Neurobehavioral Consequences of Intermittent Prenatal Exposure to High Concentrations of Toluene

Neurotoxicology and Teratology, Vol. 19, No. 4, pp. 305–313, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362/97 $17.00 1 .00

PII S0892-0362(97)00034-2

Neurobehavioral Consequences of Intermittent Prenatal Exposure to High Concentrations of Toluene HENDRÉE E. JONES AND ROBERT L. BALSTER Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0613 Received 15 November 1996; Accepted 16 March 1997 JONES, H. E. AND R. L. BALSTER. Neurobehavioral consequences of intermittent prenatal exposure to high concentrations of toluene. NEUROTOXICOL TERATOL 19(4) 305–313, 1997.—The effects of several concentrations of toluene on physical and behavioral development were examined in CD-1 mice prenatally exposed during the last week of gestation. Pregnant mice were exposed to either 200, 400, or 2000 ppm toluene (TOL) for 60 min three times a day during gestational days 12–17. A sham group was exposed concurrently to filtered air. No group differences were observed in maternal weight gain or food consumption, common measures of maternal toxicity. Initial litter characteristics including gestation length, number of litters delivered, and litter size were also similar. At birth, mean initial individual pup weight from representative male and female 2000 TOL-exposed pups was less than sham-exposed pups; however, entire litter weight did not differ. Pups were evaluated on postnatal days 1–20. Pups exposed to 2000 TOL gained less weight and performed more poorly on the behavioral tests of the righting reflex, grip strength, and inverted screen. In contrast, pups exposed to either 200 or 400 TOL did not differ from sham-exposed pups on any of the measures of development or behavior. These data provide evidence for the neurobehavioral teratogenicity of prenatal exposure to high levels of toluene late in gestation. Because this exposure regimen of intermittent high-concentration exposure was designed to simulate human exposures that might occur with toluene abuse, these results are consistent with case reports of adverse consequences of inhalant abuse by pregnant women. © 1997 Elsevier Science Inc. Toluene

Mice

Solvents

Inhalant abuse

Prenatal

Development

(32,33). From the seventh through ninth grades there are very small gender differences in inhalant use (46). However, a sex difference emerges in the tenth grade and continues until the twelfth grade with 3.5% of male seniors and 2% of female seniors reporting use of inhalants within the past year (32). It is estimated that toluene abusers may inhale from 4,000 to 12,000 ppm, taking multiple inhalations continuing for many hours (8,9). In comparison, the workplace 10-min short-term exposure limit for toluene is 500 ppm (45). Toluene acts directly on the central nervous system to produce narcosis, tremors, lack of coordination, muscular hypertonicity, and general weakness (6). Repeated sniffing of toluene vapor results in transient distal renal acidosis with hypokalemia, hypophosphatemia, hypomagnesemia, and rhabdomyolysis (56,57). Long-term chronic toluene abuse may result in permanent

TOLUENE, an aromatic hydrocarbon solvent, is used in the production of a number of industrial chemicals (benzene, phenol, benzoic acid, and vinyl toluene), paints, and rubber (43), and is a component of gasoline, which contains 5–7% toluene by volume (3). Toluene is also found in many household products including paint thinners, adhesives, and various cleaning fluids. Inhalation of toluene vapors is the main source of human exposures; it occurs in the workplace, through the everyday use of toluene-containing products, or by deliberate intoxication (14). Indeed, toluene is found in many of the most widely abused products (40). Presently, inhalant abuse is one of the most pervasive and least recognized drug problems. In the United States, the prevalence of inhalant abuse among adolescent youth is exceeded only by the use of marijuana, alcohol, and tobacco

Requests for reprints should be addressed to Dr. Robert L. Balster, Department of Pharmacology & Toxicology, Medical College of Virginia, Box 980310, Virginia Commonwealth University, Richmond, VA 23298-0310. Tel: (804) 828-8402; Fax (804) 828-2117.

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306 neurotoxic damage with cerebellar degeneration and cortical atrophy (49,50) and hearing loss (48). Although toluene abuse is recognized to result in serious health consequences, there have been very few studies of the effects of prenatal exposure to high concentrations of toluene. Toluene embryopathy has been described in the clinical literature where progeny of toluene-abusing mothers have been evaluated (5,20,26,47,56,60). The neonates are small for gestational age, microcephalic, with dysmorphic features including short palpebral fissures, deep-set eyes, small face, low-set ears, micrognathia, spatulate fingertips, and small fingernails (5,26, 56,60). With age, additional developmental and neurologic consequences of prenatal toluene exposure have been reported including developmental delay, language impairment, hyperactivity, and cerebellar dysfunction (5,26,47,56). Although these clinical reports suggest that toluene abuse by pregnant women is associated with growth and behavioral deficits in the offspring, it is difficult to attribute these effects directly to toluene because controlled studies ruling out other risk factors are difficult to perform. Animal studies provide a means to more directly examine the developmental toxicity of prenatal chemical exposure (17,55). Most animal studies of prenatal toluene exposure have focused on the teratogenic effects on fetal growth and gross abnormalities following continuous low concentration exposure over an extended period of time, similar to what might be encountered in workplace exposures (11,27,28,44,51,61). Many of these studies have reported reduction of fetal body weight and retardation of skeletal development in rodents. Unfortunately, potential methodological problems limit the generalizability of the data. The methodological issues include severe exposure parameters such as continuous 24 h/day exposure for numerous days, a lack of reporting indices of maternal toxicity and use of individual pups rather than the litter as the statistical unit for analyses. For instance, in mice, no detrimental effects on the fetuses were seen following exposure to 133 ppm toluene for a total of 12 h per day on gestational days 6–15 (61), although reductions in fetal weight were reported following 24 h/day exposure to 133 ppm of toluene on gestational days 6–13 (28). Exposure to 399 ppm for a total of 12 h/day during gestation days 6–15 resulted in abortions whereas exposure to the same concentration for 24 h/day during gestational days 6–13 was lethal to all dams (28). Convincing evidence for the developmental toxicity of toluene comes from studies that have used appropriate controls and less severe exposure parameters. It was reported that exposure to neither 200 nor 400 ppm toluene for 7 h/day on gestational days 7–16 resulted in reduced fetal body weight in mice; however, exposure to 400 ppm resulted in offspring having a greater number of rib abnormalities relative to control animals. No differences in maternal body weight occurred at either concentration (11). In another study using mice, exposure to 1000 ppm toluene for 6 h/day from gestation day 1–17 resulted in a significant increase in fetuses with an extra rib (51). Exposure to 2000 ppm 6 h/day from 80 days prior to mating through gestation day 19 results in reduced fetal body weight in rats relative to controls (30). Although it is evident from the results of the studies conducted in rodents that toluene is associated with fetotoxicity, it is less clear what impact perinatal toluene exposure may have on postnatal development and behavior. Few studies have examined postnatal maturational endpoints or behavior of animals exposed in utero to toluene. Of the three reported examinations of postnatal development and behavior following in utero exposure to toluene, two of the studies exposed

JONES AND BALSTER dams to low levels of toluene continuously from gestation until adulthood. In rats, there was little evidence for delays in the ontogeny of hair eruption and eye opening following exposure to low-levels of toluene in drinking water at 100 ppm beginning on gestation day 13 and administered to the offspring until postnatal day (PND) 48 (52). Adult neurobehavioral and cognitive development was affected by prenatal and postnatal exposure via drinking water to toluene in NYLAR mice (38) and via inhalation in Wistar rats (52). However, because the exposure period in both studies persisted far beyond gestation, it was not possible to isolate what changes were associated with in utero exposure alone. Thus far, only one report of exposure to toluene (1.2 g/kg, SC) given only during the last week of gestation examined postnatal behavior (13). Results indicated that toluene did not produce differences in spontaneous activity at 30 days of age or conditioned avoidance at 90 days old. As yet, there have been no published studies of preweaning postnatal outcome following in utero exposures to repeated short-term high concentrations of toluene via inhalation as might occur in toluene abusers. Therefore, the aim of the present experiment was to characterize in mice during the early postnatal period the developmental and behavioral consequences of prenatal exposure to various concentrations of toluene in an intermittent pattern of exposure. The exposure concentrations of 200 and 400 ppm were selected based on previous literature demonstrating fetotoxicity or embryo lethality (11,28). Although the effects following adult exposure are not necessarily predictive of the effects observed in offspring exposed prenatally, the highest concentration of 2000 ppm was selected based on the profile of behavioral effects in adult animals reported following acute exposure such as increased ease of handling, disturbances in righting reflex, decreased forelimb grip strength, and impaired coordination (58). This concentration produces acute, reversible, psychoactive drug-like effects on learned behavior of adult mice (41,42) and may be within the range of concentrations thought to be encountered by toluene abusers. The treatment conditions and study design were similar to those used in a previous study, which demonstrated behavioral teratological effects of exposure to another abused solvent 1,1,1-trichloroethane (TCE) (34). The focus on early assessment was selected for several reasons. Because most of the available clinical population of exposed offspring are still young, correlations between animal and human data might be more closely analogous by testing animal populations early in life. Examining the possible teratogenic effects of toluene in young animals may provide evidence regarding the underlying primary alterations associated with the substance especially because the primary effects could lead to secondary disruptions in the development of other neural systems that might alter the animals behavior in adulthood. Both of these consequences may alter later development as the animal matures, making it difficult to tease apart the underlying primary alterations from the compensatory or secondary changes resulting from prenatal exposure to a compound. METHOD

Animals Fifty-two timed-pregnant female CD-1 mice weighing an average of 30 g were obtained on gestation day 11 (sperm plug 5 day 0) from Charles River Laboratories (Wilmington, MA). Following the procedures detailed in the Internal Animal Care and Use Committee-approved protocol, all mice were singly housed in 28.5 3 17.5 3 12 cm clear plastic cages with hardwood

NEUROBEHAVIORAL CONSEQUENCES OF TOLUENE chip bedding material (Sani-Chips™, P. J. Murphy Forest, Montville, NJ) and food and water available ad lib. The vivarium in which the mice were housed was a light (12 h on/12 h off) and temperature (21–24°C; humidity 50 6 5%) controlled, American Association for Accreditation of Laboratory Animal Care-approved animal facility. The morning following arrival to the vivarium, dams were assigned to one of four groups using the randomization procedure of sampling without replacement (36) (N 5 13): sham exposed (sham), toluene 200 ppm exposed (TOL 200), toluene 400 ppm exposed (TOL 400), or toluene 2000 ppm exposed (TOL 2000). During the behavioral studies the temperature (21–23°C), humidity (50 6 5%), and light cycle (12L:12D, lights on 0700 h) were held constant. Animal Husbandry During Exposures All dams had access to food and water in their home cages except during periods of toluene exposure. Before each daily exposure period, the mice were removed from the home cage, weighed, and placed into the exposure chamber. Food intake was monitored daily. Inhalation Exposures Exposures to 200, 400, and 2000 ppm toluene and sham were conducted three times a day for 60 min during gestation days 12–17. Commencing at 0800 h on day 12 of gestation, mice were weighed and dams in the sham-exposed control group were placed in an identical exposure chamber at the same time as the TOL 200 exposure group, but exposed to filtered air only. The same procedure was repeated at 1100 and 1500 h. The TOL 400 and TOL 2000 groups were exposed concurrently at 0915, 1215, and 1615 h. Exposures were conducted in two 20.8-l sealed glass exposure chambers with a stainless steel grid floor and fitted with a Teflon lined lid that were housed under a chemical fume hood (Kewaunee Scientific Equipment Co., Adrian, MI). To expose the subjects, air containing toluene vapor was continually passed through the chamber. To generate the toluene vapor and introduce it into the chamber, the flow of pumped filtered air was split and passed through two flow regulators (R7630 series, Matheson Co., Dorsey, MD). Air from the lower flow rate flow regulator passed through a gas dispersion tube to a fritted bubbler (#7202-16, Ace Glass Co., Vineland, NJ) inserted in a 2-l flask containing toluene. The vapor-laden air passed to a 1-l mixing chamber where it was diluted with filtered air controlled by a second flow regulator. The two flow rates were adjusted to produce the test concentrations while maintaining a 10 l/min flow rate through the exposure chamber. At the conclusion of the exposure period, the chamber was flushed with air for 3 min and then subjects were removed to their home cages to await the next exposure period. Chamber toluene concentrations were continuously monitored by single wavelength-monitoring IR spectrometry (Miran IB, Foxboro Analytical, North Haven CT). The spectrometer was equipped with a 10-cm flow-through stainless steel cell (Model 424, Foxboro Analytical) and BaF2 windows. Concentrations were calibrated using standard closed-loop procedures. Toluene was monitored at the analytical wavelength of 13.8 mm. Manual adjustments of flow regulators were made to maintain test concentrations, although this was not needed after proper settings had been established. Recordings of chamber concentrations on an x-y plotter showed that concentrations did not vary by more than 12% over the entire experiment

307 and chamber concentrations reached about 96% of target concentration or a near 0 level within 3 min. The average temperature and humidity in the hood where the chambers were located were 20.5 6 0.5°C and 68 6 4%, respectively. Toluene (99% purity; 17996-5) was purchased from Aldrich Chemical Co. Inc. (Milwaukee, WI). Parturition Procedures Following the termination of the last exposure on gestation day 17, dams were returned to their home cages and given a paper towel for nest construction. To ensure that the person conducting tests would be naive to the treatment of the dam, an independent laboratory technician assigned each of the dams a code number that was placed on their home cage. After recording the code and treatment of the dam, all other identifying information about group assignment was removed from the home cages. The dams were checked twice daily (0830 and 1630 h) for parturition. Litters born after 0830 h and before 1630 h were deemed day 0 of life. For litters born after 1630 h, the next day was considered day 0. As soon as possible after birth, the dam was weighed and the following litter data were collected: litter size, litter weight and sex ratio, initial weight for one randomly chosen male and female pup, representing initial offspring body weight, and the number of malformed or dead offspring. Litters were culled to four of each sex. Litters remained with their biological mothers as described by the Environmental Protection Agency Guidelines for Developmental Neurotoxicity (62). Developmental and Behavioral Assessment Observations. The developmental and behavioral tests were selected to measure various CNS functions at several stages of development. The preweaning tests were selected from a number of sources and our own pilot work, which suggested that many of the tests demonstrated validity, reliability, and sensitivity (2,10,63). On PND 1, one male and one female pup from each litter were randomly assigned to one of four test categories: 1) physical development, 2) reflex development, 3) muscle strength, and 4) motor coordination using the sampling without replacement method (36). The pups were identified using an indelible nontoxic ink marker (Marker II; Precision Instruments, Van Nuys, CA) to label the pup with the designated number (1–4) of the behavioral test category to which it was assigned. One litter was tested for all measures appropriate for that day before moving to the next litter. All pups were removed from the nest and placed on a heating pad to provide warmth before and after testing. Each pup was given all of the tests from that testing category before the next pup was tested. The order of the individual tests within each testing category was as follows: 2) reflex development—righting reflex and then rooting reflex, 3) muscle strength—forelimb grip strength, and 4) motor coordination—the negative geotaxis test was followed by the inverted screen test. After all of the tests were administered to the designated pups, all pups were returned to the nest. The order of testing the litters was determined initially by chance but then reversed on alternate days for the remainder of the study. All testing was conducted between 0900 and 1630 h. Physical development. One PND 1, one male and one female pup from each litter was randomly assigned for measurement of body weight and development of physical landmarks including pinnae detachment (PNDs 2–5), incisor eruption (PNDs 8–10), and eye opening (PNDs 13–15). Pups were examined on PNDs 1–20 for weight gain.

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Reflex development. Animals were tested on a standardized surface of a surgical blue pad. For the righting reflex, the pup was placed on its side and the latency to right itself recorded in seconds. Each pup received one trial per day on PNDs 1–12. The criterion used for successful righting was the animal’s turning over to a position in which all four paws were in direct contact with the test surface and the limbs in a position so that walking was possible. A maximum of 60 s was given for the pup to right. Pups that did not right were assigned the maximum latency of 60 s. For the rooting reflex, the pup was placed with all four paws on the test surface and the facial region of the snout was stimulated bilaterally by placing the thumb and index finger on both sides of the head and pressing very lightly for up to 5 s. The most complete response was demonstrated with up and down and pushing movements of the head of the mouse as it was stimulated. If the experimenter moved her hand in a linear path, measured over a marked piece of tape, away from the animal while continuing to stimulate the snout, it was expected that the animal would follow the hand while continuing the rooting. Rooting distance (cm) was used to measure rooting reflex intensity. Each pup assigned to this category received one trial each day on PNDs 1–14. Pups failing to meet criterion were assigned a score of 0 cm. Muscle strength. Forelimb grip strength was assessed on PNDs 3–15 by the use of a modified push–pull strain gauge (DFG-2, Chatillon, Greenburough, NC). Modifications included the mounting of a horizontal bar (0.15 cm diameter) supported by a 9 3 10 cm steel bar frame. The horizontal bar was suspended 28.5 cm from a surface covered with cotton gauze. The animal was held by the nape of the neck and its forepaws placed on the bar. The animal was then pulled gently by the tail and the strength of the grasp measured in kilograms (lower limit 5 0.001 kg) and later converted into grams. The peak strength of five determinations was used for data analysis. Coordination. Coordination was examined in two behavioral tests. For the negative geotaxis test the pup was placed, head down, 20 cm from the bottom of a wire mesh screen (0.5 cm2 holes) that was tilted 45°. The latency (up to 60 s) for the pup to turn 180° (upright) was recorded. Pups were given one trial each day on PNDs 8–12. For the inverted screen test, the

pup was placed on a horizontal wire mesh square that was rotated 180° so that the mouse was upside down. The latency (up to 60 s) to climb on the top side of the screen was recorded. The apparatus consisted of a metal rod, 1.4 m long, to which were attached six evenly spaced, 13-cm2 square pieces of wire screen mesh (0.5 cm2 holes). Cotton gauze was provided under the apparatus to cushion the pup’s fall. Pups were given a single trial on PNDs 13–20 and if the pup did not perform the task a score of 60 s was assigned. Statistical Analyses The litter was utilized as the unit of measure and treatment group was the main effect for all models. To control for the use of repeated ANOVAs, the significance levels were adjusted in the negative direction using the Greenhouse–Geisser adjustment of the F-ratio (18,36). Maternal data. Maternal weight and food consumption during exposures were analyzed using a two-factor (group 3 day) repeated measures ANOVA. Initial litter characteristics and developmental landmark acquisition. Litter size, entire litter weight, initial offspring body weight, and developmental landmarks were analyzed by a one-factor ANOVAs. Behavioral and weight gain data. The behavioral tests of righting reflex, rooting reflex, grip strength, negative geotaxis, and inverted screen were analyzed using a two-factor repeated measures ANOVA. If the treatment factor was significant in the repeated measures ANOVAs, further day-specific ANOVA tests were conducted. Because two pups per litter (one male, one female) were tested for each measure, a litter mean score for each developmental and behavioral measure was obtained to avoid inflation of the sample size. Pup weight was also analyzed using a two-factor repeated measures ANOVA. When the day-specific models reached the criterion for significance, Scheffé’s post hoc test was used. Preliminary analysis of the data included sex as an additional variable in each of the ANOVAs and, in the absence of a significant sex by treatment interaction, further analyses were conducted on the data collapsed across sex as described above. The significance level was set at 0.05.

TABLE 1 EFFECTS OF PRENATAL EXPOSURE TO TOLUENE ON MATERNAL AND LITTER CHARACTERISTICS

Maternal characteristics Maternal weight gain (g)* Maternal food consumption (g)† Litter characteristics Number of litters/number of dams Number of litters examined Gestation length (days) Litter size (range) Total litter weight (g) Initial offspring body weight (g/pup)

Sham Exposure

TOL 200 Exposure

TOL 400 Exposure

TOL 2000 Exposure

(N 5 13) 13.7 6 0.6 39.2 6 1.4

(N 5 13) 11.8 6 0.7 38.0 6 2.0

(N 5 13) 11.7 6 0.9 37.2 6 1.5

(N 5 13) 11.2 6 0.3 36.5 6 1.8

13/13 13 20.0 6 0.5 12.2 6 0.6 (9–14) 17.8 6 0.9 1.7 6 .02

13/13 12‡ 20.0 6 0.1 11.5 6 0.4 (9–14) 17.2 6 0.8 1.6 6 .03

13/13 11§ 20.0 6 0.0 11.8 6 0.04 (9–14) 17.5 6 0.8 1.5 6 0.6

13/13 13 20.0 6 0.0 11.7 6 0.4 (10–14) 16.9 6 0.8 1.4 6 .06#

*Mean 6 SE maternal weight gain from gestation days 12–17. Maternal weight before exposure on day 12 subtracted from maternal weight before exposure on day 17. †Total amount of food eaten (g) over the 6 days of exposure. ‡Only 12 of 13 litters examined. One dam cannibalized litter before assessment. §Eleven of 13 litters were tested—two dams killed their litters on PND 1. # p , 0.05 Scheffé F-test vs. sham.

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FIG. 1. Effects of prenatal 200, 400, or 2000 ppm toluene exposure on pup weight gain. Mean 6 SE of pup weights (grams) for PND 1–20. *Toluene 2000 group significantly different from sham exposure group (p , 0.05). RESULTS

Maternal and Litter Results All fifty-two dams that arrived from the supplier were pregnant. Maternal and litter data for the dams included in the four experimental groups are presented in Table 1. Maternal weight gain and food consumption during gestation did not significantly differ among groups. Following parturition, one dam in the TOL 400 condition cannibalized her litter before any assessments were made, resulting in 12 of 13 litters used in the litter analyses for this group. No significant differences were found in the length of gestation or litter size. No missing limbs, gross malformations, or external soft tissue malformations were apparent in any of the newborn pups. No sex difference was observed in initial individual pup weight; therefore, the data were collapsed across sex. There was a significant difference in initial offspring body weight assessed by using the mean weight of a representative male and female pup, F(3, 47) 5 7.09, p , 0.05 (Table 1). Pups exposed to TOL 2000 weighed significantly less than sham-exposed pups. However, total litter weight did not significantly differ among groups. Two dams in the TOL 200 group killed their litters before testing on PND 1. Therefore, number of litters evaluated with developmental tests were as follows: sham exposed (N 5 13), TOL 200 (N 5 12), TOL 400 (N 5 11), and TOL 2000 (N 5 13). Body Weight and Developmental Landmarks There was a significant main effect for treatment on pup weight (Fig. 1), F(3, 45) 5 3.84, p , 0.05. Significant decreases in weight gain were observed from PND 2–7 in the TOL 2000exposed pups relative to sham-exposed pups (p , 0.05). No differences were obtained between sham-exposed and TOL 200- and TOL 400-exposed pups.

FIG. 2. Effects of prenatal 200, 400, or 2000 ppm toluene exposure on reflex development. Top: Effects on righting reflex. Mean 6 SE of pup righting times (in seconds) for PND 1–12. *Toluene 2000 group significantly different from sham exposure group (p , 0.05). Bottom: Effects on rooting reflex. Mean 6 SE of pup rooting distance (centimeters) for PND 1–14.

Table 2 shows that toluene exposure had no significant effects on ontogeny of development. No significant effects of treatment on the timing of pinnae detachment, F(3, 45) 5 0.689, p 5 0.564, incisor eruption, F(3, 45) 5 2.02, p 5 0.125, or eye opening, F(3, 45) 5 0.629, p 5 0.6, were observed. Righting Reflex There was a significant effect for treatment, F(3, 45) 5 5.19, p , 0.05, on the latency to perform the righting reflex (Fig. 2, top panel). TOL 2000-exposed pups had longer latencies to

TABLE 2 DAY AT WHICH DEVELOPMENTAL LANDMARKS WERE ACHIEVED Developmental Landmark

Pinnae detachment Incisor eruption Eye opening

Sham Exposure

TOL 200 Exposure

TOL 400 Exposure

TOL 2000 Exposure

4.0 6 0.1 10.0 6 0.8 13.6 6 0.2

4.0 6 0.2 10.6 6 0.2 13.5 6 0.2

3.7 6 0.2 10.0 6 0.1 13.7 6 0.2

3.8 6 0.2 10.7 6 0.1 13.8 6 0.1

Shown are mean 6 SE days.

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FIG. 3. Effects of prenatal 200, 400, or 2000 ppm toluene exposure on forelimb grip strength. Mean 6 SE of pup grip strength (grams) for PND 3–15. *Toluene 2000 group significantly different from sham exposure group (p , 0.05).

right relative to sham-exposed pups on PNDs 1 and 5–6. No differences in righting were observed between TOL 200 and TOL 400 when compared to sham-exposed pups. Rooting Reflex There was no effect of treatment, F(3, 45) 5 0.69, p 5 0.562, on the rooting reflex measured as distance traveled (Fig. 2, bottom panel). The rooting reflex shows a clear ontogeny with a progressive increase in rooting distance from PND 5 peaking at PND 8–10 and lost by PND 13–14. Forelimb Grip Strength There was a main effect of treatment, F(3, 45) 5 5.41, p , 0.05, on forelimb grip strength (Fig. 3). TOL 2000-exposed pups displayed weaker forelimb grip strength compared to sham-exposed pups on PNDs 5–7 and 9–11. The forelimb grip strength measure shows an ontogeny with TOL 2000-exposed pups lagging behind sham-exposed pups. TOL 200- and TOL 400-exposed pups did not differ in grip strength relative to control pups. Negative Geotaxis No main effect of treatment, F(3, 45) 5 1.16, p 5 0.34, on latency to orient towards the top of an inclined screen (Fig. 4, top panel) was observed. Inverted Screen There was a main effect of treatment, F(3, 45) 5 12.41, p , 0.05, on the latency to climb to the top of the screen (Fig. 4, bottom panel). TOL 2000-exposed pups had longer latencies to climb on top of the screen relative to sham-exposed pups on PNDs 14–17. No differences were observed between shamexposed and TOL 200 or TOL 400 pups. DISCUSSION

The present study examined the preweaning developmental and behavioral effects of intermittent (60 min, three times/day) prenatal exposure to 200, 400, and 2000 ppm toluene during gestational days 12–17. TOL 2000 offspring exhibited deficits in growth and behavioral endpoints relative to sham-exposed

FIG. 4. Effects of prenatal 200, 400, or 2000 ppm toluene exposure on negative geotaxis and the inverted screen test. A 60-s cutoff was used for each measure. Top: Effects on negative geotaxis. Mean 6 SE latency (seconds) to turn 180° for PND 8–12. Bottom panel: Effects on inverted screen test. Mean 6 SE latency (seconds) to climb on top of the screen for PND 13–20. *Toluene 2000 group significantly different from sham exposure group (p , 0.05).

pups. No differences were seen when TOL 200- and TOL 400exposed pups were compared with the performance of shamexposed offspring. The effects of 2000 ppm toluene on offspring development and behavior occurred in the absence of any obvious maternal or fetal toxicity. No differences were observed in maternal weight gain, food consumption, number of litters delivered, litter size, or entire litter weight. Previous studies of toluene exposure in mice and rats have produced conflicting results regarding indices of maternal and fetal toxicity (11,13,22,23,30). A possible explanation for the discrepancies in the results of maternal and fetal toxicity may lie in the unique methodologies used by each experimenter with regard to exposure concentration and duration, maternal age, stage of development during exposure, routes of administration, and the species used. We obtained contradictory data on pup birth weight. Comparisons of total litter weight showed no effect of treatment. On the other hand, comparisons of the initial body weight of representative male and female offspring from each litter showed an effect of TOL 2000 compared to sham-exposed animals. Similar to our representative pup’s decreased weight relative to controls, other studies have reported decreases in initial body weight following high levels of prenatal toluene exposure (22,23,30).

NEUROBEHAVIORAL CONSEQUENCES OF TOLUENE There was a significant effect of TOL 2000 on pup weight gain early in the postnatal period. Other concentrations of toluene were without effect on weight gain. By PND 8, the TOL 2000 pups in the physical development group had caught up to their age mates in weight and remained unaffected for the remainder of the preweaning period. Our data indicating no differences in pup weight gain in 200 or 400 ppm toluene-exposed pups in the physical development group relative to shams are consistent with a previous report in which exposure to either 200 or 400 ppm 6 h/day on days 7–16 of gestation did not alter mouse pup body weights (11). Effects of prenatal exposure to toluene were seen in measures of reflex development, muscle strength, and motor coordination. Pups exposed to TOL 2000 exhibited delays in the righting reflex, inverted screen test, and had weaker grip strength relative to sham-exposed pups. The righting reflex test, considered a test of subcortical maturation (4), resulted in TOL 2000-exposed pups exhibiting prolonged righting reflex latencies relative to control pups on days 1, 5, and 6. The days on which increased righting reflex latencies were observed also corresponded to the period of time during which significant differences were observed in body weights in the physical development group animals (Fig. 1). The latencies of TOL 200- and TOL 400-exposed pups did not differ from sham-exposed animals. In utero exposure to TOL 2000 resulted in decreased grip strength on PNDs 5–7 and 9–11. No differences were seen between TOL 200- and TOL 400-exposed pups and their sham counterparts. Prenatal exposure to TOL 2000 resulted in the delayed ability of the pups to successfully invert on the inverted screen task until PND 18. Although the differences in grip strength and the inverted screen tasks might be related to relatively lower body weights, it is unlikely because behavioral alterations were observed at times when there were no statistically significant differences in the body weights of the physical development group. No differences were observed on the inverted screen task when TOL 200 and TOL 400 ppm pups were compared to sham-exposed pups. Decreases in grip strength were also observed at 2000 ppm toluene. Similar to the present observations of decreased body growth in mice, postnatal growth deficiency has also been described in children born to women who abused toluene during pregnancy (21,47). Although there is a paucity of information on the long-term developmental outcome of children exposed in utero to toluene, the limited data suggest a majority of the children had delays in cognitive, motor, and speech skills and displayed a variety of behavioral disturbances (5,26,64). These cases provide evidence in humans of an association between prenatal toluene exposure and developmental toxicity; however, conclusions based on these data are limited at best by the numerous potentially confounding factors such as maternal nutrition, alcohol, cigarette smoking, or other toxic exposure. The mechanisms by which toluene’s teratogenic effects are produced have yet to be explored. The direct toxic effects of toluene on the developing fetus remains a possibility needing further exploration because toluene crosses the placenta in both humans (20) and mice (19). In vitro, toluene can be embryotoxic in rats. A dose–effect relationship was reported following exposure of 0.37–4.06 mmol/ml toluene, with higher concentrations (the effect level 5 2.25 mmol/ml or 207 mg/ml) resulting in significant reductions in embryonic growth and development and impaired yolk sac circulation (7). It is important to note that some embryos displayed retarded development in spite of well-developed yolk sacs, suggestive of a direct embryotoxic effect of toluene. It is also possible that the decreased weight gain and im-

311 paired behavioral development observed in the TOL 2000exposed pups could be a result of a number of indirect effects. For example, malnutrition has been demonstrated to be a significant factor resulting in delayed growth and maturation (54). However, in the present investigation there was no difference in maternal food consumption or weight gain, common indices of maternal toxicity that could lead to malnutrition in utero. On the other hand, a study that directly examined prenatal exposure to toluene compared to an untreated and pair-fed control group reported decreased fetal and placental weights regardless of maternal food consumption (23). Even if the mother was properly nourished, the placental transport of important nutrients may be impaired resulting in suboptimal levels of nutrition in the fetus. It is presently not known what effect that toluene may have on the diffusion or facilitated transport of critical nutrients required for growth. An investigation examining the effects of toluene on fetal development in adequately and malnourished rats reported that dams administered toluene (1.2 g/kg, SC) during days 14–20 of gestation had long-lasting effects on body weight that were augmented by severe malnutrition (13). These results support the idea that toluene interferes with nutrient transport or utilization. Impaired placental transport of essential vitamins and minerals has been demonstrated in rodent models following exposure to alcohol (37,53), another abused solvent. If there was fetal malnutrition from toluene in our study, it must have been minimal because birth weights of the pups were only minimally affected. Although it has been demonstrated that the fetus is relatively protected from hypoxia (15,31), fetal hypoxia has resulted in learning disabilities, motor disturbances, behavioral alterations (12,16), transient and long-term disruptions in neurobehavioral development, chemical changes (24), and incomplete masculinization of male sexual behavior in adulthood (25). As with prenatal exposure to alcohol (1), fetal hypoxia remains a possible factor in the etiology of tolueneassociated developmental delays. It would be of interest in the future to examine this possibility more fully. In the present experiment pups remained with their biological mother. It is recognized that this limits the ability to draw conclusions about the direct toxic effects of toluene on the fetal–placental unit; however, in this initial study it was a priority to optimize the chance of observing alterations in the offspring. Findings from other studies indicated that even when offspring prenatally exposed to toluene were fostered to untreated dams a deficit in body weight was detected (13). Furthermore, the fostering procedure was not used because the complex maternal–pup relationship is not fully understood and it is not known if the fostering manipulation is stressful for the pups and/or the foster dam and, most importantly, what impact this would have upon development and behavior of the offspring. Thus, it is possible that the effects observed in the TOL 2000 pups were due to lingering effects of toluene on maternal health and behavior. In the future, it is necessary to study the effects of prenatal and maternal exposure to toluene in a separate study using a cross-fostering experimental design to specifically examine the effects of maternal treatment on the pup’s development and behavior. It is possible that the alterations in the behavioral performance of the pups on the tests of righting reflex and grip strength were secondary to the weight deficits observed in the 2000 ppm TOL-exposed pups. However, we did not directly examine the body weight of the animals performing the tasks. Therefore, for these behavioral tasks the issue of whether the findings are primary or secondary to toluene exposure cannot

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be answered in the present study. It is important to note that the deficits that were observed in later tests of grip strength and the inverted screen task occurred in the absence of any difference in weight relative to control animals in the physical development group showing that not all the effects could have been secondary to a smaller size. The behavioral alterations observed were transient, disappearing by weaning. Perhaps the young animals learn to compensate for early effects, evidencing the plasticity of the immature nervous system. Evidence for CNS compensation in prenatally toluene-exposed offspring is found in reports of recovery over trials from initial deficits in rotorod performance (38). An abnormal splay of hindlimbs was noted even when rotorod latencies had recovered, suggestive of compensation. Tests of postweaning behavioral deficits from prenatal toluene exposure were not conducted in our study but adverse effects have been noted in some others (38,51). This would be important area of study for future investigation. Although most solvents appear to produce their neurotoxic effects due to the parent compound, it is possible that reactive metabolites may damage cells and place the rapidly changing embryo/fetus at risk (7). In rodents, most toluene is excreted unchanged with some liver metabolism via the pathway of benzyl alcohol–benzaldehyde–benzoic acid, which is excreted after conjugation with glycine as hippuric acid (29, 59). Presently, it is unknown to what extent toluene is metabolized in the embryo/fetus or what exposure to these metabolites might produce. It is known that toluene is metabolized primarily in the liver in humans and rodents and that the half-

life of elimination of toluene is 60 min for mice (39). Thus, because the metabolites are produced mostly in the liver and are highly reactive and have relatively short half-lives, it is doubtful that the metabolites reach the embryo or fetus (35). In conclusion, the results of the present experiment have shown that repeated prenatal exposure to a high concentration of toluene is associated with developmental toxicity in the progeny of exposed mice. The aim of the present study was to examine the possible early developmental consequences of exposure to toluene similar to what pregnant solvent abusers might encounter. In an attempt to mimic an exposure regimen of a solvent abuser, we repeatedly exposed dams to one of three concentrations of toluene during the last week of gestation. The highest concentration examined (2000 ppm) produced some weight reduction and a pattern of deficits in measures of neuromuscular behavior early in the postnatal period. Furthermore, our results in animals are similar to the clinical neurodevelopmental teratogenic effects seen in the offspring of toluene-abusing women. Therefore, we encourage the increased awareness of toluene abuse during pregnancy, because this presently underrecoginized form of substance abuse may have adverse consequences on the baby. ACKNOWLEDGEMENTS

Research supported by NIDA grant DA03112 and NIDA individual predoctoral fellowship DA05665. We would like to thank Dr. Scott Bowen for his helpful advice and Josiah Hamilton for his technical assistance.

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