P > 0.05).
220
SILVANA CHIAVEGATTO
and MARL~MARTHA BERNARDI
RESULTS AND D I S C U S S I O N
In the present study, indirect evaluation of milk production in mothers of both the experimental and control groups were not statistically different. Thus, the m e a n s o f the differences between the body weight of rats suckled by D (17 pups) or S (20 pups) treated mothers were, respectively, 1.3 and 1.1 g and the SEM was 0.1 g in both cases. These results show that D administration during the entire pregnancy was not capable to modify the milk production of dams; and this is an important finding since undernutrition during gestation and/or lactation results in differences in mean ages at the maturation of physical features and reflexes (Smart and Dobbing, 1971). Furthermore, a central histaminergic system controlling prolactin release has been demonstrated in rats (Hough, 1988) being this hormone able to modify milk production (Nicoll, 1974). As it is known, D induces central effects (Douglas, 1985), what would alter maternal behavior of dams treated during pregnancy, and this fact can modify pups development (Francov~, 1985). In this study, the median scores for maternal behavior at 1, 2 and 3 weeks postpartum for all treatment groups were 4.0, 4.0 and 3.0 respectively, and all medians were in the 3.0-4.0 range. No statistical differences were observed between the six groups analysed. Litters were weighed immediately after birth. An analysis of variance indicated no differences in this parameter (data not shown). Figure 1A stresses that prenatal exposure to D induced an evident decrease in the average weight gain of pups observed during lactation (SD group in relation to SSe group). At first sight, this finding suggests that in utero exposure to D resulted in (A)
40
"It
4t
t-
"~ 20 O')
/: SSe (B)
40 A
"~ e- 20
SS DD DDo Groups Fig. 1. (A) Weight gain of pups only exposed prenatally or postnatally to D or S. (B) Weight gain of pups exposed pre and postnatally to D or S. Pups of DDe and SSe groups were fostered by dams from the same treatment groups; pups of DS and SD groups were fostered by mothers from different treatments and DD and SS were raised by their biological mothers.
Table 1. The mediansand respectiveconfidencelimitsof weightgain of pups Groups No. of animals Weight gain (g) SS 16 31.0 (27.0-35.0) DD 24 28.0 (22.0-31.0) DDc 16 32.0 (28.0-34.0) SSe 16 28.5 (25.0-34.0) SD 24 24.0*(20.0-35.0) DS 24 24.5"~20.0-30.0) Prenatal x postnatal effects of diphenhydramineon weight gain of rats. Dams were treated, once a day, sc, during the entire pregnancy with 20 mg/kg diphenhydramine(D) or NaCI 0.9% (S). After birth, the offspringwere exchanged(or not) between mothers from differentor similartreatments. Weightgain was calculatedby the differencebetween the pups body weightfrom 2-21 days of age. Table 1 shows the medians and respective confidencelimits of weight gain of pups. *P < 0.05 (Kruskal-Wallismultiplecomparison test in relation to SSe group). developmental retardation of pups growing. Since D rapidly crosses the placental barrier of pregnant animals (Yoo et al., 1986), it may act on the fetus either by an interference with the placental function or directly via placental transfer. Data from Fig. IA also indicates that postnatal D effects (residual) decreased the average weight gain of pups (DS group in relation to SSe group). As shown, modification in milk production or maternal behavior cannot be involved with the present results. However, it is known that the milk contains hormone, neuropeptides and growth factors (Gupta, 1983) and from the gastrointestinal tract of suckling animals, various peptides and hormones can be absorbed with preserved biological properties (Koldovsky et al., 1986). Since these milk-borne factors may affect pituitary function of the suckling rat (Acs et al., 1977), it is reasonable to infer that residual D resulting from the treatment of dams during pregnancy might alter via milk the development of the progeny. So, pre and postnatal D effects could mediate the reduced weight gain of pups, possibly by different mechanisms. In general, pre and postnatal effects of drugs administered during pregnancy are additive (Spyker and Spyker, 1977). It was observed, however, that weight gain of animals perinatally exposed to D (the DD and DDe groups) did not show differences between those weight gains of the control groups (SS and SSe groups). These results make very difficult the acceptance of the hypothesis explaining the D pre and postnatal effects. Thus, it is possible that the reduced weight gain in DS and SD groups were mediated through an interference with postnatal motheroffspring interaction, not identified here. Finally, present observations might be important in many experimental studies about the pre or postnatal effects of drugs administered during pregnancy, usually carried with exchanged mothers. Thus, a careful examination of the results and attempts to identify all potentially experimental variables could validate some conclusions about prenatal versus postnatal drug effects studied by means of fostering procedures. REFERENCES
Acs A., Kacsoh B., Veress Z. and Toth B. E. (1988) Rat milk stimulates pituitary growth hormone secretion of neonatal pituitaries in vivo. Life Sci. 42, 2315-2321.
Prenatal versus postnatal effects of diphenhydramine Douglas W. W. (1985) Goodman and Gilman's the Pharmacological Basis of Therapeutics, pp. 396-417, Macmillan Publ. Co. Inc., New York. Francov~ S. (1985) Relationship between nutrition during lactation and maternal behaviour of rats. Activ. Nerv. Super. 13, 1-8. Gupta D. (1983) Hormones and human milk. Endocrinol. Exp. (Bratisl), 17, 359-370. Hayes A. W. (1989) Principles and Methods of Toxicology, pp. 455456. Raven Press, New York. Hough L. B. (1988) Cellular localization and possible functions for brain histamine: recent progress. Prog. Neurobiol. 30, 469-505. Johnson N. and Leone F. (1964) Engineering and Physical Sciences, pp. 241-244. John Wiley, New York. Koldovsky D., Bcdrick A. and Thornburg W. (1986) Processing of hormone-like substances from milk in the gastrointestinal tract of suckling rats. Endocrinol. Exp. (Bratisl) 20, 119-130. Morag M. (1970) Estimation of milk yield in the rat. Lab. Anim. 4, 259-272. Nicoll C. (1974) Handbook of Physiology, pp. 1253-1292. American Physiology Society, Washington.
221
Smart J. L. and Dobbing J. (1971) Vulnerability of developing brain II. Effects of early nutritional deprivation of reflex ontogeny and development of behaviour in the rat. Brain Res. 18, 85-95. Snedecor G. W. (1946) Statistical Methods, 4th edn, pp. 55--60. University Press, Iowa State. Sodersten P. and Eneroth P. (1984) Effects of exposure to pups on maternal behaviour, sexual behaviour and serum prolaetin concentrations in male rats. J. Endocrinol. 102, 115-119. Spyker J. M. (1975a) Assessing the impact of low level chemicals on development: behavioral and latent effects. Fed. Proc. 34, 1835-1844. Spyker J. M. (1975b) Behavioral Toxicology (Edited by Weiss B., Laties V. G.), pp. 311-314. Plenum Press, New York. Spyker D. A. and Spyker J. M. (1977) Response model analysis for cross-fostering studies: prenatal versus postnatal effects on offspring exposed to metilmercury dicyandiamide. Toxicol. Appl. Pharmacol. 40, 511-527. Yoo S. D., Axelson J. E., Taylor S. M. and Rurak D. W. (1986) Placental transfer of diphenhydramine in chronically instrumented pregnant sheep. J. Pharmacol. Sci. 75, 685-687.
0300-9629/91 $3.00 + 0.00 © 1991 Pergamon Press plc
PRENATAL VERSUS POSTNATAL EFFECTS ON OFFSPRING WEIGHT GAIN OF RATS EXPOSED TO DIPHENHYDRAMINE: A CRITICAL EVALUATION OF FOSTERING PROCEDURES IN RATS SILVANA CHIAVEGATTOand MARIA MARTHA BERNARDI*~" Departamento de Farmacologia, Instituto de Ci~ncias Biomrdicas, Universidade de S~o Paulo, 05508 Sho Paulo, SP, Brazil; *Departamento de Patologia, Faculdade de Medicina Veterin~ria e Zootecnia, Universidade de S~o Paulo, 05340 S~o Paulo, SP, Brazil. Telephone: 210-2122/438 (Received 10 August 1990) Abstract--1. In order to evaluate the relative contribution of fostering procedures in the analysis of the development of rats prenatally exposed to diphenhydramine (20 mg/kg/day, sc) the weight gain of litters fostered or not by their biological mothers were examined from 2-21 days of age. 2. Maternal behavior and milk production were also assessed. 3. The results showed a decreased weight gain only in offspring fostered by mothers from different prenatal treatments and a lack of effects on maternal behavior and milk produetion. 4. It was concluded that the reduced weight of cross-fostered litters was mediated through an interference with postnatal mother-offspring interaction, which was not herein identified.
INTRODUCTION
Longitudinal research into behavioral teratology is fraught with potentially confounding variables that may obscure or contribute to real effects. Litter size, changes in the mother's milk and maternal behavior due to prenatal drug administration, postnatal transmission of drugs administered prenatally, etc, might directly or indirectly affect the subsequent maturation and development of neonate (Spyker, 1975a). One of the most subtle of such influences is that o f postnatal effects induced by the mother. In mammals, any maternal treatment producing prenatal effects must also be considered capable of affecting offspring postnatally. In order to separate prenatal influences from subsequent maturation and development, fostering (exchanging offspring with similarly treated mothers) and crossfostering (exchanging treated progeny with control mothers and vice-versa) procedures should be employed prior to nursing. Additionally, control and experimental offspring should be raised by the biological mother to control the fostering variable itself (Spyker, 1975b). The present experiment was undertaken to evaluate the relative contribution of fostering procedures in the analysis of the development of rats prenatally exposed to diphenhydramine (D) an histamine-H~ antagonist. MATERIALS AND METHODS
Fifteen pregnant Wistar rats from our colony were housed in temperature-controlled (22°C) and artificially tCorrespondence should be addressed to: Dr M. M. Bernardi, Departamento de Patologia, Faculdade de Medicina Veterin~iria e Zootecnia, USP Av. Corifeu de Azevedo Marques, 2720, 05340 S~o Paulo, SP, Brazil. 219
lighted (12 hr light and 12hr dark, lights on at 6:00 a.m.) rooms with free access to food and water. Animals were treated with D (20 mg/kg) or the same volume of NaCl 0.9% solution (S), sc, daily, during the entire pregnancy. Following delivery, eight pups were left with each dam. Therefore, the progeny of two-three mothers were exchanged within the same (SSe and DDe groups) or different treatments (SD and DS groups) where the first letter indicates the mother treatment and the second the progeny prenatal treatment. The remaining litters (2-3) were raised by their own biological mothers (SS and DD groups). The body weight of all pups was measured at 2 and 21 days after birth and the weight gain was calculated by the body weight differences. The maternal behavior of the dams in their individual cages was evaluated by the scoring system of Sodersten and Eneroth (1984), which is based on the following observations: (0) absence of nest, (1) presence of a nest constructed from a sheet of paper towel available in each cage, (2) presence of all pups in the nest, (3) all pups warm, and (4) dam positioned over all pups in a nursing posture. These observations were performed once a day at 9:00 a.m., for every 21 days after delivery. Thirteen pregnant female rats, treated, as D and S groups above described with six and seven dams, respectively, were used to evaluate the milk production (Morag, 1970). Briefly, on the fourth day after the delivery, the litter of each dam was discarded at 9:00 hr. After 7 hr, the dam was anesthetised (pentobarbital, 30 mg/kg, ip), injected (sc) with oxytocin (0.5 IU) and placed on its flank in a cage. Three healthy control pups for each dam, 9-14 days old, and starved for 8 hr, were weighed and allowed to suckle for 40 min. A pup not sucking was discarded. After the first 20 min, an additional 0.5 IU of oxytocin was administered and the dam was turned on its other flank. The milk production during the 7 hr secretory period was taken as the difference between initial and final body weight of the pups. The milk production was analysed by the Student t-test (Snedecor, 1946) and the maternal behavior and weight gain by the Kruskal-Wallis analysis of variance followed by multiple comparison test (Hayes, 1989) since Bartlet's test (Johnson and Leone, 1964) showed that the data were respectively, homocedastic (P < 0.05) or not (P > 0.05).
220
SILVANA CHIAVEGATTO
and MARL~MARTHA BERNARDI
RESULTS AND D I S C U S S I O N
In the present study, indirect evaluation of milk production in mothers of both the experimental and control groups were not statistically different. Thus, the m e a n s o f the differences between the body weight of rats suckled by D (17 pups) or S (20 pups) treated mothers were, respectively, 1.3 and 1.1 g and the SEM was 0.1 g in both cases. These results show that D administration during the entire pregnancy was not capable to modify the milk production of dams; and this is an important finding since undernutrition during gestation and/or lactation results in differences in mean ages at the maturation of physical features and reflexes (Smart and Dobbing, 1971). Furthermore, a central histaminergic system controlling prolactin release has been demonstrated in rats (Hough, 1988) being this hormone able to modify milk production (Nicoll, 1974). As it is known, D induces central effects (Douglas, 1985), what would alter maternal behavior of dams treated during pregnancy, and this fact can modify pups development (Francov~, 1985). In this study, the median scores for maternal behavior at 1, 2 and 3 weeks postpartum for all treatment groups were 4.0, 4.0 and 3.0 respectively, and all medians were in the 3.0-4.0 range. No statistical differences were observed between the six groups analysed. Litters were weighed immediately after birth. An analysis of variance indicated no differences in this parameter (data not shown). Figure 1A stresses that prenatal exposure to D induced an evident decrease in the average weight gain of pups observed during lactation (SD group in relation to SSe group). At first sight, this finding suggests that in utero exposure to D resulted in (A)
40
"It
4t
t-
"~ 20 O')
/: SSe (B)
40 A
"~ e- 20
SS DD DDo Groups Fig. 1. (A) Weight gain of pups only exposed prenatally or postnatally to D or S. (B) Weight gain of pups exposed pre and postnatally to D or S. Pups of DDe and SSe groups were fostered by dams from the same treatment groups; pups of DS and SD groups were fostered by mothers from different treatments and DD and SS were raised by their biological mothers.
Table 1. The mediansand respectiveconfidencelimitsof weightgain of pups Groups No. of animals Weight gain (g) SS 16 31.0 (27.0-35.0) DD 24 28.0 (22.0-31.0) DDc 16 32.0 (28.0-34.0) SSe 16 28.5 (25.0-34.0) SD 24 24.0*(20.0-35.0) DS 24 24.5"~20.0-30.0) Prenatal x postnatal effects of diphenhydramineon weight gain of rats. Dams were treated, once a day, sc, during the entire pregnancy with 20 mg/kg diphenhydramine(D) or NaCI 0.9% (S). After birth, the offspringwere exchanged(or not) between mothers from differentor similartreatments. Weightgain was calculatedby the differencebetween the pups body weightfrom 2-21 days of age. Table 1 shows the medians and respective confidencelimits of weight gain of pups. *P < 0.05 (Kruskal-Wallismultiplecomparison test in relation to SSe group). developmental retardation of pups growing. Since D rapidly crosses the placental barrier of pregnant animals (Yoo et al., 1986), it may act on the fetus either by an interference with the placental function or directly via placental transfer. Data from Fig. IA also indicates that postnatal D effects (residual) decreased the average weight gain of pups (DS group in relation to SSe group). As shown, modification in milk production or maternal behavior cannot be involved with the present results. However, it is known that the milk contains hormone, neuropeptides and growth factors (Gupta, 1983) and from the gastrointestinal tract of suckling animals, various peptides and hormones can be absorbed with preserved biological properties (Koldovsky et al., 1986). Since these milk-borne factors may affect pituitary function of the suckling rat (Acs et al., 1977), it is reasonable to infer that residual D resulting from the treatment of dams during pregnancy might alter via milk the development of the progeny. So, pre and postnatal D effects could mediate the reduced weight gain of pups, possibly by different mechanisms. In general, pre and postnatal effects of drugs administered during pregnancy are additive (Spyker and Spyker, 1977). It was observed, however, that weight gain of animals perinatally exposed to D (the DD and DDe groups) did not show differences between those weight gains of the control groups (SS and SSe groups). These results make very difficult the acceptance of the hypothesis explaining the D pre and postnatal effects. Thus, it is possible that the reduced weight gain in DS and SD groups were mediated through an interference with postnatal motheroffspring interaction, not identified here. Finally, present observations might be important in many experimental studies about the pre or postnatal effects of drugs administered during pregnancy, usually carried with exchanged mothers. Thus, a careful examination of the results and attempts to identify all potentially experimental variables could validate some conclusions about prenatal versus postnatal drug effects studied by means of fostering procedures. REFERENCES
Acs A., Kacsoh B., Veress Z. and Toth B. E. (1988) Rat milk stimulates pituitary growth hormone secretion of neonatal pituitaries in vivo. Life Sci. 42, 2315-2321.
Prenatal versus postnatal effects of diphenhydramine Douglas W. W. (1985) Goodman and Gilman's the Pharmacological Basis of Therapeutics, pp. 396-417, Macmillan Publ. Co. Inc., New York. Francov~ S. (1985) Relationship between nutrition during lactation and maternal behaviour of rats. Activ. Nerv. Super. 13, 1-8. Gupta D. (1983) Hormones and human milk. Endocrinol. Exp. (Bratisl), 17, 359-370. Hayes A. W. (1989) Principles and Methods of Toxicology, pp. 455456. Raven Press, New York. Hough L. B. (1988) Cellular localization and possible functions for brain histamine: recent progress. Prog. Neurobiol. 30, 469-505. Johnson N. and Leone F. (1964) Engineering and Physical Sciences, pp. 241-244. John Wiley, New York. Koldovsky D., Bcdrick A. and Thornburg W. (1986) Processing of hormone-like substances from milk in the gastrointestinal tract of suckling rats. Endocrinol. Exp. (Bratisl) 20, 119-130. Morag M. (1970) Estimation of milk yield in the rat. Lab. Anim. 4, 259-272. Nicoll C. (1974) Handbook of Physiology, pp. 1253-1292. American Physiology Society, Washington.
221
Smart J. L. and Dobbing J. (1971) Vulnerability of developing brain II. Effects of early nutritional deprivation of reflex ontogeny and development of behaviour in the rat. Brain Res. 18, 85-95. Snedecor G. W. (1946) Statistical Methods, 4th edn, pp. 55--60. University Press, Iowa State. Sodersten P. and Eneroth P. (1984) Effects of exposure to pups on maternal behaviour, sexual behaviour and serum prolaetin concentrations in male rats. J. Endocrinol. 102, 115-119. Spyker J. M. (1975a) Assessing the impact of low level chemicals on development: behavioral and latent effects. Fed. Proc. 34, 1835-1844. Spyker J. M. (1975b) Behavioral Toxicology (Edited by Weiss B., Laties V. G.), pp. 311-314. Plenum Press, New York. Spyker D. A. and Spyker J. M. (1977) Response model analysis for cross-fostering studies: prenatal versus postnatal effects on offspring exposed to metilmercury dicyandiamide. Toxicol. Appl. Pharmacol. 40, 511-527. Yoo S. D., Axelson J. E., Taylor S. M. and Rurak D. W. (1986) Placental transfer of diphenhydramine in chronically instrumented pregnant sheep. J. Pharmacol. Sci. 75, 685-687.