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Lack of behavioural teratogenic effect of phenytoin in rats
39
Toxicology Letters, 22 (1984) 39-46 Elsevier
TOXLett.
1232
LACK OF BEHAVIOUBAL BATS (Behavioural
teratology;
TEBATOGENIC
test development;
EFFECT OF PHENYTOIN
phenytoin;
acrylamide;
IN
rat)
S.L. CASSIDY, G.P. ROSE* and R.W. HEND Shell Research Ltd., Shell Toxicology Laboratory, Sittingbourne Research Centre. Sittingboume, ME9 8AG (U.K.)
Kent
(Received. February 28th. 1984) (Accepted March 12th, 1984)
SUMMARY A small battery of tests (static and air-righting reflexes, eye opening and a narrowing bridge test) was used to investigate the behavioural teratogenic potential of prenatal oral exposure (day 7-19 of gestation) of up to 100 mg phenytoin/kg/day to female rats. No treatment-related effects upon mother or offspring were detected. In order to validate our narrowing bridge test, neonatal old rats were tested over 4 weeks following early postnatal exposure to acrylamide (5 x 50 mg/kg/day i.p.). These animals showed a severe transient deficit compared with saline-treated controls. Despite the absence of an effect using phenytoin, the narrowing bridge test is considered worthy of further validation.
INTRODUCTION
A programme of work was initiated to investigate the functional development of rat neonates during the pre- and postweaning period with the intention of evolving a battery of tests to assess the behavioural teratogenic potential of development chemicals. Three preweaning tests were selected; the static righting reflex, eye opening and the air righting reflex [l, 10, 121. One postweaning test was included in our battery, the narrowing bridge test, adapted from that used for testing adult rats [7, 91. Initial studies on the litters of female rats dosed on days 15 and 16 p.c. with up to 500 mg/kg/day of phenytoin or hydroxyurea showed no effects upon the off*To whom correspondence should be addressed. Abbreviations: p.c., post coitum; p.p., post partum. 0378-4274/84/$ 03.00 0 Elsevier Science Publishers B.V.
40
spring even in the presence of marked bodyweight reduction in the phenytointreated mothers. In a small study on litters of female rats treated i.p. with 500 mg hydroxyurea/kg on day 12 p.c., the pups showed no deficits on the preweaning tests (including a forward locomotion test) but, owing to the presence of structural teratogenicity (tail deformity, missing forepaw digits), they performed poorly in the narrowing bridge test. The aims of the present study were twofold: first to establish these methods in our laboratory and to use them to detect any possible functional changes following prenatal phenytoin exposure. Phenytoin was finally chosen, since it has been shown to induce developmental deficits in prenatally exposed Wistar rats in a treatment regime similar to the one used in our study [8]. Our second aim was to investigate the usefulness of the narrowing bridge test for behavioural teratogenicity studies in both the phenytoin-treated rats and also in rat pups treated directly with acrylamide, a substance known to cause peripheral neuropathy [4] and functional deficits in adult rats undergoing this test [7, 91. METHODS
The animals used in this study were Wistar rats bred and maintained in specific pathogen-free conditions in Shell Toxicology Laboratory. They were housed singly (except during mating) in solid-bottom polypropylene cages lined with sawdust (and woodwool during the last week of pregnancy in Experiment I). The room was maintained at 18-25°C with a relative humidity of 4060% and an automated light-darkness cycle (12 h : 12 h). The animals were maintained on LAD 2 powdered feed (Spratts Patent Ltd., Barking, Essex) and water ad lib. througout the study. Phenytoin (5,5’-diphenylhydantoin, sodium salt, 99% pure) was received from Sigma (London) Chemical Company, Ltd., Poole, Dorset (CAS number 630-93-3). It was formulated as aqueous suspensions in 0.5% (w/v) carboxymethylcellulose at concentrations such that the dosing volume for each group was 2.5 ml suspension/kg bodyweight. Acrylamide (CAS number 79-06-1, 97% pure) was received from BDH, Poole, Dorset. It was formulated as a 10% (w/v) solution in physiological saline for dosing at a daily rate of 0.5 ml/kg/day. Experiment
I
Each of 51 female rats (aged 15 weeks) was mated with a male (the day on which sperm was detected was designated day 0 p.c.). 42 females were successfully mated, housed singly and allocated to one of 3 treatment groups: 0, 50 or 100 mg phenytoin/kg bodyweight/day (14 per group). The rats were orally dosed from day 7-19 p.c. inclusive (each daily dose being based on the most recent bodyweight), and at the end of gestation, were allowed to deliver their litters (the day of birth being considered as day 0 p.p.). The females were weighed regularly throughout gestation and at the end of the weaning period. All females and offspring were observed daily for
41
clinical abnormalities. Various reproduction parameters of gestation length, percentage of pregnant rats, litter size and sexes, number and sex of preweaned deaths and litter weights were measured. One day after birth, each litter was culled to a maximum of 5 males and 5 females and on day 3 p.p., the neonates were identified by toe clipping. Postnatal behavioural testing of offspring For the eye-opening and both righting reflexes, each neonate was tested once from the appropriate day and the reflex was scored as present or absent. If the neonate did not respond, it was retested on subsequent days until a positive reflex was achieved. (i) Static righting reflex. From 4 days of age, each neonate was placed on its back on a flat surface and its ability to return to a normal position with all four feet on the ground within 30 s was measured. In our studies almost every neonate achieved a static righting reflex on day 4. Earlier examination was not possible because the neonates were toe-clipped on day 3. To toe-clip earlier would increase the possibility of cannibalism by the dam and to toe-clip and perform the reflex test on the same day was impracticable. (ii) Eye opening, Each neonate was examined for the start of eye opening from the age of 12 days. Results from our previous work show the first occurrence of eye opening between days 12 and 16. (iii) Air-righting reflex. A solid-bottomed polypropylene cage was threequarters filled with sawdust. The 14-day-old neonate was held feet upwards, 50 cm above the sawdust bed, dropped and its ability to land with all four feet on the sawdust was measured. In earlier pilot studies when neonates were dropped from 25 cm, this height was found to be insufficient for the rats to right themselves correctly and was increased to 50 cm. Our results showed that over 90% of neonates achieved an air-righting reflex on either day 14 or 15. (iv) The narrowing bridge test (30-day-old animals). The test is used in our laboratory as an aid in the investigation of compounds suspected of causing peripheral nerve damage in the adult rat. Using this test it is possible to detect any neuromuscular dysfunction, before the appearance of obvious clinical signs of nervous system disturbance. Apparatus. For adult rats (1 l-13 weeks old) the apparatus consists of three 1.5-m long wooden bridges 23 cm above the ground, 2.5 cm, 2.0 cm and 1.8 cm wide arranged at right angles. For neonate rats (approx. 30 days old) the bridges are 2.0, 1.8 and 1.6 cm wide and arranged in a straight line. The rat is placed at the beginning of the widest bridge and has to transverse the three bridges to get to the home cage. The number of slips off the bridges are counted and the scores weighted as follows. Score on 2.0 cm wide bridge = number of slips x 3 Score on 1.8 cm wide bridge = number of slips x 2 Score on 1.6 cm wide bridge = number of slips x 1
42
original procedure used for adult rats. When adult rats were tested, they were first trained on the apparatus until they consistently scored under 1 before being exposed to the test chemical. This usually took 3 days, training consisting of 3 runs per animal per day. However, it was not possible to use this approach in our behavioural teratology studies and, therefore, it had to be modified. Procedure for neonates. On the day of testing the neonates were given 3 training runs followed immediately by 3 test runs. For each of the 3 test runs the total score is calculated (as described above) and a mean score for the 3 runs determined for individual neonates. For males and females from each litter, separate mean slip scores were calculated and finally a treatment group mean slip score was calculated using male and female scores for all available litters. Experiment if A group of 5 male and 5 female lo-day-old neonates from untreated female rats were treated i.p. with acrylamide, 50 mg/kg/day for 5 consecutive days (days lo-14 p.p.). A control group of neonates received 0.5 ml physiological saline/kg/day over the same period. The rats were then tested on the narrowing bridge test once a week for 4 weeks starting 7 days after the first dose (week 2 score), the procedure for this test being as described under Exp. I. Statistical analysis Visual inspection of maternal bod~eights, reproduction parameters and narrowing bridge test data of the phenytoin experiment revealed no major inter-group differences. For each preweaning reflex, a two-way contingency table was drawn up of treatments and the day the reflex was first observed. Under the hypothesis that there was no association between treatment and day of reflex a x2 goodness of fit statistic was calculated. If the hypothesis of no association was rejected, then Fisher’s Exact Probability Test was used to compare each treatment against control, testing to see if the treatment had effectively caused the reflex to occur on or before a given day. Narrowing bridge test results from Exp. II were statistically analysed using twoway analysis of variance. RESULTS
Experiment I paternal effects Females treated with phenytoin displayed no clinical signs of intoxication or reduction in bodyweight gains compared with the controls. One female died from an accidental dosing injury on day 16 p.c.
43 TABLE I MEAN RESULTS OF POSTNATAL BEHAVIOURAL TOIN TREATMENT Phenytoin treatment (mg/kg/ day)
TESTING OF OFFSPRING AFTER PHENY-
Mean number of days (and range) taken to achieve: Narrowing bridge slip scoresa
Static righting reflex
Eye opening
Dynamic righting reflex
Males
Females
Males
Females
Males
Females
Males
Females
4.0
4.1
14.4 (14-16)
14.4 (14-16)
1.8
(4-7)
13.4 (12-15)
2.0
(4-5)
13.5 (12-15)
50
4.0 (4-5)
4.1 (4-5)
13.5 (12-16)
13.3 (12-15)
14.2 (14-15)
14.4 (14-16)
2.0
1.6
100
4.0 (4-5)
4.2 (4-6)
13.1 (12-16)
13.0 (12-15)
14.3 (14-18)b
14.4 (14-16)
1.9
1.7
0
a Using the litter as the experimental unit. b One animal only; the values for other males in this group were in the range 14-16 days.
Reproduction parameters Phenytoin exerted no adverse effect upon the number of pregnancies and live litters produced, litter size, sex ratios, pup survival or litter weights. Behavioural effects (Table I) No treatment-related delays in the achievement of the static or air-righting reflexes or the occurrence of eye opening were observed. The offspring of phenytoin-treated females showed no performance deficit in the narrowing bridge test compared with the controls. Experiment II The results of the narrowing bridge are shown in Table II. A clear increase in the TABLE II NARROWING BRIDGE SLIP SCORES AFTER ACRYLAMIDE TREATMENT Treatment/sex
Narrowing bridge slip score (standard error) at week 2-5 after dosing” 2
Males Control Acrylamide
1.8 (i 0.7) 4.4 (* 0.8)
Females Control Acrylamide
2.8 (* 0.7) 3.4 (f 0.8)
3 2.0 (+ 1.5 (*
0.8) 0.7)
2.0 (* 0.5) 31.4 (* 18.8)s
’ Week 2 = 7 days after first dose. b Significantly higher than control (P < 0.05).
4
5
2.0 (* 0.8) 25.0 (+ 16.5)b
1.2 (* 0.5) 2.0 (f 0.4)
0.2 (i 0.2) 63.6 (& 24.0)b
0.8 (k 0.5) 2.6 (* 1.8)
44
slip scores is shown in both sexes of the acrylamide-treated neonates achieving a maximum at week 4 with apparent recovery on week 5 (23 days after the last dose). The females showed earlier and more severe effects than did males, with the most clinical effects in animals showing the most marked functional deficit. DISCUSSION
The results of Exp. I show that prenatal oral treatment with phenytoin up to 100 mg/kg/day on days 7-19 p.c. produced no behavioural changes in rat offspring, detectable in this test battery. This conclusion does not concur with those of Elmazar and Sullivan [S], who detected several neurological deficits (including delay in achievement of the air righting reflex and neonatal bodyweight depression) in a set of experiments using phenytoin (100 mg/kg/day) in an exposure regimen very similar to our own. Their negative results in the elevated narrow path at postnatal age 21 agree with our failure to detect any deficits in the narrow bridge test. However, when Elmazar and Sullivan challenged their animals with the more difficult parallel rods test, they discovered a performance deficit in the phenytointreated offspring. Desor et al., failed to produce any changes in rat neonatal motor coordination, attributable to phenytoin in a dosage regime of 50 and 100 mg/kg/day throughout gestation and lactation to female rats [6]. These apparently differing results indicate, in some cases, the difficulties in reproducing effects in behavioural teratogenicity experiments but also the need to create sufficiently challenging tests. In an attempt to find a dose level of phenytoin which would produce an effect upon neonatal behaviour, we performed a small ancillary experiment using dose levels of 250 and 500 mg/kg/day in a regime identical to Exp. I. However both these dose levels induced severe maternal toxicity and deaths together with total litter losses, probably due to cannibalisation as a result of maternal stress and teratogenic effects. It is recommended that a further experiment, with a dose level intermediate between 100 and 250 mg/kg/day be carried out. The results of Exp. II demonstrate that the narrowing bridge test, as used in this laboratory, is capable of detecting muscular incoordination in neonatal rats exposed to a known neurotoxin-acrylamide. The neonatal animals administered acrylamide showed clinical evidence of neurotoxicity (abnormal gait), whose time course correlated with that of performance deficits in the narrowing bridge test. This was also observed for acrylamide-treated adult rats [7]. The recovery from the behavioural deficit seen in neonates after acrylamide treatment was quicker than that previously found (6 weeks) in adult rats 171. The probable reason for this can be explained in terms of the difference in the speed of the axonal repair processes taking place, between neonates and adult rats. An alternative possibility is that acrylamide in some way caused a transient inhibition or arrest of the natural maturation processes occurring in the peripheral nervous system of the treated neonates, since acrylamide would have been administered at a time when myelination of the peripheral nerves
45
was taking place [S]. However, with the cessation of dosing the effect of acrylamide on these processes would have been dissipated and thus, the normal functioning of the nerve would resume with minimum delay. The usefulness of the test is being further investigated using subclinical doses of agents with marginal neurotoxic effects in parallel with neurochemical and morphological assessments of neuromuscular damage. The aim of a behavioural teratogenicity screening program is to detect subtle behavioural or functional changes in offspring which have been exposed prenatally or early postnatally to toxic agents at levels below those causing overt maternal toxicity, embryo/foetal toxicity or gross structural teratogenicity. The inconsistency of results obtained in experiments with phenytoin, together with the difficulties in obtaining behavioural changes in the absence of the other above-mentioned effects with hydroxyurea [2, 1l], aspirin [13], and others suggest that the identification of a reliable positive control agent giving reproducible results in an established test battery has not yet been achieved. However, Butcher et al. [3] demonstrated behavioural effects in rat offspring exposed developmentally to low levels of brominated soybean oil and showed that careful understanding of test variability and sensitivity may greatly aid the design and interpretation of behavioural test programs. In summary, our preweaning test battery did not detect behavioural changes in offspring of female rats treated during gestation with up to 100 mg phenytoin/kg/day. Postweaning, the narrowing bridge test also showed no effects of prenatal phenytoin treatment but was effective in detecting muscular dysfunction in neonatal rats treated with acrylamide. ACKNOWLEDGEMENTS
The authors would like to acknowledge the assistance of Mr. S. Watts, in carrying out the statistical analysis of the preweaning test data, Mrs. H. Turnball and Miss J. Kemp for skilled technical assistance, Mr. R.G. Pickering for advice and the provision of facilities and Mrs. S. Marshall for preparation of the manuscript.
REFERENCES 1 J. Altman Behav.,
and K. Sudarshan,
Post-natal
development
2 R.E. Butcher, W.J. Scott, K. Kazmaier and E.J. Ritter, ment with hydroxyurea, Teratology, 7 (1973) 161-166. 3 R.E.
of locomotion
in the laboratory
rat, Anim.
23 (1975) 8%-920.
Butcher,
V. Wootten
and C.V. Vorhees,
variability and sensitivity, Teratogen. 4 J. Cavanagh, Peripheral neuropathy
Standards
Post-natal
effects
in behavioural
in rats of pre-natal teratology
Carcinogen. Mutagen., I (1980) 49-61. caused by chemical agents, C.R.C. Crit.
testing:
treatTesting
Rev. Toxicol.,
2/3
(1973) 365-417. 5 E. De Robertis,
A.P.
De Iraldi,
G.R.
Arnaiz
and L. Salganicoff,
Cholinergic
and non-cholinergic
46 nerve endings in rat brain, I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase, J. Neurochem., 9 (1962) 23. 6 D. Desor, R.J. Royer, P. Netter, J.C. Guedenet, G. Faure, B. Krafft and G. Grignon, Action de la dephenylhydantoine administree pendant la gestation et I’allaitement, sur le dtveloppement moteur et I’histologie du cervelet du jeune rat, C.R. Sot. Biol., 172 (1978) 1057-1062. 7 A.J. Dewar and A. McPherson, Development and evaluation of a new functional test for neuromuscular dysfunction in rats, Shell Toxicol. Lab., Special Series Report (1978). 8 M.M.A. Elmazar and F.M. Sullivan, Effect of pre-natal phenytoin administration on post-natal development of the rat: A behavioural teratology study, Teratology, 24 (1981) 115-124. 9 G.P. Rose and A.J. Dewar, A biochemical and functional appraisal of misonidazole-induced neurotoxicity in the rat, Workshop on Neurotoxic Properties of Misonidazole and other Radiosensitizers, Ludwig Inst., Sutton, U.K., 1980. 10 W.M. Fox, Reflex ontogeny and behavioural development of the mouse, Anim. Behav., 13 (1965) 234-24 I. 11 H. Fritz and R. Hess, Effect of hydroxyurea on post-natal growth and behaviour of rats, Agents Actions, 10/4 (1980) 389-393. 12 J.M. Spyker and D.L. Avery, Neurobehavioural effects of pre-natal exposure to the organophosphate Diazinon in mice, J. Toxicol. and Env. Health, 3 (1977) 989-1002. 13 C.V. Vorhees, K.L. Klein and W.J. Scott, Aspirin-induced psychoteratogenesis in rats as a function of embryonic age, Teratogen. Carcinogen. Mutagen., 2 (1982) 77-84.
Toxicology Letters, 22 (1984) 39-46 Elsevier
TOXLett.
1232
LACK OF BEHAVIOUBAL BATS (Behavioural
teratology;
TEBATOGENIC
test development;
EFFECT OF PHENYTOIN
phenytoin;
acrylamide;
IN
rat)
S.L. CASSIDY, G.P. ROSE* and R.W. HEND Shell Research Ltd., Shell Toxicology Laboratory, Sittingbourne Research Centre. Sittingboume, ME9 8AG (U.K.)
Kent
(Received. February 28th. 1984) (Accepted March 12th, 1984)
SUMMARY A small battery of tests (static and air-righting reflexes, eye opening and a narrowing bridge test) was used to investigate the behavioural teratogenic potential of prenatal oral exposure (day 7-19 of gestation) of up to 100 mg phenytoin/kg/day to female rats. No treatment-related effects upon mother or offspring were detected. In order to validate our narrowing bridge test, neonatal old rats were tested over 4 weeks following early postnatal exposure to acrylamide (5 x 50 mg/kg/day i.p.). These animals showed a severe transient deficit compared with saline-treated controls. Despite the absence of an effect using phenytoin, the narrowing bridge test is considered worthy of further validation.
INTRODUCTION
A programme of work was initiated to investigate the functional development of rat neonates during the pre- and postweaning period with the intention of evolving a battery of tests to assess the behavioural teratogenic potential of development chemicals. Three preweaning tests were selected; the static righting reflex, eye opening and the air righting reflex [l, 10, 121. One postweaning test was included in our battery, the narrowing bridge test, adapted from that used for testing adult rats [7, 91. Initial studies on the litters of female rats dosed on days 15 and 16 p.c. with up to 500 mg/kg/day of phenytoin or hydroxyurea showed no effects upon the off*To whom correspondence should be addressed. Abbreviations: p.c., post coitum; p.p., post partum. 0378-4274/84/$ 03.00 0 Elsevier Science Publishers B.V.
40
spring even in the presence of marked bodyweight reduction in the phenytointreated mothers. In a small study on litters of female rats treated i.p. with 500 mg hydroxyurea/kg on day 12 p.c., the pups showed no deficits on the preweaning tests (including a forward locomotion test) but, owing to the presence of structural teratogenicity (tail deformity, missing forepaw digits), they performed poorly in the narrowing bridge test. The aims of the present study were twofold: first to establish these methods in our laboratory and to use them to detect any possible functional changes following prenatal phenytoin exposure. Phenytoin was finally chosen, since it has been shown to induce developmental deficits in prenatally exposed Wistar rats in a treatment regime similar to the one used in our study [8]. Our second aim was to investigate the usefulness of the narrowing bridge test for behavioural teratogenicity studies in both the phenytoin-treated rats and also in rat pups treated directly with acrylamide, a substance known to cause peripheral neuropathy [4] and functional deficits in adult rats undergoing this test [7, 91. METHODS
The animals used in this study were Wistar rats bred and maintained in specific pathogen-free conditions in Shell Toxicology Laboratory. They were housed singly (except during mating) in solid-bottom polypropylene cages lined with sawdust (and woodwool during the last week of pregnancy in Experiment I). The room was maintained at 18-25°C with a relative humidity of 4060% and an automated light-darkness cycle (12 h : 12 h). The animals were maintained on LAD 2 powdered feed (Spratts Patent Ltd., Barking, Essex) and water ad lib. througout the study. Phenytoin (5,5’-diphenylhydantoin, sodium salt, 99% pure) was received from Sigma (London) Chemical Company, Ltd., Poole, Dorset (CAS number 630-93-3). It was formulated as aqueous suspensions in 0.5% (w/v) carboxymethylcellulose at concentrations such that the dosing volume for each group was 2.5 ml suspension/kg bodyweight. Acrylamide (CAS number 79-06-1, 97% pure) was received from BDH, Poole, Dorset. It was formulated as a 10% (w/v) solution in physiological saline for dosing at a daily rate of 0.5 ml/kg/day. Experiment
I
Each of 51 female rats (aged 15 weeks) was mated with a male (the day on which sperm was detected was designated day 0 p.c.). 42 females were successfully mated, housed singly and allocated to one of 3 treatment groups: 0, 50 or 100 mg phenytoin/kg bodyweight/day (14 per group). The rats were orally dosed from day 7-19 p.c. inclusive (each daily dose being based on the most recent bodyweight), and at the end of gestation, were allowed to deliver their litters (the day of birth being considered as day 0 p.p.). The females were weighed regularly throughout gestation and at the end of the weaning period. All females and offspring were observed daily for
41
clinical abnormalities. Various reproduction parameters of gestation length, percentage of pregnant rats, litter size and sexes, number and sex of preweaned deaths and litter weights were measured. One day after birth, each litter was culled to a maximum of 5 males and 5 females and on day 3 p.p., the neonates were identified by toe clipping. Postnatal behavioural testing of offspring For the eye-opening and both righting reflexes, each neonate was tested once from the appropriate day and the reflex was scored as present or absent. If the neonate did not respond, it was retested on subsequent days until a positive reflex was achieved. (i) Static righting reflex. From 4 days of age, each neonate was placed on its back on a flat surface and its ability to return to a normal position with all four feet on the ground within 30 s was measured. In our studies almost every neonate achieved a static righting reflex on day 4. Earlier examination was not possible because the neonates were toe-clipped on day 3. To toe-clip earlier would increase the possibility of cannibalism by the dam and to toe-clip and perform the reflex test on the same day was impracticable. (ii) Eye opening, Each neonate was examined for the start of eye opening from the age of 12 days. Results from our previous work show the first occurrence of eye opening between days 12 and 16. (iii) Air-righting reflex. A solid-bottomed polypropylene cage was threequarters filled with sawdust. The 14-day-old neonate was held feet upwards, 50 cm above the sawdust bed, dropped and its ability to land with all four feet on the sawdust was measured. In earlier pilot studies when neonates were dropped from 25 cm, this height was found to be insufficient for the rats to right themselves correctly and was increased to 50 cm. Our results showed that over 90% of neonates achieved an air-righting reflex on either day 14 or 15. (iv) The narrowing bridge test (30-day-old animals). The test is used in our laboratory as an aid in the investigation of compounds suspected of causing peripheral nerve damage in the adult rat. Using this test it is possible to detect any neuromuscular dysfunction, before the appearance of obvious clinical signs of nervous system disturbance. Apparatus. For adult rats (1 l-13 weeks old) the apparatus consists of three 1.5-m long wooden bridges 23 cm above the ground, 2.5 cm, 2.0 cm and 1.8 cm wide arranged at right angles. For neonate rats (approx. 30 days old) the bridges are 2.0, 1.8 and 1.6 cm wide and arranged in a straight line. The rat is placed at the beginning of the widest bridge and has to transverse the three bridges to get to the home cage. The number of slips off the bridges are counted and the scores weighted as follows. Score on 2.0 cm wide bridge = number of slips x 3 Score on 1.8 cm wide bridge = number of slips x 2 Score on 1.6 cm wide bridge = number of slips x 1
42
original procedure used for adult rats. When adult rats were tested, they were first trained on the apparatus until they consistently scored under 1 before being exposed to the test chemical. This usually took 3 days, training consisting of 3 runs per animal per day. However, it was not possible to use this approach in our behavioural teratology studies and, therefore, it had to be modified. Procedure for neonates. On the day of testing the neonates were given 3 training runs followed immediately by 3 test runs. For each of the 3 test runs the total score is calculated (as described above) and a mean score for the 3 runs determined for individual neonates. For males and females from each litter, separate mean slip scores were calculated and finally a treatment group mean slip score was calculated using male and female scores for all available litters. Experiment if A group of 5 male and 5 female lo-day-old neonates from untreated female rats were treated i.p. with acrylamide, 50 mg/kg/day for 5 consecutive days (days lo-14 p.p.). A control group of neonates received 0.5 ml physiological saline/kg/day over the same period. The rats were then tested on the narrowing bridge test once a week for 4 weeks starting 7 days after the first dose (week 2 score), the procedure for this test being as described under Exp. I. Statistical analysis Visual inspection of maternal bod~eights, reproduction parameters and narrowing bridge test data of the phenytoin experiment revealed no major inter-group differences. For each preweaning reflex, a two-way contingency table was drawn up of treatments and the day the reflex was first observed. Under the hypothesis that there was no association between treatment and day of reflex a x2 goodness of fit statistic was calculated. If the hypothesis of no association was rejected, then Fisher’s Exact Probability Test was used to compare each treatment against control, testing to see if the treatment had effectively caused the reflex to occur on or before a given day. Narrowing bridge test results from Exp. II were statistically analysed using twoway analysis of variance. RESULTS
Experiment I paternal effects Females treated with phenytoin displayed no clinical signs of intoxication or reduction in bodyweight gains compared with the controls. One female died from an accidental dosing injury on day 16 p.c.
43 TABLE I MEAN RESULTS OF POSTNATAL BEHAVIOURAL TOIN TREATMENT Phenytoin treatment (mg/kg/ day)
TESTING OF OFFSPRING AFTER PHENY-
Mean number of days (and range) taken to achieve: Narrowing bridge slip scoresa
Static righting reflex
Eye opening
Dynamic righting reflex
Males
Females
Males
Females
Males
Females
Males
Females
4.0
4.1
14.4 (14-16)
14.4 (14-16)
1.8
(4-7)
13.4 (12-15)
2.0
(4-5)
13.5 (12-15)
50
4.0 (4-5)
4.1 (4-5)
13.5 (12-16)
13.3 (12-15)
14.2 (14-15)
14.4 (14-16)
2.0
1.6
100
4.0 (4-5)
4.2 (4-6)
13.1 (12-16)
13.0 (12-15)
14.3 (14-18)b
14.4 (14-16)
1.9
1.7
0
a Using the litter as the experimental unit. b One animal only; the values for other males in this group were in the range 14-16 days.
Reproduction parameters Phenytoin exerted no adverse effect upon the number of pregnancies and live litters produced, litter size, sex ratios, pup survival or litter weights. Behavioural effects (Table I) No treatment-related delays in the achievement of the static or air-righting reflexes or the occurrence of eye opening were observed. The offspring of phenytoin-treated females showed no performance deficit in the narrowing bridge test compared with the controls. Experiment II The results of the narrowing bridge are shown in Table II. A clear increase in the TABLE II NARROWING BRIDGE SLIP SCORES AFTER ACRYLAMIDE TREATMENT Treatment/sex
Narrowing bridge slip score (standard error) at week 2-5 after dosing” 2
Males Control Acrylamide
1.8 (i 0.7) 4.4 (* 0.8)
Females Control Acrylamide
2.8 (* 0.7) 3.4 (f 0.8)
3 2.0 (+ 1.5 (*
0.8) 0.7)
2.0 (* 0.5) 31.4 (* 18.8)s
’ Week 2 = 7 days after first dose. b Significantly higher than control (P < 0.05).
4
5
2.0 (* 0.8) 25.0 (+ 16.5)b
1.2 (* 0.5) 2.0 (f 0.4)
0.2 (i 0.2) 63.6 (& 24.0)b
0.8 (k 0.5) 2.6 (* 1.8)
44
slip scores is shown in both sexes of the acrylamide-treated neonates achieving a maximum at week 4 with apparent recovery on week 5 (23 days after the last dose). The females showed earlier and more severe effects than did males, with the most clinical effects in animals showing the most marked functional deficit. DISCUSSION
The results of Exp. I show that prenatal oral treatment with phenytoin up to 100 mg/kg/day on days 7-19 p.c. produced no behavioural changes in rat offspring, detectable in this test battery. This conclusion does not concur with those of Elmazar and Sullivan [S], who detected several neurological deficits (including delay in achievement of the air righting reflex and neonatal bodyweight depression) in a set of experiments using phenytoin (100 mg/kg/day) in an exposure regimen very similar to our own. Their negative results in the elevated narrow path at postnatal age 21 agree with our failure to detect any deficits in the narrow bridge test. However, when Elmazar and Sullivan challenged their animals with the more difficult parallel rods test, they discovered a performance deficit in the phenytointreated offspring. Desor et al., failed to produce any changes in rat neonatal motor coordination, attributable to phenytoin in a dosage regime of 50 and 100 mg/kg/day throughout gestation and lactation to female rats [6]. These apparently differing results indicate, in some cases, the difficulties in reproducing effects in behavioural teratogenicity experiments but also the need to create sufficiently challenging tests. In an attempt to find a dose level of phenytoin which would produce an effect upon neonatal behaviour, we performed a small ancillary experiment using dose levels of 250 and 500 mg/kg/day in a regime identical to Exp. I. However both these dose levels induced severe maternal toxicity and deaths together with total litter losses, probably due to cannibalisation as a result of maternal stress and teratogenic effects. It is recommended that a further experiment, with a dose level intermediate between 100 and 250 mg/kg/day be carried out. The results of Exp. II demonstrate that the narrowing bridge test, as used in this laboratory, is capable of detecting muscular incoordination in neonatal rats exposed to a known neurotoxin-acrylamide. The neonatal animals administered acrylamide showed clinical evidence of neurotoxicity (abnormal gait), whose time course correlated with that of performance deficits in the narrowing bridge test. This was also observed for acrylamide-treated adult rats [7]. The recovery from the behavioural deficit seen in neonates after acrylamide treatment was quicker than that previously found (6 weeks) in adult rats 171. The probable reason for this can be explained in terms of the difference in the speed of the axonal repair processes taking place, between neonates and adult rats. An alternative possibility is that acrylamide in some way caused a transient inhibition or arrest of the natural maturation processes occurring in the peripheral nervous system of the treated neonates, since acrylamide would have been administered at a time when myelination of the peripheral nerves
45
was taking place [S]. However, with the cessation of dosing the effect of acrylamide on these processes would have been dissipated and thus, the normal functioning of the nerve would resume with minimum delay. The usefulness of the test is being further investigated using subclinical doses of agents with marginal neurotoxic effects in parallel with neurochemical and morphological assessments of neuromuscular damage. The aim of a behavioural teratogenicity screening program is to detect subtle behavioural or functional changes in offspring which have been exposed prenatally or early postnatally to toxic agents at levels below those causing overt maternal toxicity, embryo/foetal toxicity or gross structural teratogenicity. The inconsistency of results obtained in experiments with phenytoin, together with the difficulties in obtaining behavioural changes in the absence of the other above-mentioned effects with hydroxyurea [2, 1l], aspirin [13], and others suggest that the identification of a reliable positive control agent giving reproducible results in an established test battery has not yet been achieved. However, Butcher et al. [3] demonstrated behavioural effects in rat offspring exposed developmentally to low levels of brominated soybean oil and showed that careful understanding of test variability and sensitivity may greatly aid the design and interpretation of behavioural test programs. In summary, our preweaning test battery did not detect behavioural changes in offspring of female rats treated during gestation with up to 100 mg phenytoin/kg/day. Postweaning, the narrowing bridge test also showed no effects of prenatal phenytoin treatment but was effective in detecting muscular dysfunction in neonatal rats treated with acrylamide. ACKNOWLEDGEMENTS
The authors would like to acknowledge the assistance of Mr. S. Watts, in carrying out the statistical analysis of the preweaning test data, Mrs. H. Turnball and Miss J. Kemp for skilled technical assistance, Mr. R.G. Pickering for advice and the provision of facilities and Mrs. S. Marshall for preparation of the manuscript.
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