Neurotoxicology

Pergamon 0892-0362(94)00060-3

and Teratology, Vol. 17, No. 2, pp. 121-130, 1995 Copyright o 1995 ElsevierScienceLtd Printed in the USA. All rights reserved 0892-0362/95 $9.50 + .04l

Effect of Prenatal Exposure to Styrene on the Neurobehavioral Development, Activity, Motor Coordination, and Learning Behavior of Rats
REIKO KISHI, BING QING CHEN, YOHKO KATAKURA, AND HIROTSUGU MIYAKE TOSHIKO IKEDA

Department

of Public Health, Sapporo Medical College, Sapporo 060 Japan Received 6 July 1994; Accepted 15 August 1994

KISHI R., B. Q. CHEN, Y. KATAKURA, T. IKEDA AND H. MIYAKE. Effects of prenatal exposure to styrene on the neurobehavioral development, activity, motor coordination, and learning behavior of rats. NEUROTOXICOL TERATOL 17(2) 121-130, 1995. -Maternal Wistar rats were exposed via inhalation to 0,50, or 300 ppm styrene for 6 h/day during gestation days 7 to 21, and offspring were subsequently evaluated in several neurobehavioral tests. Preliminary results with a small number of litters revealed significant dose-dependent effects in tests performed prior to weaning (surface righting, pivoting locomotion, and bar holding), as well as in tests performed after weaning (motor coordination, open-field behavior, and motor activity). Exposure to low concentrations of styrene (50 ppm) caused disturbances in motor coordination in addition to delaying some motor and reflex developments. Large doses (300 ppm) led to changes in open-field behavior and increases in spontaneous activity in addition to the delay in neurobehavioral developments. Exposure of dams to styrene did not clearly affect the learning behavior of the offspring. It was also observed that age played a role in the differences in styrenes effects on neurobehavioral function. Only subtle effects were found in both open-field behavior and motorcoordination function when compared with control rats at 120 days of age. These results suggest that the functional neurobehavioral development of progeny of dams exposed to styrene (or other solvents) should be further investigated. Functional teratology Learning Styrene Neurobehavioral development Prenatal exposure Open-field behavior Motor coordination

BECAUSE of their extensive use, organic solvents have received much attention in the past decade as possible factors in the causation of embryotoxic effects in humans. Maternal occupational exposure to organic solvents has been strongly suggested to be linked with congenital abnormalities, spontaneous abortions, and fetal death (5,22,23). The organic solvent styrene has been used in the manufacture of plastics and

has a wide variety of other industrial applications (17,32). Lemasters et al. (19), studied reproductive outcomes of pregnant workers at 31 companies producing reinforced plastics. These workers generally were exposed to styrene under the level of 30 ppm. Although there was no significant increase in any menstrual disorder of women workers, women who worked at the most highly exposed jobs had offspring with an adjusted birthweight of 4Vo less than the offspring of unexposed women. In addition, there are conflicting reports from

Scandinavia of an association between exposure to styrene, congenital birth defects of the central nervous system and fetal loss (7,8,20). The styrene monomer is a lipophilic low-molecular-weight compound that is easily absorbed and that crosses the maternal placental barrier (14). This study on the placental transfer of styrene showed that styrene was transmitted to the embryo. The concentration in fetal tissue was almost the same as in the maternal brain, though it was about half of that in the maternal blood. The slower rate of styrene elimination from the fetus compared with maternal tissue after exposure is also important, especially in the case of chronic exposure, because long retention of styrene in the fetal tissue may place the fetus at greater risk due to prolonged exposure. A few studies have investigated the teratological effects of styrene or styrene oxide after single or repeated exposures.

Requests for reprints should be addressed to Reiko Kishi, Department of Public Health, Sapporo Medical College, Minami-1, Nishi-17, Chuo-Ku, Sapporo 060 Japan. 121

122 Teratogenic effects have been reported in chick embryos (11,29) and mice (12), while negative results were reported in other species (2,4,25,27). Recently, we investigated neurochemical effects in the offspring of styrene-exposed rats. Although reproductive toxicity and fetotoxicity were not evident following gestational styrene exposure, analysis of neurotransmitter studies showed dose-dependent decreases of neuroamines (15). Evaluation of neurological function via behavioral testing may be a more sensitive technique for assessing prenatal toxicity than standard teratological procedures (30,31). As an extension of our earlier neurochemical investigation, the present study investigated the postnatal functional effects of prenatal exposure to styrene. A variety of functions were evaluated: (a) Physical growth and maturation, (b) Reflex and motor development, (c) Sensory function, (d) Activity levels, and (e) Learning abilities.
METHOD

KISHI ET AL. feet. Age at eye opening was recorded when both eyes were completely opened. Incisor eruption was examined daily to determine the first day of the bilateral appearance of the upper and lower incisors (scored separately). Female offspring were examined for patency of the vaginal canal. Preweaning tests of behavior. We applied five behavioral tests: 1. 2. 3. 4. 5. Surface righting Pivoting locomotion Bar holding ability Negative geotaxis Cliff drop avoidance to assess neurobehavioral ment before weaning (1).

develop-

Subjects Virgin female and male Wistar rats were acclimated to a 12L : 12D cycle; the dark periods beginning at 9 a.m. After mating, the presence of sperm within the vaginal tract marked gestation day (GD) 0. A total of 24 pregnant rats were assigned to styrene or used as controls, i.e., 14 litters to 0 ppm (air), 3 litters to 50 ppm styrene, and 7 litters to 300 ppm styrene. However, due to the limited number of inhalation chambers available, only 12 litters exposed at the same periods were evaluated in the neurobehavioral study (5 at 0 ppm; 2 at 50 ppm; 5 at 300 ppm). Purina Lab Chow and tap water were available ad lib, except when pregnant animals were in exposure chambers. At 1 day of age, litters were culled to six pups (except for one litter which consisted of only 5 pups). Animals were weaned at 22 days and given food and water ad lib. The body weights of animals were checked every morning before neurobehavioral tests. Apparatus and Exposure Procedures The pregnant rats were placed alone in 38 x 33 x 17 cm polycarbonate cages with bedding of heat-treated sawdust from a local suppher. Static exposure to styrene was carried out in five similar stainless steel gas chambers (70 x 70 x 120 cm) (24). A calculated volume of styrene was introduced from an upper inlet by using a vaporizer. Pregnant rats were exposed for 6 h to 0 (ambient air), 50 or 300 ppm of styrene on GD 7 to 21. Styrene vapor concentration was determined hourly with a Shimazu 4 BPTF gas chromatograph. The mean concentration of styrene in the gas chamber during exposure periods was 60.1 f 1.9 (SD) ppm for exposed group 1 (50 ppm) and 292.7 f 7.2 (SD) ppm for exposed group 2 (300 ppm). Behavioral Procedures Development of physical milestones and postnatal neurological function. Pups were evaluated daily for development of the startle reflex, the righting reflex, the age of eye opening, incisor eruption, and vaginal patency. The startle response was evoked by a clicking sound, and a positive startle response consisted of muscle jerk in any of the extremities. The righting reflex was elicited by holding the rat upside down by the nape of the neck and tail and dropping it from approximately 30 cm above some wood shavings. A response was considered positive if the animal righted in mid-air and landed on all four

Surface righting: Surface righting was a test for reorientation from a supine to a prone position on GDs 3-7. The time from release to reaching a prone position with all four feet touching the table surface was recorded. Pivoting locomotion: The time spent in pivoting during a I-min observation period was recorded on GDs 5 through 12. Bar holding ability: The animal was held by the nape of the neck and its forepaws placed on a bar, and then released. Latency for falling from the bar was recorded on GDs 3-12. Negative geotaxis: The pups were tested for geotaxis on a 25 inclined plane made of plywood on GDs 3-12. The time (up to 60 s) needed to rotate from a head-down to a head-up orientation through at least 180 was recorded. Cliff drop avoidance: Each pup was placed on the edge of a platform such that most of its head and forelimbs extended over the edge. The latency for moving its entire body away from the cliff edge was recorded on GDs 3-12. Postweaning tests of behavior. After weaning (22 days of age) offspring were evaluated for open-field behavior, motor coordination, activity, and operant conditioning learning. For both spontaneous activity and operant conditioning learning, only males were tested because the time required for testing each animal was very long in each test.
Open-field behavior (30 and 31,60 and 61, and 127 and 128 days of age). Animals were tested individually in a modified

open-field device (see ref. 3). On 2 consecutive days, each animal was placed into the device, which consisted of an enclosed round white wall (diameter: 800 mm) with a black grid, that was divided into 18 segments with a circle in the center. The animals behavior in this mild stress situation was evaluated for 2 min for the following behavioral categories: (a) ambulation, i.e., number of segments entered with all 4 feet within 2 min; (b) rearing; (c) grooming behavior; (d) number of fecal boluses deposited.
Motor coordination measurement (30, 60, and 120 days of age). To investigate motor coordination, individual rats were

placed on a 70 mm diameter cylinder of a Rota-rod Treadmill (Ugo Basile Co.), which was rotated at 8, 12, or 16 cycles/ min. When a rat fell off its cylinder section onto the plate below, a counter was tripped, and this recorded the animals endurance time in seconds. The time of each animal on the rod was recorded up to a limit of 120 s. The tests were performed three times at each of three test speeds on each of 3 test days, and the mean endurance times were calculated. Activity measurement (48 to 50 days of age). Spontaneous activity of each rat in a plastic cage was automatically determined using a 2-channel Automex (Columbus Instruments) for 24 h. Activity scores, accumulated on digital counters, were recorded every 15 min. Measurements commenced at 10: 00 a.m. Data were statistically analyzed separately in both the D (dark) and L (light) periods, and the first hour was

PRENATAL

STYRENE

EXPOSURE

AND DEVELOPMENT

123 in the tables, results of the statistical comparisons were described in each section with F values. The repeated measures ANOVA (28) was used for data collected for several consecutive days in the same developmental stage (some preweaning tests, e.g., surface righting etc.).
RESULTS

regarded as the exploratory response to the new apparatus. Food and water were available ad lib during activity measurement . Operant conditioning procedures (78 to 118 days of age). A 25 x 30 x 30 cm rat operant chamber (BRS/LVE: rodent and small primate test cage, No. 143-21) with a lever was used. Following 77 days of free access to food, all animals were slowly deprived to 90% of body weight and trained over 15 days to lever press on a continuous reinforcement schedule (CRF) (6) in which every lever response was reinforced with a single food pellet. Experimental sessions lasted 20 min and were conducted daily for 7 days. Animals were then trained to lever press with a subsequent shift to a DRL 12-s schedule. During a differential reinforcement at a low rate of responding in 12 s (DRL 12 s) schedule, only responses preceded by a 12-s pause were reinforced (6). If the rat made a second response within 12 s of the first, the clock was reset and the animal had to wait 12 s from the second response. A precise and steady rate within a narrow range of inter-response intervals was required and a very high or very low rate of responding reduced the reinforcement received. The DRL-12 s test was conducted for 40 min/day for 26 sessions. Acquisition of the CRF schedule was begun at 78 days of age, and the DRL-12 s schedule ended at 118 days of age. The CRF and DRL 12-s schedules were used as indicators of learning ability and cognitive function. Pharmacological challenge. Offspring were evaluated at 150 days of age for their response to a pharmacological challenge (1). Rats were injected with sodium pentobarbital (30 mg/Kg) IP, and sleeping time was measured with an activity meter (Obara Medical Instrument Co., Ltd.). Histopathological examination. Mother rats and pups of either 0 (day of birth), 21, or 160 days of age were examined for histological analysis. Brain, liver, lung, and kidney tissues were fixed in 10% buffered formalin and blocks were embedded in paraffin, after which the sections were cut and stained. Brain sections were stained with cresyl violet for Nissl staining and the other tissues were stained with hematoxylin and eosin. Tissues were then examined by light microscopy. Data analysis. Basically, the data obtained for all animals within a litter were analyzed by analysis of variance (ANOVA). Differences were calculated only between controls and 300 ppm-exposed, because neither the significance value nor SD of the 50 ppm styrene-exposed group were calculated because of the small number of litters. When a significant difference between controls and 300 ppm-exposed animals was observed by ANOVA using litters as the unit of analysis, ANOVA using the individual offspring was carried out, and Scheffes statistics (26) was employed to investigate the multiple comparison, especially to clarify the effects of 50 ppm styrene exposure. Although results based on individual offspring are not listed

Adult Findings and Reproductive Effects The concentration of styrene exposures used did not result in overt signs of toxicity. Mean maternal body weight gains in rats exposed to styrene were not significantly different from controls but were slightly affected by the 300 ppm concentration. There were no significant differences found in the length of gestation between groups. No significant group effect was found for the number of offspring delivered. The mean numbers of pups per litter for the 0 ppm, 50 ppm and 300 ppm exposure groups were 9.4 + 2.6, 9.5 + 3.4, and 9.4 + 3.0, respectively (Table 1). Offspring Mortality and Weight Gain One pup of the 0 ppm group, and two pups from the 300 ppm group were found dead, when litters were culled to six pups at 1 day of age. Early postnatal mortality was not related to styrene exposure, because there was no difference across groups. Postnatal body weights of offspring of rats are presented in Table 2. Body weights at 1 day of age of the 300 ppm rats were not significantly lower than those of the control group. At 21 and 77 days of age, body weights of 300 ppm rats were significantly lower than the controls, whereas at 125 days of age no differences between the controls and styreneinhaling offspring were observed. Developments Development of physicar milestones and postnatal neurological function. Styrene-exposed offspring demonstrated delayed development of physical milestones and postnatal neurological function. Significant effects were found for the development of incisor eruption and eye-opening, auditory startle reflex, and righting reflex, when the calculations were based on the mean of the data obtained for all offspring within a litter (Table 3). Scheffes comparisons based on the data of individual offspring implied that even the 50 ppm exposure group had delayed auditory startle reflex, and righting reflex compared to the control group, F(2,66) = 42.3, p < 0.05, and F(2,66) = 44.4, p < 0.05, respectively. Preweaning tests of behavior. Table 4 shows the results of the neurobehavioral development of the five different measures as calculated by repeated measures ANOVA. Develop-

TABLE 1
RESULTS OF REPRODUCTIVE TOXICITY OF STYRENE EXPOSURE ON CDs 7-21 IN RATS styrene (ppm) No. of Mother (14) Maternal wt. Gain During Exposure (g) Length of Gestation (Day) No. of Live-Born Offspring

50 300

(3) (7)

48.1 f 14.6 45.0 f 18.0 39.1 + 5.5 SD.

21.1 + 0.9 22.0 + 1.0

21.0 + 0.0

9.4 + 2.6 9.5 f 3.4

9.4 + 3.0

All values expressed as mean i

TABLE 2
POSTNATAL BODY WEIGHT OF OFFSPRING OF RATS INHALING STYRENE

NO. Offspring l-Day-Old* F Mt Ft Ft. Mf F* M M* 21-Days-Old 77-Days-Old 125-Days-Old M (15)

Styrene

@pm) 5

No. Litter

F*

50 300

2 5

(8) (15)

(13) (4) (14) 6.1 + 0.4 5.0 - 6.0 5.6 f 0.4 304.5 f 18.7 265.0 - 275.0 273.3 +I 11.5 F; = 7.1 207.4 + 1.6 191.0 - 196.0 192.3 + 1.6 F; = 67.3 6.1 + 0.5 5.0 - 6.0 5.7 k 0.6 46.9 37.8 40.1 F; + 2.1 - 41.8 t 1.6 = 17.8 48.2 + 1.7 39.0 - 42.5 40.7 * 0.4 F; = 27.5

360.9 + 30.3 325.0 - 332.0 341.1 k 11.6

243.5 i 9.2 226.0 - 232.0 233.7 + 6.2

Values in the controls and 300 ppm styrene exposed are expressed as mean (g) + SD, based on the data obtained for all offspring within a litter. Only the range (min-max) of the data of 50 ppm group is listed because the mean and SD of the 50 ppm exposed group is not calculated because of the small number of litters. Significance (p value) was calculated between controls and groups exposed to 300 ppm styrene. *ns; tp < 0.01; $p < 0.05.

TABLE 3
EFFECTS Incisor Eruption styrene @pm) NO. Litter Upper Lower No. Offspring Eye Opening (Day) (Day) Auditory Startle Reflex (Day) Righting Reflex (Day) Vaginal Opening OF PRENATAL STYRENE EXPOSURE ON POSTNATAL PHYSIOLOGICAL DEVELOPMENTS

50 300
F; = 5.7

5 2 5

28 12 29

9.2 k 0.5 9.8 - 10.0 9.9 f 0.5

14.3 _+ 0.8 15.9 - 16.0 16.1 f 1.3

F; = 4.7

38.6 + 2.1 40.5 - 42.0 35.2 f 3.2

F; = 5.4

8.1 k 0.5 8.0 - 9.0 9.4 f 0.6 F: = 6.8

15.0 + 0.5 15.2 - 15.5 15.8 zk 0.4 F; = 5.0

11.9 * 0.3 12.5 - 12.9 13.2 f 0.5 F; = 14.0

Values in the controls and the 300 ppm exposed group are expressed as mean (day) f SD, based on the data obtained for all offspring within a litter. Only the range (min-max) of the data of 50 ppm group is listed because mean and SD of the 50 ppm exposed group were not calculated because of the small number of litters. Significance (p value) was calculated between controls and groups of exposed to 300 ppm styrene. p < 0.05;p < 0.005.

TABLE 4
SUMMARY OF THE RESULTS OF NEUROBEHAVIORAL DEVELOPMENT Dose Effect FValue df F:, F;, F:, Pr>F= TESTS ANALYZED BY REPEATED Time (Days) Effect FValue df Pr>F= ANOVA Time x Dose Effect F Value df Pr>F=

Measures
No. Litter No. Offspring Observed (Day-Day)

Neurobehavioral Development

0.03
O.oool O.oool

Ft F:, F;,

0.0001
O.oool

Surface righting Pivoting Bar holding Negative geotaxis Cliff drop avoidance 12 12 12 12 12 69 69 69 69 69

FZ,
F&

FL FL: G G, F261 FL 0.63 0.13 0.0001 O.OcQl 0.0001

Fg

days days days days days

3-7 5-12 3-12 3-12 3-12

3.67 12.86 50.44 0.25 2.13

28.20 48.19 7.29 12.37 37.68

0.79 1.62 1.98 0.97 0.83

0.61 0.07 0.03 0.48 0.62

Degrees of freedom are different because of missing values. Pr = probability.

PRENATAL

STYRENE

EXPOSURE

AND DEVELOPMENT

125 endurance time at all drum speeds used in 30-day-old rats and in 60-day-old rats between the controls and 300 ppm styreneexposed offspring (Table 6). Scheffes comparison based on the data using the individual offspring as the unit also revealed that even the 50 ppm group had shortened endurance time at 8 rpm and 12 rpm at 30 days of age, F(2, 66) = 44.5, p < 0.05 and, F(2,66) = 11.7, p < 0.05, respectively, and 12 rpm and 16 rpm in 60-day-old rats, F(2, 66) = 15.4, p < 0.05 and F(2, 66) = 32.7,~ < 0.05, respectively. It was also observed that age played a role in the differences in styrenes effect on motor coordination. No reductions of endurance time were found in any of the offspring of dams of the styrene-inhaling groups when compared with control rats at 120 days of age. Spontaneous activity. Table 7 shows the mean activity levels at 48 to 50 days of age in light and dark conditions for all groups. The male offspring of 300 ppm styrene-exposed

IO 9-

o-----O control b---d. 50ppm 300ppm

(Air)

73 8 P .E g 65432lI I 1 I I

o----o control b---n 50ppm 300ppm

(Air)

Age

(Days)

FIG. 1. Mean surface righting time (s) on CDs #-7.

ment of both the pivoting and bar holding were significantly

2 co
e i= p .K L ? 20 30

delayed in the offspring of the 300 ppm styrene-inhaling group. The results of the surface righting also showed a significant dose effect, whereas neither cliff drop avoidance tested on days 3 to 12 nor negative geotaxis revealed major differences among the groups. There was also a significant time x dose interaction in the neurobehavioral development of bar holding. Figures 1 through 3 present the results of the mean time of surface righting, pivoting, and bar holding, respectively. Post weaning tests of behavior. Open-field test. The 300 ppm styrene-inhaling offspring, as compared to the age-matched controls, exhibited increased open-field activity. Many offspring showed hyperactivity that exceeded the incidence among controls. This difference, however, seemed to become smaller with age. At 30 to 31 days of age and 60 to 61 days of age, the offspring of rats that had been exposed to 300 ppm styrene showed significantly higher ambulation and rearing scores when compared to their controls, especially the male offspring. However, at 127 and 128 days of age, i.e., approximately 4 months after delivery, there were only small differences between the controls and styreneexposed offspring, which showed similar ambulation and rearing scores (Table 5). Rota-rod performance. As an index of neurotoxicity, Rotarod performance revealed a deficit in the motor coordination of the styrene 300 ppm-exposed offspring at 30 and 60 days of age (Table 6). ANOVA indicated a significant decrease in the

10

7 Age

9 10 11 12 (Days)

FIG. 2. Mean pivoting time (s) on CDs 5-12.

126

KISHI

ET AL.

40 o--o p----n control 50ppm 300ppm (Air)

bital. The mean sleeping times of controls were 68.6 f 28.6 min, and 216.6 + 70.0 min (males and females, respectively), whereas in the 50 ppm group they were 43.7 + 22.7 min and 197.5 + 58.6 min (males and females, respectively) and in the 300 ppm group they were 63.7 f 31.9 min and 171.0 + 64.7 min (males and females, respectively). The big differences between males and females were mainly because of the apparatus used. Sleeping time was measured in steel cages, that were equipped with microswitches activated by tilting of the cage floor as the rats moved around. The sleeping time of males was more sensitively counted because of their body weight. Pathological study. No overt microscopic abnormalities were detected in the serial sections of the brain, lung, liver, and kidney of mother rats or 21-day-old and 160-day-old offspring exposed to styrene during gestation.
DISCUSSION

10 11 12

Age (Days)
FIG. 3.

Mean bar holding time(s) on GDs 3-12.

groups showed an initial high level of exploratory activity during the first 1 hour in the testing apparatus. The total 24-h activity and the dark phase activity in the rats exposed to 300 ppm styrene during gestation were significantly higher than those of controls. These data suggest that styrene exposure during prenatal periods alters animal reactivity as the groups receiving higher doses exhibited higher activities in the developmental stage. The data also suggest that dark phase (active period) in rats was a more sensitive time for detecting effects. Operant behavior. The operant behavior CRF data of males are presented in Table 8. There were significant differences between 300 ppm exposure and the controls in acquisition of the CRF schedule only in the 3rd session (Table 8). The Scheff6s statistics based on the data of individual offspring also showed the mean response rate of the 50 ppm exposed group in the 3rd session to be lower than that of the control, F(2, 35) = 5.3. The overall response rate on the DRL 12 schedule for male offspring of dams that inhaled styrene was similar to that of the control values. Postnatal sensitivity to barbiturates. There were no group differences in sleeping time after injection with sodium pentobar-

No apparent reproductive toxicity was evident following gestational styrene exposure, i.e., no significant effect was found for the number of offspring delivered at the 50 ppm or 300 ppm styrene concentrations studied. These results are in agreement with a previous report on the teratogenicity of inhaled styrene in rats and rabbits (25). In the present study, some marked delays in neurobehavioral development, differing open-field behavior, hyperactivity, deficits of motor coordination, and delays of learning acquisition were observed. Large doses (300 ppm) of styrene led to changes in openfield behavior and an increase in spontaneous activity in addition to delays in neurobehavioral development. In addition, although results from the 50 ppm group are based on data from individual offspring, it appears that this lower exposure concentration may also affect developmental milestones, rotarod performance, and learning acquisition (CRF). Locomotor activity is often used as an index of an animals reactivity and adjustment to novel and, to some extent, stressful situations (3). One of the significant findings of this study is the observation that maternal exposure to styrene during pregnancy alters the manner in which the progeny react and adapt to novel test environments, as observed in both open-field tests and Automex spontaneous activity tests. The major goals of our experiment were to investigate the effects of gestational styrene exposure on the developing nervous system and compare the results of neurobehavioral tests with our neurochemical study (15). Under the same styrene exposure conditions as in the present study, we previously observed a significant decrease in the levels of some neurotransmitters such as 5-HT (serotonin) in the cerebrum at 1 day of age. It is possible that some of the functional (neurobehavioral) abnormalities observed in the offspring of mothers exposed to styrene may be attributable to the decrease in the levels of neurotransmitters, because the role of serotonin (5HT) and other neurotransmitters as differentiation signals during brain organogenesis is well documented (18,21). It is also reported that certain drugs and chemicals that interfere with synthesis, release or receptor interactions of these neurotransmitters are able to disturb the processes of blastoderm growth, primitive streak formation, neurulation, brain formation, and somatogenesis (10). Although both the route and duration of exposure were different from our present study, Khanna et al. (13) reported that pups born to dams which were administered styrene solution orally from GD 6 until weaning of the pups at the concentration of 100 mg/kg body weight/day and received a low protein diet showed a delay in

TABLE5
EFFECTS PRENATAL 30-31 Days of Age 60-61 Days of Age No. NO. 127-128 Days of Age STYRENE EXPOSURE ON POSTNATAL OPEN-FIELD BEHAVIOR

Styrene (ppm)

litter Ambulation* Ambulation? Rearing Ambulationt

Offspring

Rearing

Rearin&

Male 5 2 5 F; = 21.6
p < 0.01 ns ns

0 50 300 F; = 15.9 JJ < 0.01 5 2 5 7.4 47.6 t 40.5 - 59.0 59.1 + 9.4 ns F; = 16.3 p < 0.01 ns F; = 7.9 p < 0.05 5.5 + 1.6 10.5 - 12.0 8.8 * 0.3 42.1 + 7.1 48.0 - 49.4 58.7 +I 15.6 5.1 + 7.4 9.5 + 1.5 7.6 0.8 13 4 14 F; = 15.9 p < 0.01 F; = 6.1 p < 0.05

15 8 15

46.2 + 7.6 51.4 - 63.3 76.4 + 8.3 42.0 f 5.0 48.8 - 49.4 65.7 f 14.7

5.6 + 0.8 7.8 - 11.3 11.6 + 2.1 5.6 f 7.4 20.4 + 1.5 7.6 2.4

44.3 * 15.1 22.3 - 43.7 47.6 r 11.6

4.4 + 5.1 6.4 f

2.2 5.5 1.3

Difference @ value)

Female 0 50 300

47.8 + 14.2 22.3 - 43.7 35.4 + 10.8

5.1 + 5.1 6.4 k

1.7 5.5 2.1

Difference (p value)

n.9

nS

The numbers of grooming and defecation are not listed because there was no significant difference. Values in the controls and 300 ppm styrene exposed are expressed as mean + SD, based on the data obtained for all offspring within a litter. Only the range (min-max) of the data of 50 ppm group is listed because the mean and SD of the 50 ppm exposed group were not calculated because of the small number of litters. Significance (p value) was calculated between controls and groups exposed to 300 ppm styrene.

TABLE6
DURATION 30 Days Old OF TIME FOR THREE ROTATION SPEEDS OF THE ROTA-ROD 60 Days Old 12 RPM 8 RPM 12 RPM 16 RPM 8 RPM TEST OF MOTOR COORDINATION 120 Days Old 12 RPM 16 RPM

Styrene

No.

No.

(kw)

litter 28 12 29 6.2 112.1 6.2 89.5 + F; = 16.3

p < 0.01

Offspring 80.2 + 13.5 47.8 - 61.0 47.6 + 5.5 112.4 t 100.9 -

8 RPM

12 RPM

0 50 300

5 2 5

113.8 + 5.4 96.7 - 104.6 89.4 + 4.1

115.6 k 1.9 98.0 - 104.7 91.7 f 4.7 F; = 30.8 F; = 31.5 p < 0.01
p < 0.01

111.3 f 4.1 72.3 - 87.3 88.9 k 8.0 F; = 20.1 p < 0.01 F; = 22.3 p < 0.01

114.3 * 4.9 79.5 - 98.5 86.5 k 4.6 F: = 26.8

p < 0.01

109.2 + 11.4 91.7 - 109.2 105.7 rf- 19.2

114.1 + 5.5 109.4 - 117.7 113.6 + 7.2

109.3 + 9.4 100.0 - 107.6 113.6 + 4.7

Difference @ value)

nS

ns

ns

Data reported combined males and females as no significant gender differences were found. Values in the controls and 300 ppm styrene exposed are expressed as mean (s) and SD, based on the data obtained for all offspring within a litter. Only the range (min-max) of the data of 50 ppm group is listed because the mean and SD of the 50 ppm exposed group were not calculated because of the small number of litters. Significance @value) was calculated between controls and groups exposed to 300 ppm styrene.

TABLE I
EFFECTS OF PRENATAL STYRENE EXPOSURE ON SPONTANEOUS (48-50 DAYS OLD, MALE OFFSPRING) ACTIVITY FOR 24 H

Styrene (ppm) 0

NO. litter

NO. Offspring

Exploratory (First lh) Activity Total 24 h Activity Dark Phase Activity

Light Phase Activity

50 300 Difference @ value) ns F; = 5.7 p < 0.05

5 2 5 36133 rt 3480 35930 - 42483 42833 f 2126 22157 + 2916 23435 - 24670 27506 + 3277 F; = 4.2 0.05 < p < 0.01

15 8 15

3512 + 666 3173 - 5273 5080 + 874

13979 + 2977 12490 - 17810 15325 f 2696 Its

Values on the controls and 300 ppm styrene exposed are expressed as mean + SD, based on the data obtained for all offspring within a litter. Only the range (min-max) of the data of 50 ppm group is listed because the mean and SD of the 50 ppm exposed group were not calculated because of the small number of litters. Significance @ value) was calculated between controls and groups exposed to 300 ppm styrene.

TABLE 8
RESPONSE RATE FOR CONTINUOUS REINFORCEMENT SCHEDULE Session No. litter No. Offspring (MALE OFFSPRING)

styrene (ppm)

1
2

50 300

5 2 5 15 8 15 43.8 + 30.3 1.5 - 18.3 28.7 + 13.0 92.0 + 39.6 6.0 - 58.3 44.1 f 23.0

150.7 + 22.4 43.0 - 81.7 68.2 + 41.4 F; = 9.7

134.6 k 49.0 92.0 - 101.0 85.8 i 74.3

132.6 f 55.3 109.3 f

87.3 119.3 64.5

154.2 k 51.0 127.8 - 168.0 137.0 ?I 72.3

166.3 f 49.6 107.0 - 182.0 138.5 k 58.4

Difference 0, value) KS ns

<

0.01

ns

fLS

KY

fZ.7

Values in the controls and 300 ppm styrene exposed are expressed as mean f SD, based on the data obtained for all offspring within a litter. Only the range (min-max) of the data of 50 ppm group is listed because the mean and SD of the 50 ppm exposed group were not calculated because of the small number of litters. Significance (p value) was calculated between controls and groups exposed to 300 ppm styrene.

PRENATAL

STYRENE

EXPOSURE

AND DEVELOPMENT

129
We should consider, however, the limitations in our present findings. The principal problem in any neuroteratological study of this kind is the need to avoid the influence that maternal factors may have on offspring performance by treating litter as the smallest statistical unit and testing a large number of litters. Because of the small number of litters, especially in the lower-dose group (50 ppm), it was rather difficult to assess the maternal or genetic influences (9). In addition, the observed effects cannot be definitely attributed to a prenatal effect because a cross-fostering design was not employed. Whereas the findings of this study should be regarded as preliminary, they are important because little or no attention has previously been directed toward the potential effects that exposure to styrene, or to solvents in general, may have on the functional development of offspring. Organic solvents may not be totally without effect simply because no apparent structural damage is ascertained during the teratological examination.
ACKNOWLEDGEMENT

development and an increase in the levels of dopamine and serotonin receptors in comparison to those born to dams receiving only a low protein diet. The behavioral effects of in utero styrene exposure were task- and age-specific. For example, both the ambulation score and rearing frequency of gestationally exposed rats were higher than controls on CDs 30 and 31 and on GDs 60 and 61, whereas significant differences were observed neither in rearings nor in ambulation on GDs 127 and 128. Deficits in motor coordination were seen in 30- and 60-day-old offspring exposed to styrene although these deficits did not persist when animals were tested at 120 days of age. Catch up or rehabilitative effects might account for these findings, because the differences in body weight of the offspring among the groups became gradually smaller with time after birth. In our previous study we observed the same age-specific effect, i.e., behavioral effects of lead acetate administered to rats during the first 3 weeks of life disappeared over time and could not be distinguished at approximately 10 months after intubation (16). In addition, neurohistological examination revealed no evidence of overt brain damage in styrene-exposed animals. Therefore, our findings provide presumptive evidence that the behavioral changes encountered after prenatal styrene exposure occurred in the absence of overt neuropathology.

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (No. 61570277) of Japan.

REFERENCES 1. Adams, J. Methods in behavioral teratology. In Riley E. P.; Vorhees C. V., eds. Handbook of behavioral teratology. New York: Plenum Press; 1986:67-97. 2. Beliles, R. P.; Butala, J. H.; Stack, C. R.; Makris S. Chronic toxicity and three-generation reproduction study of the styrene monomer in the drinking water of rats. Fundam. Appl. Toxicol. 5855-868; 1985. 3. Broadhurst, P. L. Application of biometrical genetics to the inheritance of behavior. In: Eysench, H. J. ed. Personality, Vol. 1. London: Routledge & Kegan Paul; 1960:3-102. 4. Chernoff, N.; Woodrow, S.; Miller, D. B.; Rosen, M.; Rogers, J. M. Effects of chemically induced maternal toxicity on prenatal development in rats. Teratology 42:651-656; 1990. 5. Fabro, S.; Brown, N. A.; Scialli, D. R. Is there a fetal solvent syndrome? Reproductive Toxicology 2:17-20; 1983. 6. Ferster, C. B.; Skinner, B. F. Schedules of reinforcement. New York: Appleton-Century-Crofts; 1957. 7. Harkonen, H.; Holmberg, P. C. Obstetric histories of women occupationally exposed to styrene. Stand. J. Work. Environ. Health 8:74-77; 1975. 8. Holmberg, P. C. Central nervous defects in two children of mothers exposed to chemicals in the reinforced plastics industry: Chance or causal relation? Stand. J. Work Environ. Health 3: 212-214; 1977. 9. Holson, R. R.; Pearce, B. Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol. Teratol. 14:221-228; 1992. 10. Jurand, A. Malformations of the central nervous system induced by neurotropic drugs in mouse embryos. Dev. Growth Diff. 22: 61-70; 1980. 11. Kankaanpaa, J. T. J.; Hemminki, K.; Vainio, H. Embryotoxicity and teratogenicity of styrene and styrene oxide on chick embryos enhanced by trichloropropylene oxide. Acta Pharmacol. Toxicol. 45:399-402; 1979. 12. Kankaanpaa, J. T. J.; Elovarra, E.; Hemminki, K.; Vainio, H. The effects of maternally inhaled styrene on embryonal and foetal development in mice and Chinese hamsters. Acta Pharmacol. Toxicol. 47:127-129; 1980. 13. Khanna, V. K.; Husein, R.; Hanig, J. P.; Seth, P. K. Increased neurobehavioral toxicity of styrene in protein-malnourished rats. Neurotoxicol. Teratol. 13:153-159; 1991. 14. Kishi, R.; Katakura, Y.; Okui, T.; Ogawa, H.; Ikeda, T.; Miyake, H. Placental transfer and tissue distribution of 14C-Styrene:A radioautographic study in mice. Br. J. Ind. Med. 46:376-383; 1988. 15. Kishi, R.; Katakura, Y.; Ikeda, T.; Chen, B. Q.; Miyake, H. Neurochemical effects in rats following gestational exposure to styrene. Toxicol. Lett. (in press). 16. Kishi, R.; Ikeda, T.; Miyake, H.; Uchino, E.; Tsuzuki, T.; Inoue, K. Effects of low lead exposure on neuro-behavioral function in the rat. Arch. Environ. Health 38:25-33; 1983. 17. Kostinen, K.; Hemminki, K.; Experimental teratogenicity and embryotoxicity of occupational chemicals. In: Hemminki K.; Sorsa, M.; Vainio, H., eds. Occupational hazards and reproduction. Sapporo, Japan: Washington Hemisphere Publishing Corp.; 1985:127-144. 18. Lauder, J. M.; Helmet, K. Neurotransmitters in development as possible substrates for drugs of use and abuse. In: Yanai, J., ed. Neurobehavioral teratology. New York: Elsevier Science; 1984: 289-328. 19. Lemasters, G. K.; Samuels, S.; Morrison, J. A.; Brooks, S. M. Reproductive outcomes in women exposed to solvents in 36 reinforced plastics companies. II Lowered birth weight. JOM 31:115120; 1989. 20. Lindbohm, M. L. Effects of styrene on the reproductive health of women: A review. In: Sorsa, M.; Peltone, K.; Vainio, H.; Hemminki, K., eds. Butadiene and styrene: Assessment of health hazards. IARC 1993:163-169. 21. Loizou, L. A. The postnatal ontogeny of monoamine-containing neurons in the central nervous system of the albino rat. Brain Res. 40:395-418; 1970. 22. McDonald, J. C.; Lavoie, J.; Cote, R.; McDonald, A. D. Chemical exposures at work in early pregnancy and congenital defects: A case-referent study. Br. J. Ind. Med. 44:527-533; 1987. 23. McDonald, A. D.; McDonald, J. C.; Armstrong, B.; Cherry, N. M.; Cote, R.; Lavoie, J.; Nolin, A. D.; Robert, D. Fetal death and work in pregnancy. Br. J. Ind. Med. 45:148-157; 1988. 24. Miyake, H.; Ikeda, T.; Maehara, N.; Harabuchi, I.; Kishi, R.; Yokota, H. Slow learning in rats due to long-term inhalation of toluene. Neurobehav. Toxicol. Teratol. 5:541-548; 1983. 25. Murray, F. J.; John, M. F.; Balmer, M. F.; Schwetz, B. A. Teratological evaluation of styrene given to rats and rabbits by inhalation or by gavage. Toxicology 11:353-343; 1978.

130
26. Scheffe, H. A method for judging all contrasts in the analysis of variance. Biometrika 40:87-104; 1953. 27. Sikov, M. R.; Canon, W. C.; Carr, D. B.; Miller, R. A. Reproductive toxicology of inhaled styrene oxide in rats and rabbits. J. Appl. Toxicol. 6:155-164; 1986. 28. Tamura, R. N.; Buelke-Sam, J. The use of repeated measures analyses in developmental toxicology studies. Neurotoxicol. Teratol. 14:205-210; 1992. 29. Vainio, H.; Hemminki, K.; Elovaara, E. Toxicity of styrene and styrene oxide on chick embryos. Toxicology 8:319-325; 1977.

KISHI ET AL.
30. Vorhees, C. V. Origins of behavioral teratology. In: Riley E.; Vorhees, C. V. eds. Handbook of behavioral teratology. New York: Plenum Press; 1986:3-22. 31. Weiss, B.; Spyker, J. M. Behavioral implications of prenatal and early postnatal exposure to chemical polhrtants. Pediatrics 53: 851-859; 1974. 32. Zielhuis, R. L.; Stijkel, A.; Verberk, M. M.; van de Poel-Bot, M. Plastic Monomers. In: Health risks to female workers in occupational exposure to chemical agents. Berlin: Springer-Verlag; 1984: 5-14.