Prenatal bupropion exposure enhances the cocaine reward and stress susceptibility in adult mice

Although a growing body of evidence supports the notion that certain antidepressant treatments in pregnancy produce earlier delivery and minor behavioral teratogenesis in infants, the long-term effects of such treatments in adulthood remain ill-defined. Recently, postnatal exposure to psychotropic drugs was found to affect the emotional development and susceptibility to abused drugs. Thus, this study aimed to examine whether prenatal exposure of four frequently-used antidepressants, bupropion, fluvoxamine, citalopram, and trazodone, altered the responsiveness to stress and cocaine in the adulthood. Dams received daily injection of bupropion (25 or 12.5 mg/kg), citalopram (5 mg/kg), fluvoxamine (10 mg/kg), trazodone (20 mg/kg) or saline throughout their third trimester of gestation, and several birth outcome indices were then examined. Locomotor activity, naive anxiety levels, and the sensitivity to the cocaine reinforcing effects were observed in pups at their day 56-60 post partum. We found that trazodone treatment produced a high mortality rate in pups after weaning. Mice, prenatally treated with bupropion at 25 mg/kg, exhibited lower rearing numbers and ambulatory activity as compared to the saline-treated mice. More importantly, such treatment enhanced the mouse sensitivity to the reinforcing effects of cocaine. Taken together, these results suggest that use of bupropion in the late pregnancy may run a risk of enhancing the offspring's susceptibility to stress and cocaine reward in adulthood.     

Prenatal cocaine exposure disrupts the dopaminergic system and its postnatal responses to cocaine

Impaired attention is the hallmark consequence of prenatal cocaine exposure (PCE), affecting brain development, learning, memory and social adaptation starting at an early age. To date, little is known about the brain structures and neurochemical processes involved in this effect. Through focusing on the visual system and employing zebrafish as a model, we show that PCE reduces expression of dopamine receptor Drd1, with levels reduced in the optic tectum and other brain regions, but not the telencephalon. Organism‐wide, PCE results in a 1.7‐fold reduction in the expression of the dopamine transporter (dat), at baseline. Acute cocaine administration leads to a 2‐fold reduction in dat in drug‐naive larvae but not PCE fish. PCE sensitizes animals to an anxiogenic‐like behavioral effect of acute cocaine, bottom‐dwelling, while loss of DAT due to genetic knockout (DATKO) leads to bottom‐dwelling behavior at baseline. Neuronal calcium responses to visual stimuli in both PCE and DATKO fish show tolerance to acute cocaine in the principal regions of visual attention, the telencephalon and optic tectum. The zebrafish model can provide a sensitive assay by which to elucidate the molecular mechanisms and brain region‐specific consequences of PCE, and facilitate the search for effective therapeutic solutions.     

Prenatal exposure to glucocorticoids and adult disease

There is evidence to suggest that an individual's susceptibility to cardiovascular disease cannot be entirely explained by differences in life style factors (i.e., low physical activity, high fat/salt diet), or genetic causes, but may also be influenced by factors encountered during intrauterine life. Epidemiological studies found the link between low birth weight for gestational age (a broad index of sub-optimal intrauterine environment) and increased incidence of cardiovascular and metabolic diseases in adulthood. Many animal models in which the intrauterine environment was altered during early/late or throughout gestation demonstrated long-term effects on adult health. In general stress in early gestation is more likely to be associated with adult cardiovascular disease including hypertension, whereas late gestation stress may also be associated with adult hypotension in addition to metabolic/endocrine abnormalities. Two systems have been widely hypothesised to serve as mechanisms via which adverse prenatal influences impinge on adult cardiovascular and metabolic disease; hippocampal-hypothalamo-pituitary-adrenal axis (HHPA) and renin-angiotensin system (RAS). Interestingly, at least in our animal model of adult hypertension after brief/early prenatal glucocorticoid exposure, HHPA axis is not altered when studied either in late gestation or at several stages during adulthood. However, our more recent results, using the same animal model, suggest a major role for the central and renal RAS. This review will mainly focus on animal models and potential mechanisms via which a perturbed intrauterine environment (undernutrition or steroid exposure) lead to adult cardiovascular and/or metabolic disease.     

Prenatal exposure: The effects of prenatal cocaine and methamphetamine exposure on the developing child

Prenatal substance use remains a significant issue in the United States. Initial reports regarding prenatal cocaine and methamphetamine exposure suggested profound adverse effects on child development. However, subsequent prospective, longitudinal investigations have found more subtle effects. What follows is a brief review of the health, growth, behavioral, and intellectual outcomes for children exposed to prenatal cocaine and prenatal methamphetamine. Factors that may mitigate or intensify subtle adverse effects manifested in exposed children will also be discussed. Birth Defects Research (Part C) 108:142–146, 2016. © 2016 Wiley Periodicals, Inc.     

Prenatal ethanol exposure in mice: teratogenic effects.

C57BL/6J mice were fed a liquid diet in which 17, 25, or 30% of the calories were derived from ethanol from the fifth through the tenth day of gestation. Control mice were fed lab chow or pair-fed identical diets, except that sucrose substituted isocalorically for ethanol. At term the fetuses were removed and, following fixation, examined by microdissection. The incidence of fetal resorptions and congenital malformations increased in a dose-related manner. Anomalies included skeletal, neurological, urogenital, and cardiovascular systems. These data indicate that in mice, an alcohol diet which is adequate in vitamins and protein results in increased fetal wastage and birth defects.     

Prenatal effects of transplacental exposure to ifosfamide in rats

Ifosfamide is an alkylating chemotherapeutic agent that exhibits activity against a wide range of tumors. Exposure to such agent just prior to mating (preconception period) may have adverse effects on developing embryos. I investigated the rate of apoptosis and the histological changes in both placenta and developing fetal tissues after exposure to ifosfamide of young female rats before mating. I clarified the roles of the drug and the placenta in causing fetal developmental toxicity. Rats were divided into four groups: (1) untreated controls, (2) rats administered saline, (3 and 4) rats administered 25 mg/kg and 50 mg/kg ifosfamide, respectively. After treatment of females with ifosfamide, the treated females were allowed to mate with normal untreated males. All pregnant animals were sacrificed on day 18 of gestation. Treatment with high doses of ifosfamide caused small placentas, fewer viable fetuses, greater post-implantation losses and more resorbed fetuses. Reduced progesterone and increased prolactin levels also were found. Immunohistochemical staining, the TUNEL technique and histological studies showed increased apoptotic cells and many histological changes in the placenta, and in fetal brain, liver and kidney tissues. Ifosfamide treatment increased apoptosis and caused hypoplasia of placental basal and labyrinth zones, which resulted in pathological changes in developing fetal tissue.     

Chronic prenatal exposure to cocaine alters cerebrovascular responses in newborn pigs

Maternal cocaine abuse may increase the incidence of perinatal asphyxia. In nonexposed asphyxiated neonates, decreased cerebrospinal fluid (CSF) cAMP concentrations are associated with poor neurological outcome. On the other hand, cocaine increases central nervous system (CNS) cAMP. Therefore, we hypothesized that in utero cocaine exposure may increase brain cAMP and thereby preserve cerebrovascular responses to cAMP-dependent stimuli following asphyxia. Pregnant pigs received either cocaine (1 mg/kg, i.v.) twice weekly during the last trimester or normal saline vehicle (sham-control) and were allowed to deliver vaginally at term. Cranial windows were implanted in the newborn pigs within the first week of life and used to collect CSF for cAMP determinations and to assess changes in pial arteriolar diameters (PAD). In the first part of the study, pial arteriolar responses to different vasodilator and vasoconstrictor stimuli were evaluated in piglets prior to asphyxia (n = 20). In newborn pigs exposed to cocaine, cerebrovascular responses to hypercapnia and norepinephrine were significantly exaggerated compared to controls. Then, piglets were randomly selected for the second part of the study that involved prolonged asphyxia (n = 12). In cocaine-exposed but not sham-control piglets, CSF cAMP increased markedly during asphyxia. In the sham piglets, but not the cocaine-exposed piglets, CSF cAMP fell progressively below the baseline during recovery. Cerebrovascular reactivity to cAMP-dependent stimuli (hypercapnia and isoproterenol) was preserved during recovery from asphyxia in the cocaine-exposed piglets but significantly attenuated in the sham controls. We conclude that piglets with chronic prenatal exposure to cocaine show exaggerated cerebrovascular responses to vasogenic stimuli and preserved cAMP-dependent cerebral vasoreactivity following asphyxia.     

Prenatal exposure to a 50 Hz magnetic field has no effect on spatial learning in adult mice

Male CD1 mice were exposed in utero to a 50 Hz sinusoidal magnetic field at 5 mT (rms) for the period of gestation and were raised subsequently without applied fields. At 82-84 days of age, they began a radial-arm-maze experiment that was designed to test for deficits in spatial learning in memory. Mice exposed in utero and sham-exposed mice exhibited no statistically significant differences in performances. © 1996 Wiley-Liss, Inc.     

Prenatal exposure to ethanol causes partial diabetes insipidus in adult rats

Chronic consumption of ethanol in adult rats and humans leads to reduced AVP-producing neurons, and prenatal ethanol (PE) exposure has been reported to cause changes in the morphology of AVP-producing cells in the suprachiasmatic nucleus of young rats. The present studies further characterize the effects of PE exposure on AVP in the young adult rat, its hypothalamic synthesis, pituitary storage, and osmotically stimulated release. Pregnant rats were fed a liquid diet with 35% of the calories from ethanol or a control liquid diet for days 7-22 of pregnancy. Water consumption and urine excretion rate were measured in the offspring at 60-68 days of age. Subsequently, the offspring were infused with 5% NaCl at 0.05 ml.kg(-1).min(-1) with plasma samples taken before and at three 40-min intervals during infusion for measurement of AVP and osmolality. Urine output and water intake were approximately 20% greater in PE-exposed rats than in rats with no PE exposure, and female rats had a greater water intake than males. The relationship between plasma osmolality and AVP in PE-exposed rats was parallel to, but shifted to the right of, the control rats, indicating an increase in osmotic threshold for AVP release. Pituitary AVP was reduced by 13% and hypothalamic AVP mRNA content was reduced by 35% in PE-exposed rats. Our data suggest that PE exposure can cause a permanent condition of a mild partial central diabetes insipidus.     

Perinatal effects of exposure to PCBs on social preferences in young adult and middle-aged offspring mice

In social species, social interactions between conspecifics constitute a fundamental component to establish relations, provide best chances to reproduce, and even improve survival rates. In this study, a three-chambered social approach test was used to estimate the level of sociability and level of preference for social novelty in both male and female young adult (postnatal day (PND) 50) and middle-aged (PND 330) offspring mice (n = 10 per group) that were perinatally exposed to a mixture of six polychlorinated biphenyls (PCBs), 28, 52, 101, 138, 153, and 180, at environmentally low doses (10 and 1000 ng/kg b.w. for dams during gestation and lactation), a profile that closely mimics human exposure to contaminated fish. Our results showed that PCBs bidirectionally modulated social preferences in offspring mice, and the effects were sex and age dependent. However, increased levels of social interactions were rather frequently detected in both assays of the three-chambered test. Reduced social interaction was only induced in 1000 ng/kg PCB-exposed middle-aged males, which exhibited similar preferences to social and non-social stimuli when compared to middle-aged controls. Furthermore, results showed that plasma levels of both corticosterone and acetylcholinesterase activity were higher in all PCB-exposed middle-aged males and females than in their control counterparts. In summary, although the effects of PCBs were only of moderate magnitude, our results suggest that a PCB mixture can act as an endocrine disruptor in offspring mice, disturbing the formation of normal social habits.     

Abdominal radiation exposure elicits inflammatory responses and abscopal effects in the lungs of mice

An inflammatory reaction is a classical feature of radiation exposure and appears to be a key event in the development of the acute radiation syndrome. We have investigated the radiation-induced inflammatory response in C57BL6/J mice after total abdominal or total-body irradiation at a dose of 15 Gy. Our goal was to determine the radiation-induced inflammatory response of the gut and to study the consequences of abdominal irradiation for the intestine and for the lungs as a distant organ. A comparison with total-body irradiation was used to take into account the hematopoietic response in the inflammatory process. For both irradiation regimens, systemic and intestinal responses were evaluated. A systemic inflammatory reaction was found after abdominal and total-body irradiation, concomitant with increased cytokine and chemokine production in the jejunum of irradiated mice. In the lungs, the radiation-induced changes in the production of cytokines and chemokines and in the expression of adhesion molecules after both abdominal and total-body irradiation indicate a possible abscopal effect of radiation in our model. The effects observed in the lungs after irradiation of the abdomino-pelvic region may be caused by circulating inflammatory mediators consequent to the gut inflammatory response.     

Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster

The trace biogenic amines tyramine and octopamine are found in the nervous systems of animals ranging in complexity from nematodes to mammals. In insects such as Drosophila melanogaster, the trace amine octopamine is a well-established neuromodulator that mediates a diverse range of physiological processes, but an independent role for tyramine is less clear. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC). We previously reported the identification of two Tdc genes in Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, we examined normal and cocaine-induced locomotor activity in flies that lack both neural tyramine and octopamine because of mutation in Tdc2 (Tdc2(RO54)). Tdc2(RO54) flies have dramatically reduced basal locomotor activity levels and are hypersensitive to an initial dose of cocaine. Tdc2-targeted expression of the constitutively active inward rectifying potassium channel Kir2.1 replicates these phenotypes, and Tdc2-driven expression of Tdc1 rescues the phenotypes. However, flies that contain no measurable neural octopamine and an excess of tyramine due to a null mutation in the tyramine beta-hydroxylase gene (TbetaH(nM18)) exhibit normal locomotor activity and cocaine responses in spite of showing female sterility due to loss of octopamine. The ability of elevated levels of neural tyramine in TbetaH(nM18) flies to supplant the role of octopamine in adult locomotor and cocaine-induced behaviors, but not in functions related to female fertility, indicates mechanistic differences in the roles of trace amines in these processes.     

Developmental exposure to environmental estrogens alters anxiety and spatial memory in female mice

Humans and wildlife are exposed to numerous anthropogenic drugs and pollutants. Many of these compounds are hormonally active, and recent evidence suggests that the presence of these endocrine disruptors permanently alters normal development and physiology in a variety of vertebrate species. Here, we report on the effects of developmental exposure to two common estrogenic pollutants, bisphenol A and ethinyl estradiol on sexually dimorphic, non-reproductive behavior. Mice (Mus musculus domesticus) were exposed to environmentally relevant levels of these chemicals (2 and 200 microg/kg/day for bisphenol A and 5 microg/kg/day for ethinyl estradiol) throughout prenatal and early postnatal development. As adults, the animals were observed in a variety of tests measuring sexually dimorphic behaviors including short-term spatial memory (in a radial-arm maze and a Barnes maze) and anxiety (in an elevated-plus maze and a light/dark preference chamber). Developmental exposure to ethinyl estradiol was found to masculinize behavior in all of the assays used. Bisphenol A increased anxious behavior in a dose-dependent fashion but had no effect on spatial memory. These results indicate that non-reproductive, sexually dimorphic behavior is sensitive to endocrine disruption. In addition, these experiments suggest that both humans and wildlife are being exposed to levels of these endocrine disrupting compounds that are sufficient to disrupt the development of the nervous system and that may have permanent consequences on sexually dimorphic behaviors.     

Assessment of adult skeletons to detect prenatal exposure to acetazolamide in mice

A skeletal variant assay system (SVAS) consisting of a group of 88 spontaneously occurring qualitative variations of the adult mouse skeleton was applied to CD-1 animals that had been exposed in utero to 0, 200, or 1,000 mg/kg/day of the sodium salt of acetazolamide dissolved in distilled water, presented by SC injection of the dam during day 8 or days 9-11 of gestation. Two separate series of experiments were performed, and skeletons were examined at postnatal 62 +/- 2 days. Variation occurred in 62 and 67 characters in the two series. Frequencies of occurrence differed from untreated (UNTD) and vehicle-treated (VEH) values of substantial numbers of variants in a dose related manner for both series in both treatment regimes as did the number of variants which showed significantly different frequencies (P less than .01) in comparisons of experimental with either UNTD or VEH. At the high doses 12 and 16 variants occurred with significantly different frequencies from UNTD in day 8 treatments in the two series, and 15 and 19 variants differed in the days 9-11 treated group. Contrasting high-dose animals with appropriate vehicle controls revealed differences in 13 and 12 variants in day 8 treatment groups and in 18 and 15 variants in days 9-11 groups. Agreement between the two series was good, especially in the D9-11 treatments. Several variants differed significantly from both UNTD and VEH in both series of experiments. Among these were a number which appeared more or less specific to acetazolamide exposure. They include: day 8 treatments--accessory parietal, frontal extension, and 27 presacral vertebrae; day 9-11 treatments--sacral fusions in dorsal processes and vertebral bodies, and caudal fusions and malformations; both sets of treatments--lumbar fusions, and fusions of the transverse processes of the sacral vertebrae. Other importantly affected variants, also seen in exposure to other compounds include: day 8 treatments--abnormal metoptic roots; day 9-11 treatments--accessory mental foramen, foramina transversaria imperfecta of the atlas, arch foramen of the fifth cervical (C) vertebra, malformed sternebrae, fossa olecrani perforata, and fewer than 30 caudal vertebrae; both treatment regimes--parted frontals, accessory transverse foramina in C3-C6, reduced articular processes on the thoracic vertebrae, and 14 ribs. By all criteria applied, the SVAS is able to detect prenatal exposure to acetazolamide in adult skeletons even in the absence of any gross morphological abnormalities.     

Prenatal exposure to androgen influences morphology and aggressive behavior of male and female mice

To assess the effects of prenatal exposure to androgen on adult aggressiveness in mice, pregnant mice were given injections of 1.5 mg testosterone propionate (TP) or oil from Days 12 to 16 of pregnancy. All offspring were gonadectomized on the day of birth. Neonatal treatment occurred on the day following birth and consisted of one-half of the animals from each prenatal treatment group being injected with 100 μg TP while the other half were injected with oil, yielding four Prenatal/Neonatal treatment groups for each sex. On postnatal Day 60, all offspring were given subcutaneous implants of encapsulated testosterone (T) and tested for 10 min every other day against a male opponent until aggression was observed. Female offspring of TP-treated mothers were indistinguishable from males on external examination at birth. The duration of exposure to T required to induce aggression provides an index of the sensitivity of the neural substrate to T. When arranged from the most sensitive to the least sensitive to the aggression inducing action of T, the four Prenatal/Neonatal treatment groups of females were significantly different from each other: Group TP/TP > Group OIL/TP > Group TP/OIL > Group OIL/OIL. A similar pattern was observed for the male offspring. There were no differences in the proportion of animals per group that exhibited aggression (virtually all animals fought) or the intensity of aggression once exhibited. The results demonstrate that morphological and behavioral masculinization can occur in response to exposure to androgen during prenatal as well as neonatal life in mice.     

Splenocyte subsets in normal and protein malnourished mice after long-term exposure to cocaine or morphine

An experimental model which resembles human drug addiction was developed to study the effect of chronic drug (cocaine or morphine) administration on the immune system. As malnutrition has been associated with drug use, a low protein diet has been evaluated for its contribution to the impairment of the immune system during cocaine/morphine addiction. Female C57BL/6 mice that received a 20% or 4% casein diet were studied. Both drugs were administered intraperitoneally daily for 11 weeks and drugs were administered in increasing daily doses, beginning after 3 weeks of diet consumption. Doses of cocaine began with 5 mg/kg body weight and reached the maximum dose of 40 mg/kg/day at the fourth week. Doses of morphine gradually increased from 10 mg/kg to 75 mg/kg body weight with the maximum dose reached after 5 weeks of treatment. Cocaine administration reduced body weight, particularly in the low protein diet group, and spleen weight in protein malnourished mice. Cocaine as well as saline injected mice showed a decrease in the percentage of CD4+ CD8+ and Mac-1+ cells and an increase in B cells in the spleens of well nourished mice. Morphine-treated mice showed similar results to those observed in cocaine or saline treated mice. These results suggest that cocaine, morphine or saline injection can alter the percentage of cells that express a defined phenotype independently of the nutritional status of the subject. Moreover, the effect appears dependent on a stress mediated process.     

Comparison of cerebrovascular effects of intravenous cocaine injection in fetal, newborn, and adult sheep

Cocaine may cause stroke, intracranial hemorrhage, seizures, and neurobehavioral abnormalities in fetuses, newborns, and adults, and there could be developmental and/or species differences in mechanisms for these cocaine-induced cerebrovascular effects. To evaluate developmental differences in responses to cocaine, we compared the cerebrovascular and metabolic responses to a 2 mg/kg iv cocaine dose in unanesthetized fetal (n = 8, previously reported, direct fetal injection), newborn (n = 6), and adult (n = 12) sheep. We measured cerebral blood flow, mean arterial blood pressure, and arterial and venous O(2) content, and we calculated cerebral O(2) consumption and cerebral vascular resistance at baseline and at 30 s and at 5, 15, and 60 min after cocaine injection. Cerebral blood flow increased 5 min after injection in the fetus and newborn, but not until 15 min in the adult. In the fetus, cocaine caused a transient cerebral vasoconstriction at 30 s; in all three groups, cocaine caused cerebral vasodilation, which was delayed in the adult. Cerebral metabolic O(2) consumption increased 5 min after injection in the fetus and newborn, but not until 15 min after injection in the adult. Arterial O(2) content decreased 5 min after injection in the fetus and 15 min after injection in the adult. We speculate that clinical differences in response to cocaine injection may be explained, in part, by these developmental differences in the cerebrovascular and metabolic responses to cocaine.     

Prenatal inflammation exacerbates hyperoxia-induced functional and structural changes in adult mice

Maternally derived inflammatory mediators, such as IL-6 and IL-8, contribute to preterm delivery, low birth weight, and respiratory insufficiency, which are routinely treated with oxygen. Premature infants are at risk for developing adult-onset cardiac, metabolic, and pulmonary diseases. Long-term pulmonary consequences of perinatal inflammation are unclear. We tested the hypothesis that a hostile perinatal environment induces profibrotic pathways resulting in pulmonary fibrosis, including persistently altered lung structure and function. Pregnant C3H/HeN mice injected with LPS or saline on embryonic day 16. Offspring were placed in room air (RA) or 85% O(2) for 14 days and then returned to RA. Pulmonary function tests, microCTs, molecular and histological analyses were performed between embryonic day 18 and 8 wk. Alveolarization was most compromised in LPS/O(2)-exposed offspring. Collagen staining and protein levels were increased, and static compliance was decreased only in LPS/O(2)-exposed mice. Three-dimensional microCT reconstruction and quantification revealed increased tissue densities only in LPS/O(2) mice. Diffuse interstitial fibrosis was associated with decreased micro-RNA-29, increased transforming growth factor-β expression, and phosphorylation of Smad2 during embryonic or early fetal lung development. Systemic maternal LPS administration in combination with neonatal hyperoxic exposure induces activation of profibrotic pathways, impaired alveolarization, and diminished lung function that are associated with prenatal and postnatal suppression of miR-29 expression.     

Assessment of adult skeletons to detect prenatal exposure to trypan blue in mice

A Skeletal Variant Assay System (SVAS) consisting of a group of 88 spontaneously occurring qualitative variations of the adult mouse skeleton was studied in CD-1 mice which had been exposed in utero by way of three daily ip injections of their dams on days 7-9 of gestation with trypan blue. Treatment groups received daily doses of 0.25 cc of 0, .037, .075, .15, or .30% trypan blue dissolved in 0.9% NaCl. Two separate series of experiments were performed, and skeletons were examined at 62 +/- 2 days postnatal. Sixty-six and 58 of the variants occurred in the two series, respectively. Frequencies of occurrence of substantial numbers of variants differed from Untreated (UNTD) and Vehicle-Treated (VEH) values in a dose-related manner for both series. At the high dose 18 and 22 variants occurred with significantly different (P less than .01) frequencies from UNTD in the two series. Contrasting high-dose animals with vehicle controls revealed significant differences in 24 and 17 variants. There were 13 and 14 variants in the two series, respectively, which differed from both UNTD and VEH. If one considers differences at P less than .01 in one comparison and P less than .05 in the other, then 22 and 18 variants qualify as being significantly different from both controls in the two series. Agreement between the two series was excellent regarding which variants were affected. Several differed significantly from both UNTD and VEH in both series of experiments. Among these were a number which appeared more or less specific to trypan blue exposure. They include Dyssymphysis of the Atlas, Carpal Fusions, and Tarsal Fusions. Although increased frequency of an Interfrontal bone is seen with several treatments, the magnitude of the response and the low doses at which it is elicited are unique to trypan blue exposure. Numerous low-dose effects are striking in this set of experiments, making the SVAS a very sensitive indicator of trypan blue exposure. In addition to the variants mentioned, a large cluster of cervical (C) vertebrae variants, including dyssymphyses, fusions, imperfect transverse foramina of C1 and C2, and accessory transverse foramina of C3-C6, as well as vertebral fusions at various levels (especially cervical, sacral, and caudal), appear to be the principal effects of exposure to this compound. In addition, rib malformations at the high dose level, and increased frequency of occurrence of 27-presacral vertebrae at all dose levels, were important effects.(ABSTRACT TRUNCATED AT 400 WORDS)