* First Department of Anatomy, Kinki University School of Medicine, Osakasayama, Osaka, 589-8511, Japan; and
Division of Animal Morphogenesis, Department of Biology, Faculty of Mathematics and Sciences, Bandung Institute of Technology, Bandung, Indonesia; and
Kinki University, Osakasayama, Osaka, 589-8511, Japan
Received November 29, 2000; accepted February 15, 2001
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ABSTRACT |
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Key Words: rubratoxin B; mycotoxin; prenatal exposure; behavioral teratology; developmental neurotoxicity; mouse.
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INTRODUCTION |
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The developmental toxicity of RB has been studied in 3 species (for review see, Hood, 1979; Hood and Szczech, 1983). RB has been reported to be teratogenic and/or embryotoxic in mice (Evans and Harbison, 1977; Hayes and Hood, 1976
; Hood, 1986
; Hood et al., 1973
; Koshakji et al., 1973
; Wilson and Harbison, 1973
), in hamsters (Hayes and Hood, 1976
), and in chick embryos (Gilani et al., 1979
). A previous study in our laboratory demonstrated that RB induced gross malformations, internal anomalies, skeletal malformations, embryo lethality, intrauterine growth retardation, increased skeletal variations, and delayed ossification in the mouse fetuses of dams injected intraperitoneally with doses 0.6 or 1.2 mg/kg/day on one of days 69 of gestation (Surjono, et al., 1985
).
In the past 20 years, the importance of postnatal evaluation for studying behavioral teratology has received increasing recognition, and the test battery system for assessment of behavioral teratogenic potential in reproductive and developmental toxicity studies has been widely used (Meyer, 1998; Riley and Vorhees, 1986
; Tanimura, 1992
; Ulbrich and Palmer, 1996
; Voorhees, 1997). However, there are no published data known to us on the behavioral teratogenic effects of RB in animals and humans.
The present study, therefore, was conducted to determine the behavioral teratogenic effects on the mouse offspring of dams injected intraperitoneally with 2 macroscopically subteratogenic doses of RB during early or mid organogenesis, by using a behavioral test battery system for functional evaluation (Fox, 1965; Kihara, 1991
; Kihara et al., 1995
; Vorhees et al., 1979a
,b
).
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MATERIALS AND METHODS |
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Pregnant mice were injected intraperitoneally with 0.1 (low dose) or 0.2 (high dose) mg/kg/day of RB (Makor Chemicals Ltd., Jerusalem, Israel) dissolved in propylene glycol (Wako Pure Chemical Industries, Ltd., Osaka)-water (1:1) solution on days 79 (Group A) or 1012 (Group B) of gestation. Control animals received vehicle-only intraperitoneally on days 712 of gestation. The treatment volume was 10-ml/kg body weight. Fresh solutions of RB were prepared on the day of use.
The dams were allowed to deliver spontaneously and rear their offspring until weaning. At 4 days after birth, litters were culled randomly to groups of 8 offspring, with the same number of males and females, as far as possible. At 21 days, the offspring were weaned, separated by sex, and housed with 4 littermates of the same sex. Markings were used to identify all offspring with dry ink before weaning, and with picric acid-ethanol solution after weaning.
Body weight and physical landmarks.
The dams were weighed on 0, 14, and 18 days of gestation, daily during treatment, and on days 0, 4, 7, 14, and 21 after delivery. After weaning their litters (21 days after delivery), all the dams were killed by ether overdose, and were examined for number of implantation sites and any abnormalities of the reproductive organs. At birth, all live and dead offspring were counted. Live offspring were weighed, sexed, and examined for external malformations. The live offspring were again counted and weighed on days 4, 7, 14, and 21 after birth. After weaning, all offspring were counted and weighed weekly until 10 weeks of age.
The following physical landmarks were noted: bilateral pinna unfolding at 46 days of age, abnormal hair emergence and low incisor eruption at 79 days, eye opening at 1416 days, descent of both testes in males at 2123 days, and vaginal opening at 2830 days in females.
Behavioral test battery.
The behavioral tests and ages at testing are listed in Table 1. All the behavioral testing procedures were conducted blind with regard to the treatment groups, and tests were performed between 9:00 A.M. and 1:00 P.M. Only post-weanling males were used in order to exclude possible influence of the estrus cycle on behavioral performance.
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Cliff avoidance.
Each animal was placed on a table edge with the forepaws and nose over the edge. The amount of time required to complete backing and turning away from the edge was recorded. Each offspring was tested in 1 trial. The maximum time allowed per trial was 60 s (Fox, 1965; Kihara, 1991
; Vorhees et al., 1979a
,b
). The number of animals with successful responses within 30 s was recorded.
Negative geotaxis.
The time taken to complete a 180-degree turn when placed in a head-down position on a 25-degree inclined plywood surface was measured. Each animal was given one trial (Fox, 1965; Kihara, 1991
; Vorhees et al., 1979a
,b
).
Swimming development.
This procedure has been described elsewhere (Fox, 1965; Kihara, 1991
; Rice and Millan, 1986
; Vorhees et al., 1979,b
). Each mouse was individually placed in a tank of water (28°C) for 510 s and the angle in the water (head position), direction, and limb usage were observed and scored.
Rotarod test.
The apparatus consisted of a rod 3 cm in diameter, which had a hard rubber surface and was linked to a variable-speed motor. The top of the rod was 34 cm above the back of the apparatus (Shinano Seisakusho Ltd., Tokyo, SN-498). Animals were placed individually on the rod for at least 5 s, and the rod was rotated at speeds of 5 or 15 rpm. Animals were tested in 2 trials per day for 2 consecutive days. The maximum trial duration was 180 s, and the inter-trial interval was about 30 min. The time that each animal remained on the rod at each rotation speed was recorded (Kihara, 1991).
Open-field test.
Mice were tested in a circular open field (100-cm diameter circular black polyvinyl chloride) on 3 consecutive days for 180 s per day. The time to leave the start area (latency), number of sections entered with all 4 legs (ambulation), number of rearings, and number of fecal boluses were recorded (Kihara, 1991).
Conditioned avoidance learning test.
The apparatus used was a 2-way shuttle box (48WX22DX20.5H, Takei Kiki Kogyo, Co., Tokyo). The box was divided into equal compartments by a metal partition. The grid floor of each compartment consisted of stainless steel rods spaced 0.7 cm center-to-center. An electric shock could be delivered though the grid floor.
As an auditory warning device, a commercial buzzer (about 90 dB) was placed in a central position between the 2 boxes. The conditioned-avoidance schedule was as follows: after adaptation for 1 min in one side-compartment, a 10-s warning duration (conditioned stimulus; CS), and electric shock (approximately 85 V, 0.5 mA, 60 Hz, Ac; unconditioned stimulus; UCS) for a maximum of 10 s, and a 20-s inter-trial interval (ITI). Crossing to the opposite side compartment during the CS period immediately terminated the stimuli and foot shock was avoided. Both crossing to the other compartment at times other than the CS presentation period, and no-crossing to the other compartment were ineffective. Each session consisted of 20 consecutive trials per day, and animals were tested on consecutive days for 4 sessions. The number of conditioned avoidance responses and first start latency time were recorded, and the mean percent of avoidance responses per day was calculated.
Water E-maze test.
The E-shaped maze apparatus consisted of a gray polyvinyl chloride, 50 cm in breadth (back wall), middle arm (start point) 50 cm from front to back wall, both right and left arms each measuring 30 cm long and 10 cm in alley width. The escape ramp (wire mesh, on the ends of the arms) was not visible from the choice point. The maze was filled to a depth of about 20 cm with water maintained at a temperature of 22°C.
On the first day of testing, at the first trial, an escape ramp was placed in each of the right and left arms. From the second to the sixth trial, the escape ramps were placed in opposite positions from the first trial. On the second and third days, the escape ramps were in the same position, and on the fourth day, the escape route was reversed. The inter-trial interval was approximately 30 min. The swimming time and the number of errors in the maze were recorded for each trial.
Brain weight.
One male offspring from each litter was killed at 11 weeks of age by ether overdose and autopsied, and the brains were removed and weighed.
Statistical analysis.
The number of implantation sites and live offspring, as well as the body weight of dams and offspring, were analyzed by 1-way analysis of variance (ANOVA), followed by Scheffe`s test if differences were found. The survival-index data were analyzed by the Chi-square test. The behavioral measures, and physical developmental observations were evaluated by using the Mann-Whitney U test for nonparametric comparison of group means (Siegel, 1956; Sokal and Rohlf, 1973
). Before weaning, the data from individual subjects were averaged, and the litter was used as the unit of analysis. After weaning, the analyses were performed on the basis of individual animals from each litter. The accepted level of significance was p < 0.05.
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RESULTS |
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Growth and Physical Landmarks of the Offspring
There were no significant differences between the RB groups and the control group with regard to the number of implants or live births, sex ratio of offspring, offspring with external malformations, or survival rates at 4 or 21 days of age (at weaning), or at 10 weeks of age. At birth, the body weights of both male and female offspring in the 0.2-mg/kg B group, and at weaning, the body weight of female mice in the 0.2-mg/kg B group were significantly lower than those of controls. However, there were no significant differences between the RB groups and the control group at 4 and 7 days of age (data not shown), or at 21 days or 10 weeks of age in the male offspring (Table 2).
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Behavioral Testing
There were no sex differences on any preweaning tests; therefore, males and females were combined for data analysis. The values are presented as the mean of the litter means in Tables 3 and 4.
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Swimming development.
The results are shown in Table 4. With regard to swimming angle in the water, the 0.2-mg/kg B group scored significantly lower than the control group at 10 and 12 days of age, but at 8 days of age, there was no significant effect of exposure. There were no significant differences between the RB-exposed groups and the control group for swimming direction and limb usage at 8, 10, or 12 days of age.
Rotarod performance.
There were no significant differences between the RB-exposed groups and the control group for remaining on the rotarod at 5 or 15 rpm on either the first or second test day (data not shown).
Open field activity.
There were no significant differences between the RB-exposed groups and the control group for ambulation counts, rearing and defecation frequencies, or the start-latency time on any of the test days (data not shown).
Conditioned avoidance learning.
The mean avoidance rates in the RB-exposed groups were lower than those in the control group at the second, third and fourth training sessions. The conditioned-avoidance test is presented in Figure 1. The RB-exposed groups showed significantly lower avoidance rates than the control group at the third and fourth sessions.
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Brain Weight
There were no significant differences in brain weights between the RB-exposed groups and the control group (data not shown).
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DISCUSSION |
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With regard to the avoidance-learning task, it is of considerable interest that mouse offspring exposed to RB at both high and low doses during both early and mid organogenesis showed impaired learning ability. Generally, learning ability is a very important parameter for assessment of developmental neurotoxicity (Cuomo et al., 1996; Riley and Vorhees, 1986
; Tanimura, 1992
). In this respect, the most important finding in the present study is that prenatal exposure of mice to RB severely impaired performance in the conditioned avoidance-learning test.
The most important conclusion drawn from the present study is that prenatal exposure of mice to RB at a dose lower than that which causes gross malformations or growth retardation altered neurobehavioral performance in the offspring in the preweaning and postweaning periods. Therefore, the behavioral changes observed here were the results of persistent effects of RB on the fetal nervous system.
Furthermore, the motor developmental interference of RB is greater when RB is administered during mid-organogenesis than when it is administered during early organogenesis. Several studies in addition to this one have demonstrated that mid-organogenesis in mice and rats is a highly sensitive period for some behavioral teratogens (Kihara, 1991; Rodier, 1976
, 1980
; Rodier et al., 1979
; Vorhees, 1983
, 1987
). Vorhees (1983, 1987) also suggested that mid-organogenesis is the most vulnerable period. Mid-organogenesis, at approximately days 9.512.5 in the mouse, is an extremely active phase of neurogenesis for the visual areas, cerebral cortices, basal ganglia and forebrain, and for the thalamic, hypothalamic and limbic regions (Rodier, 1980
).
At present, we can only speculate about potential mechanisms for the observed effects of RB which produced inhibition of adenosine triphosphatase activities in the mouse brain (Desaiah et al., 1977). This toxic action altered brain development, and affected early response development and learning abilities in the offspring of mice exposed to RB during middle pregnancy. Furthermore, RB produced central nervous system malformations (Evans and Harbison, 1977
; Hayes and Hood, 1976
; Hood et al., 1973
; Koshakji et al., 1973
; Surjono et al., 1985
; Wilson and Harbison, 1973
), immunosuppression (Sharma, 1993
), inhibition of mitochodrial function (Hayes, 1976
) and deceases of cyclic AMP (Hayes et al., 1978
), malfunction of DNA-dependent RNA polymerse activity and polysomal disaggregation (Watson and Hayes, 1977
). Some of the actions of RB mentioned above also are developmentally toxic, including actions resulting in behavioral teratogenicity.
However, further studies are required to test for any behavioral effects of developmental RB exposure in postweanling female mice, behavioral effects in progeny of dams treated during late organogenesis or during the lactation period, neurobiochemical effects of RB, and the mechanisms of the effects of RB on developmental neurotoxicity.
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NOTES |
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