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Human exposure to the organic solvent toluene may produce a variety of acute and chronic toxic effects(1–3). A clinically significant form of human toluene exposure occurs due to inhalant abuse. This form of substance abuse is common among adolescents of lower socioeconomic groups. Adolescent and young adult women represent a significant proportion of abusers(4, 5), with toluene considered to be the preferred solvent among abusers(6).

Several cases of clinical toluene-abuse embryopathy have been reported in offspring of toluene-abusing women(3, 7–12). As newborns, the affected infants are growth-retarded and microcephalic. Dysmorphic features including short palpebral fissures, deep set eyes, small face, low set ears, micrognathia, spatulate fingertips, and small fingernails have been described(8–11). As these children mature, developmental delay, language impairment, hyperactivity, cerebellar dysfunction, and postnatal growth retardation become evident(3, 9, 11, 12). Although these adverse effects of maternal toluene abuse were not seen in all exposed offspring, the majority were affected. Fifty-four percent of newborns were small for gestational age, 67% developed postnatal microcephaly, and developmental delay was documented in 38 to 80% of at-risk offspring(11, 12).

Although human teratogenic effects of toluene secondary to solvent abuse are apparent, the risks of occupational toluene exposure to the developing fetus have not been established. From a limited number of studies, it is suggested that maternal occupational exposure to toluene may increase the rate of spontaneous abortions(13) and result in “small babies”(14) and an increase in genitourinary and gastrointestinal defects(15). Neurodevelopmental evaluations of these children have not been reported.

The presence of a more profound teratogenic syndrome (including microcephaly and craniofacial anomalies) in the offspring of toluene abusers is likely due to inhalation dose. Toluene exposure dosages encountered by solvent abusers and workers may differ by up to two orders of magnitude. It is estimated that abusers may inhale from 4 000 to 12 000 ppm toluene, taking multiple inhalations over a several minute period with repetitive dosing continuing for many hours(16, 17). In the industrial setting, such as in a plastic processing factory, measured toluene levels ranged from 184 to 332 ppm(18), whereas the threshold limit value is 100 ppm measured as a time-weighted average over an 8-h day(19). Industrial exposure levels have been noted to increase to 30 000 ppm after an accident(20).

Previous animal research has primarily examined the teratogenic effects of maternal exposure to low dose (133-1596 ppm) toluene(21–26). In a preliminary study, we initiated an evaluation of the teratogenic effects of a higher dose of toluene(27). In that study, pregnant rats received gavage doses of toluene which produced blood tolueneversus time profiles which simulated the profiles achieved after a 3-h inhalation exposures to 3290 ppm toluene, a level somewhat below the range encountered by toluene abusers. The offspring of these toluene-exposed dams demonstrated a generalized growth retardation, with statistically significant reductions in fetal liver and kidney weights. Toluene exposure also resulted in a significant reduction in maternal gestational weight gain and a small reduction in maternal food consumption.

Because the preliminary study did not demonstrate a significant reduction in fetal brain growth due to maternal toluene exposure, a subsequent set of experiments were designed. In this second study, reported here, a higher dose of toluene was administered, and a pair-feeding model was used to evaluate the effects of any toluene-induced reduction in maternal food consumption on fetal growth.

METHODS

The procedures used in this study were approved by the Animal Use and Care Advisory Committee of the University of California, Davis. Female Sprague-Dawley rats (160-180 g) were purchased from Simonsen Laboratories, Inc. (Gilroy, CA) and housed in wire mesh cages. They were provided with water and a diet consisting of dextrose, 62%; casein, 24%; corn oil, 8%; mineral mix, 6%; and vitamin mix(28, 29) ad libitum, and they were maintained in a temperature-controlled room at 22°C under a 12-h light/dark cycle. For breeding, male rats obtained from the same vendor were placed with the females overnight. The presence of copulation plugs the following morning was taken as evidence of successful mating, and that day was designated as pregnancy d 0. For this study, there were eight control, eight pair-fed, and eight toluene-exposed dams. Throughout gestation, daily maternal weight and food consumption were monitored in all dams. Each dam in the pair-fed group was matched with a dam in the toluene-exposed group. On every day of the treatment period (d 6-19), each pair-fed dam received the same amount of food per day of gestation as its matched toluene-exposed dam consumed on that particular day of gestation.

From d 6 through 19 of gestation, toluene-exposed dams were given a daily dose of toluene by transoral gavage. Toluene (Fisher, San Francisco, CA) was diluted in corn oil to give a final concentration of 650 mg/mL. Animals in the treatment group received 1 mL/kg of this toluene/corn oil solution whereas animals in the control and pair-fed groups received an identical daily gavage dose of corn oil. Previous studies in rats have demonstrated that this gavage dose of toluene will produce blood toluene versus time profiles which, after integration, are equivalent to the integrated profiles obtained after a 3-h inhalation exposure to 4168 ppm toluene(30).

On d 19, 2 h after receiving toluene or corn oil, the dams were killed by CO2 vapor inhalation. A heparinized blood specimen was obtained by cardiac puncture and was frozen for later determination of toluene concentration by gas chromatography/mass spectroscopy(31). The gravid uterus and maternal liver were then removed and weighed. The uterus was then dissected and examined for live, stillborn, and resorbed fetuses.

Each fetus was then identified by its intrauterine location, removed along with its placenta, and sexed. The placenta and associated membranes were weighed separately from the fetus. One male and one female fetus from each litter were placed in 95% ethanol, and two male and two female fetuses from each litter were placed in formalin. The remainder of the fetuses were examined for major craniofacial and external malformations and were then dissected. The heart, liver, and kidneys were removed and weighed. The brain was removed and divided into forebrain and hindbrain at the mesencephalicpontine junction, and each portion was weighed.

The fetuses which had been placed in 95% ethanol were then processed for evaluation of skeletal ossification. After 1 wk of fixation, the specimens were stained with Alcian blue and Alizarin Red S via the method described by McLeod(32). After staining, ossification centers in the sternum, metacarpus, metatarsus, and caudal vertebrae were counted via the method of Aliverti et al.(33).

The fetuses which had been placed in formalin were examined externally and internally for malformations using the method of Barrow and Taylor(34). The heads of these fetuses were embedded in paraffin using standard techniques. These specimens were then sectioned in the coronal plane at 6 μm from the level of the anterior horns of the lateral ventricles to the level of the fourth ventricle. Every 25th section was affixed to a glass microscope slide and stained with hematoxylin and eosin. These sections were examined for malformations and neuropathologic changes. Morphometric analysis of the coronal section at the level of the anterior commissure, corresponding to level E19-16 of the atlas of Paxinos et al.(35), was accomplished with the use of a camera lucida, a digitizing tablet, and Sigma scan software (Jandel Scientific, San Rafael, CA). The cross-sectional areas of the whole brain, ventricular system, cerebral cortex, germinal matrix, caudate nucleus, and lateral septal nucleus were measured.

All numerical data presented are expressed as the mean ± SD. One-way ANOVA was used to compare the control, pair-fed, and toluene-exposed groups. For the evaluation of the effects of prenatal toluene exposure on the offspring, two different methods were used. For the majority of the measurements, the litter was chosen as the statistical unit for the analyses. However, because fewer fetuses per litter were selected for evaluation of skeletal ossification and brain morphometry, the fetus was chosen as the statistical unit for these analyses. A p value <0.05 was selected for determining statistical significance. When a statistically significant finding was documented, the values for the three groups were then compared with a Student-Newman-Keuls test.

RESULTS

The maternal effects of toluene exposure during d 6-19 of pregnancy are shown in Table 1. Toluene exposure did not result in any maternal deaths. Maternal toluene exposure resulted in a 13.7% reduction in food consumption between d 6 and 19 of gestation (p < 0.001). Maternal gestational weight gain was reduced in both the toluene-exposed group and in the pair-fed group. When compared with the control dams, the toluene-exposed dams gained 36% less weight, whereas the weight gain in the pair-fed dams was 15.5% less. The gestational weight gains in each of the three groups were significantly different from one another (p < 0.05). There was no significant difference in the weights of the gravid uteruses, suggesting that the reduced maternal gestational weight gains of both the toluene-exposed and pair-fed dams were due to a decrease in overall body weight gain. There was no significant difference in either maternal liver weight or liver/body weight ratio. All toluene-exposed dams had detectable blood toluene levels at the time of sacrifice (15.2 ± 13.1 μg/mL); these levels were in the range predicted by our previous studies of toluene uptake and elimination kinetics(30).

Table 1 Maternal effects of toluene exposure during d 6-19 of gestation (mean ± SD)
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A total of 312 fetuses were examined (102 control, 113 pair-fed, and 97 toluene-exposed). There were no differences in the number of implantations or resorbed fetuses per pregnant dam. Gestational exposure to toluene resulted in statistically significant reductions in placental weights and in fetal body and organ weights (Table 2). For each of these significant observations, the toluene-exposed group differed from both the control and pair-fed groups, whereas the latter two groups were not different from one another. For example, when compared with the control group, fetal body weight and placental weight in the toluene-exposed group were reduced by 21.6 and 21.9%, respectively (p < 0.001). Brain weight was reduced by 11.9% (p < 0.001), whereas kidney weight was reduced by 38% (p = 0.006). Ratios of fetal organ weight/body weight were not affected by toluene exposure, except for the brain weight/body weight ratio. This ratio was increased by 12% (p < 0.001,F2,21 = 28.30) in the toluene-exposed group (data not shown).

Table 2 Fetal effects of toluene exposure during d 6-19 of gestation (mean ± SD)
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Maternal toluene-exposure caused a delay in skeletal ossification. In the sternum, metacarpus, and metatarsus, a significantly smaller number of ossification centers was noted in the toluene-exposed group when compared with the control and pair-fed groups (Table 3). In the caudal vertebrae, the number of ossification centers in the toluene-exposed group was significantly less than the number of centers in the pair-fed group.

Table 3 Effects of prenatal toluene exposure on the number of ossification centra (mean ± SD)
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The effect of toluene exposure on inducing malformations was minimal. The only malformation detected was dilation of the renal collecting system. This malformation was noted in a few fetuses from each of the three groups, and the rate was not increased in the toluene-exposed group (χ2 = 1.60, NS).

Toluene-exposure did result in alterations of brain growth. Examination of serial coronal sections of the brain revealed dilation of the ventricular system in a number of the toluene-exposed fetuses (Fig. 1). No other neuropathologic findings including heterotopia, necrosis, inflammation, or hemorrhage were noted. Morphometric analysis of brain sections (Table 4) indicated that prenatal exposure to toluene resulted in smaller brains with an increase in the size of the ventricular system and a reduction in the cross sectional area of the caudate nucleus. Although the ANOVA indicated that there was a difference in whole brain surface areas between the three groups, the Student-Newman-Keuls test demonstrated that the significance was due primarily to the difference between the pair-fed group and the toluene-exposed group. Statistical analyses of the values for the ventricular system and the caudate nucleus cross-sectional areas indicated that the differences between the three groups were due to toluene exposure. The control and pair-fed groups were not different. In particular, prenatal toluene exposure increased the size of the ventricular system and the ratio of the ventricular system/whole brain both by 31%(p = 0.05 and p = 0.008, respectively) whereas this treatment reduced the cross-sectional area of the caudate nucleus by 16.5%(p = 0.002).

Figure 1
figure 1

Coronal sections of the fetal head and brain at the level of the anterior commissure obtained from control (A) and toluene-exposed (B) 19-d rat fetuses. Maternal exposure to toluene during gestation reduces the cross-sectional area of the brain and caudate nucleus (arrowhead), whereas the size of the ventricular system is increased. In this particular specimen from the toluene-exposed group, note the dilation of the dorsal third ventricle (arrow). Magnification = 4×.

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Table 4 Effects of prenatal toluene exposure on the cross sectional area of specific brain structures (mean ± SD)
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DISCUSSION

This study has demonstrated several effects of maternal oral exposure to toluene administered once daily on d 6-19 of gestation. Toluene treatment caused a significant decrease in fetal and placental weights, together with reductions in fetal organ weights and a delay in skeletal ossification. These reductions in fetal organ weights were not associated with a change in the organ weight/body weight ratio. These observations suggest that prenatal toluene exposure caused generalized growth retardation, rather than organ specific developmental toxicity. The fetal brain weight/body weight ratio was actually increased; however, this was likely due to the much larger reduction in body weight seen in the toluene-exposed fetuses.

Maternal gestational weight gain was also reduced by toluene treatment. Despite these effects, toluene exposure did not cause maternal deaths, stillbirths, or fetal resorptions. Toluene exposure also significantly reduced maternal food consumption, a change that was suggested by our earlier experiments(27). The pair-fed model used in the present study provides evidence to support the conclusion that the fetal growth retardation observed in the toluene-exposed group was due to prenatal toluene treatment, rather than to decreased maternal food consumption. It should be emphasized that certain degrees of maternal malnutrition may augment the teratogenic effects of toluene. In a previous study by da Silva and et al.(36), the teratogenic effects of this solvent were augmented when the dam's food intake was restricted to 50%. As poor nutrition is a common feature of solvent abusers(5), this finding clearly has important clinical significance.

In our previous study, in which a lower dose of toluene was administered in an identical manner, generalized fetal growth retardation also was produced. However, in that study, the degree of intrauterine growth retardation was less, and toluene exposure did not significantly reduce the weights of the fetal brain and heart(27). The toluene doses used in that study were slightly less than those encountered by toluene abusers(16, 17). As significant neurodevelopmental deficits are present in children with clinical toluene-abuse embryopathy, a legitimate animal model of this condition must include CNS teratogenic effects. The present study, which used a higher dose of toluene, and which was in the pharmacokinetic range encountered by solvent abusers, produced offspring with significant alterations in brain growth and development. Although the brain weight/body weight ratio was increased in the toluene-exposed pups, suggesting that toluene exposure reduced systemic growth more than brain growth, e.g. brain-sparing(37, 38), the morphometric studies clearly demonstrated that toluene exposure did not spare the development of the brain. Given the anatomic changes described, these toluene exposures may also have resulted in bahavioral teratologic (neurologic) deficits in these pups.

In this study, the dams were exposed to toluene during d 6-19 of gestation. During this period of time, the vast majority of forebrain neurons are being generated(39). This is true not only for the caudate nucleus, the cross-sectional area of which was reduced by toluene exposure, but also for the cerebral cortex, septal nuclei, and various other forebrain and hypothalamic nuclei. From this study, it cannot be firmly concluded that toluene has a specific teratogenic effect on caudate nucleus development. Because only one coronal section of the brain was evaluated by morphometry, the singular reduction of the cross-sectional area of the caudate nucleus by toluene may have been an artifact. Morphometric analysis of serial sections with the calculation of the volumes of specific nuclei and brain regions may shed light on this question.

The dilation of the ventricular system was striking in the toluene-exposed animals (Fig. 1,Table 4). These changes were due primarily to dilation of the third ventricle, rather than to a diffuse enlargement. Although early hydrocephalus cannot be excluded, this alteration may be due to either a loss of tissue or a reduction of neuronal generation in the region of the hypothalamus. These changes may lead to neuroendocrine dysfunction in the affected animals.

Past animal research by other investigators on the teratogenic effects of toluene in rats, rabbits, and mice has focused primarily on fetal growth and gross abnormalities(21–26). The results of these studies have been completely reviewed in our previous report(27). These past studies did not focus on the CNS, and the experimental designs would not have detected any toluene-induced alterations in brain weight or morphometry. In general, prenatal toluene exposure resulted in a decrease in fetal growth and skeletal development, both of which are confirmed by the present experiments.

The mechanisms by which toluene produces teratogenic effects have not been examined. Renal tubular acidosis has been seen frequently in toluene abusers(3), and similar biochemical abnormalities have been detected in two affected newborns(7). It has been proposed that the resultant alteration in serum electrolytes and acid-base balance may, in part, affect the developing fetus(7). Direct toxic effects of toluene on the developing fetus cannot be ruled out. With its high lipid solubility, toluene is transferred across the placenta. Transplacental transfer of toluene has been demonstrated in one newborn infant(7) as has accumulation of toluene in the placenta of pregnant mice and developing fetal tissues(40). In a rat in vitro 10-d-old embryo model, toluene at a concentration of 2.25 μmol/mL (207 μg/mL) has been shown to be embryotoxic(41). This concentration of toluene is over 13 times greater than the maternal blood toluene levels obtained in our study. In our study, we did not observe an increase in stillbirths, fetal resorptions, or a change in litter size. However, fetal growth retardation was demonstrated. This suggests that although not being fetotoxic, lower levels of toluene adversely affect the organogenic phase of fetal development.

It has been suggested that toluene abusers are exposed to inhalation toluene levels of 4 000 to 12 000 ppm, or higher(16, 17). In this study, we exposed pregnant dams to oral doses of toluene. Although this mode of toluene administration differs from the repetitive inhalations taken by toluene abusers, this single oral dose of toluene is similar, in a pharmacokinetic manner, to 3-h toluene inhalations at the lower end of this range. This study clearly shows a systemic growth retardation effect of prenatal toluene exposure, together with a reduction in brain weight and abnormal subcortical development. The rat brain undergoes significant development during the final days of gestation through the 2nd wk of postnatal life. This period of rat brain development corresponds to the third trimester of human brain development (brain growth spurt)(37, 42). As all of the pups were evaluated on d 19 of gestation, it is not known if the effects of prenatal toluene exposure described in this study (both neurologic and systemic) would persist, or whether catch-up growth might ensue. Further experiments will be needed to examine the teratogenic effects of toluene on this latter stage of brain development.