Association of alterations in energy metabolism with lead-induced delays in rat cerebral cortical development

Fifteen day-old rat pups taken from dams exposed to 200 mg of lead (Pb) in drinking water were observed to have reduced synaptic densities in the cerebral cortex as determined by ethanol-phosphotungstic acid staining. Synaptic figures of animals in the Pb groups were also observed to possess a less mature synaptic profile.Cerebral cortical slices taken from pups of Pb-treated dams exhibited enhanced respiratory responses to a 3 to 30 mM increase in K⁺ concentration. This response was associated with a higher rate of glucose uptake by tissues of Pb-treated pups unaccompanied by a change in lactic acid output. Cerebral cortical concentrations of amino acids were not altered by Pb-treatment. Serum thyroid hormone levels were identical in 15-day old animals from the control and Pb-treated groups.These data further substantiate delayed cerebral cortical development as a result of low-level Pb exposures, and suggest that such delays may be associated with a direct effect of Pb on cerebral energy metabolism.     

Development Neurotoxicity Evaluation of Orally Administered Isopropanol in Rats

Developmental Neurotoxicity Evaluation of Orally Administered Isopropanol in Rats. Bates, H. K., McKee, R. H., Bieler, G. S., Gardiner, T. H., Gill, M. W., Strother, D. E., and Masten, L. W. (1994). Fundam. Appl. Toxicol. 22, 152-158.Isopropanol was administered by gavage to timed-mated rats from Gestation Day (GD) 6 through Postnatal Day (PND) 21. Doses administered were 0, 200, 700, or 1200 mg/kg/day in a volume of 5 ml/kg. The dams were allowed to deliver and body weights and food consumption were recorded during gestation and lactation. Pups were counted, examined, sexed, and weighed on PND 0, 4, 7, 13, 17, 21, 36, 49, and 68. Litters were culled to eight pups (4:4 or 5:3 sex ratio) on PND 4 and litters without acceptable numbers of male and female pups were eliminated from the study. Pups were weaned on PND 22, and two pups from each litter and their dams were killed. Six of these pups from each dose group were perfused in situ for histopathological examination of the central and peripheral nervous system. Brains of the remaining pups were divided into four regions and weighed. Maternal liver and kidney weights were recorded. Weaned pups were assessed for day of testes descent or vaginal opening and for motor activity on PNDs 13, 17, 21, 47, and 58; auditory startle on PNDs 22 and 60; and active avoidance on PNDs 60-64. These pups were euthanized and examined on PND 68. One high-dose dam died on PND 15, but there were no other clinical observations or effects on maternal weight, food consumption, or gestation length. Pup survival, weight, sex ratio, and sexual maturation were unaffected. There were no biologically significant findings in the behavioral tests, no changes in organ weights, and no pathological findings that could be attributed to isopropanol exposure. In conclusion, there was no evidence of developmental neurotoxicity associated with isopropanol exposure as high as 1200 mg/kg/day.     

Maternal lead exposure during lactation persistently impairs testicular development and steroidogenesis in male offspring

Lead (Pb) is a testicular toxicant. In the present study, we investigated the effects of maternal Pb exposure during lactation on testicular development and steroidogenesis in male offspring. Maternal mice were exposed to different concentration of lead acetate (200 or 2000 ppm) through drinking water from postnatal day (PND) 0 to PND21. As expected, a high concentration of Pb was measured in the kidneys and liver of pups whose mothers were exposed to Pb during lactation. In addition, maternal Pb exposure during lactation elevated, to a less extent, Pb content in testes of weaning pups. Testis weight in weaning pups was significantly decreased when maternal mice were exposed to Pb during lactation. The level of serum and testicular T was reduced in Pb‐exposed pups. The expression of P450scc, P450_17α and 17β‐HSD, key enzymes for T synthesis, was down‐regulated in testes of weaning pups whose mothers were exposed to Pb during lactation. Interestingly, the level of serum and testicular T remained decreased in adult offspring whose mothers were exposed to Pb during lactation. Importantly, the number of spermatozoa was significantly reduced in Pb‐exposed male offspring. Taken together, these results suggest that Pb could be transported from dams to pups through milk. Maternal Pb exposure during lactation persistently disrupts testicular development and steroidogenesis in male offspring. Copyright © 2012 John Wiley & Sons, Ltd.     

Developmental Neurotoxicity Evaluation of Orally Administered Isopropanol in Rats

Isopropanol was administered by gavage to timed-mated rats fromGestation Day (GD) 6 through Postnatal Day (PND) 21. Doses administeredwere 0, 200, 700, or 1200 mg/kg/day in a volume of 5 ml/kg.The dams were allowed to deliver and body weights and food consumptionwere recorded during gestation and lactation. Pups were counted,examined, sexed, and weighed on PND 0, 4, 7, 13, 17, 21, 36,49, and 68. Litters were culled to eight pups (4:4 or 5:3 sexratio) on PND 4 and litters without acceptable numbers of maleand female pups were eliminated from the study. Pups were weanedon PND 22, and two pups from each litter and their dams werekilled. Six of these pups from each dose group were perfusedin Situ for histopatho logical examination of the central andperipheral nervous sys tem. Brains of the remaining pups weredivided into four regions and weighed. Maternal liver and kidneyweights were re corded. Weaned pups were assessed for day oftestes descent or vaginal opening and for motor activity onPNDs 13, 17, 21, 47, and 58; auditory startle on PNDs 22 and60; and active avoidance on PNDs 60–64. These pups wereeuthanized and examined on PND 68. One high-dose dam died onPND 15, but there were no other clinical observations or effectson maternal weight, food consumption, or gestation length. Pupsurvival, weight, sex ratio, and sexual maturation were unaffected.There were no biologically significant findings in the behavioraltests, no changes in organ weights, and no pathological findingsthat could be attributed to isopropanol exposure. In conclusion,there was no evidence of developmental neurotoxicity associatedwith isopropanol exposure as high as 1200 mg/kg/day.     

Developmental triclosan exposure decreases maternal, fetal, and early neonatal thyroxine: a dynamic and kinetic evaluation of a putative mode-of-action

This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0-300mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman(?) qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid hormone-responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300mg/kg/day). Hepatic PROD activity increased 2-3 fold in PND4 pups and PND22 dams, and UGT activity was 1.5 fold higher in PND22 dams only (300mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS-induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia.     

Critical analysis of potential body temperature confounders on neurochemical endpoints caused by direct dosing and maternal separation in neonatal mice: a study of bioallethrin and deltamethrin interactions with temperature on brain muscarinic receptors

The present investigation was conducted to understand better possible confounding factors caused by direct dosing of neonatal mice during the pre-weaning developmental period. By direct dosing, pups might encounter thermal challenges when temporarily removed from their 'natural habitat'. Typically, this leads to a cold environment and food deprivation (impaired lactation) and modulation of the toxic potency of the substance administered. Growth retardation as a consequence of such behavioural changes in pups makes it increasingly difficult to differentiate specific from non-specific mechanisms. Neonatal NMRI mice were dosed daily by gavage (0.7 mg kg(-1) body wt.) from postnatal day (PND) 10-16 with S-bioallethrin, deltamethrin or the vehicle. Then the pups, including their non-treated foster dams, were subjected temporarily for 6 h day to a hypo-, normo- or hyperthermic environment, which was followed by normal housing. The measured temperatures in the environmental chambers were ca. 21, 25 and 30 degrees C, respectively. Thus, temperatures in the hypo- and normothermic groups are comparable to the temperatures commonly present in testing laboratories, whereas the hyperthermic condition is that temperature typically present in the 'natural habitat' of pups. A deviation from the normal behaviour of both pups and dams was observed in the hypo- and normothermic groups. In these groups the rectal temperatures of pups were markedly decreased, especially in the early phase of the study (PND 10-12). Neonates that received either test substance displayed changes in body weights and brain weights at terminal sacrifice (PND 17) when subjected temporarily to a non-physiological environment. An enormous influence of environmental temperature on the density of muscarinic receptors in the crude synaptosomal fraction of the cerebral cortex was ascertained. In summary, these results demonstrate that the direct dosing of thermolabile neonatal mice by gavage is subject to significant artefacts that render the interpretation of findings from such studies difficult. It appears that if direct dosing of neonatal pups is mandated, and inhalation is a relevant route of exposure, the combined inhalation exposure of dams with their litters is an alternative procedure that does not cause disruption of the 'natural habitat' of pups. However, owing to their higher ventilation, under such conditions the pups may receive dosages at least double those of the dams.     

Secalonic acid D mycotoxin affects ontogeny of brain catecholamines and swimming in mice

Suckling mice exposed to secalonic acid D (SAD) mycotoxin postnatally (by gavaging dams with 0, 15 or 25 mg/kg on postgestational days 1 to 10) or prenatally (by gavaging with 25 mg/kg on gestational day 13 to produce a positive behavioral teratogenic control group) manifested subtle preweaning neurobehavioral, neurochemical and growth deficits. Gestational length, maternal weight gain, neonatal sex ratio and physical appearance of pups at birth were unaffected by treatment. Prenatal SAD (25 mg/kg) delayed (p less than 0.05) ontogeny of swimming on postnatal days (PND) 11 and 13 and reduced norepinephrine (NE) and dopamine (DA) levels in prosencephalon on PND 7 and in cerebellum-pons on PND 7-16. In the postnatal treatment groups, pup body weight gains were decreased from PND 9-22. Swimming was delayed in the 15 mg/kg postnatal exposure group on PND 11 and 13, while 25 mg/kg delayed swimming on PND 11-15. Postnatal exposure to 25 mg/kg also reduced NE and DA levels in prosencephalon and cerebellum-pons on PND 7-16. SAD thus caused concomitant ontogenetic delays in growth, swimming behavior and brain catecholamine levels following either prenatal (transplacental) or early postnatal (transmammary) exposure. These data indicate that both in utero and lactational exposures must be considered when assessing potential risks posed to developing mammals by environmental neurotoxicants.     

Perinatal Methanol Exposure in the Rat: I. Blood Methanol Concentration and Neural Cell Adhesion Molecules

Although the acute toxicity of methanol is well documented,few studies have addressed the consequences of perinatal exposuresto the low concentrations that are expected to arise from itsproposed use as a component of automobile fuel. This reportdescribes the general research design of a series of studies,the effects of methanol exposures on blood concentrations indams and neonates, and indices of brain development. Four cohortsof Long-Evans pregnant rats, each cohort consisting of an exposure(n=12) and a control (n=12) group, were exposed whole-body to4500 ppm methanol vapor or air for 6 hr daily beginning on GestationDay 6. Both dams and pups were then exposed through PostnatalDay 21 (PND 21). Blood methanol concentrations determined bygas chromatography from samples obtained immediately followinga 6-hr exposure reached approximately 500–800 µg/mlin the dams during gestation and lactation. Average concentrationsfor pups attained levels about twice those of the dams. Selectedoffspring from Cohort 4 were exposed for one additional 6-hrsession at ages that extended out to PND 52. Regression analysesshowed that the blood methanol concentrations of the pups declineduntil about PND 48, at which time their levels approximatedthose of their dams. Such pharmacokinetic differences mightincrease the risks posed to developing organisms. Light-microscopicanalysis showed no significant abnormalities in the brains ofthe methanol-treated animals. However, assays of neural celladhesion molecules (NCAMs) in brains of pups sacrificed on PND4 showed staining for both the 140 and the 180 kDa isoformsto be less intense in the cerebellum of exposed animals. NCAMdifferences were not apparent in animals sacrificed 15 monthsafter their final exposure.     

Concentration and persistence of tin in rat brain and blood following dibutyltin exposure during development

Dibutyltin (DBT), a widely used plastic stabilizer, has been detected in the environment as well as human tissues. Although teratological and developmental effects are well documented, there are no published reports of DBT effects on the developing nervous system. As part of a developmental neurotoxicity study of DBT, tissue samples were periodically collected to determine the distribution of total tin (Sn) in brain and whole blood. Pregnant Sprague-Dawley rats were exposed to 0, 10, or 25 ppm DBT in drinking water from gestational day (GD) 6 to weaning at postnatal day (PND) 21. Beginning on PND 3, half of the litters were directly dosed every 2 to 3 d via oral gavage with 0, 1, or 2.5 mg/kg DBT such that the dose level matched the water concentration (for example, litters with 25 ppm DBT in the water received 2.5 mg/kg). For Sn analysis, brain and blood samples were collected from culled pups on PND2 (males and females pooled), from pups (males and females separately) as well as dams at weaning (PND21), and from adult offspring (males and females) at PND93. Total Sn was quantified using inductively coupled plasma-mass spectroscopy (ICP-MS). At all ages, brain Sn levels were higher than blood. At culling, in the directly dosed pups at weaning, and in dams at weaning, Sn levels in both tissues were linearly related to dose. Weanling pups without direct dosing showed lower levels than either culled pups or dams, indicating that lactational exposure was minimal or negligible even while maternal exposure is ongoing. In the adults, Sn levels persisted in brains of directly dosed rats, and the high-dose females had higher levels than did high-dose males. No Sn was detected in adult blood. Thus, during maternal exposure to DBT in drinking water, Sn is placentally transferred to the offspring, but lactational transfer is minimal, if any. Furthermore, Sn is concentrated in brain compared to blood, and its elimination is protracted, on the order of days to months after exposure ends.     

Tissue manganese concentrations in lactating rats and their offspring following combined in utero and lactation exposure to inhaled manganese sulfate.

There is little information regarding the tissue distribution of manganese in neonates following inhalation. This study determined tissue manganese concentrations in lactating CD rats and their offspring following manganese sulfate (MnSO4) aerosol inhalation. Except for the period of parturition, dams and their offspring were exposed to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m3) for 6 h/day, 7 days/week starting 28 days prior to breeding through postnatal day (PND) 18. Despite increased manganese concentrations in several maternal tissues, MnSO4 inhalation exposure did not affect body weight gain, terminal (PND 18) body weight, or organ weights in the dams. Exposure to MnSO4 at 1 mg Mn/m3 resulted in decreased pup body weights on PND 19 and decreased brain weights in some PND 14 to PND 45 pups. Exposure to MnSO4 at > or =0.05 mg Mn/m3 was associated with increased stomach content, blood, liver, and skull cap manganese concentrations in PND 1 pups, increased brain, lung, and femur manganese concentrations in PND 14 pups, and elevated olfactory bulb, cerebellum, and striatum manganese concentrations in PND 19 pups. When compared to controls, MnSO4 exposure to > or =0.5 mg Mn/m3 increased liver and blood manganese concentrations in PND 14 pups and increased liver, pancreas, and femur manganese concentrations in PND 19 pups. Manganese concentrations returned to control values in all offspring tissues by PND 45 +/- 1. Our data demonstrate that neonatal tissue manganese concentrations observed following MnSO4 inhalation are dependent on the MnSO4 exposure concentration and the age of the animal.     

A detailed study of developmental immunotoxicity of imidacloprid in Wistar rats

Human exposure to imidacloprid is likely to occur during its use as an acaricide or an ectoparasiticide. Accordingly, the developmental immunotoxic potential of imidacloprid was investigated. Oral exposure was initiated in timed pregnant female Wistar rats on gestation day 6 (GD 6) till GD 21. On GD 20, half of the gravid dams were sacrificed, and in utero fetal development was assessed. In the other half of the dams, administration was continued till weaning on postnatal day 21 (PND 21) and maternal toxicity was investigated. A subgroup of weaned pups was sacrificed to assess immunotoxicity parameters. The other half of the pups were exposed to imidacloprid till PND 42, and immunotoxicity was assessed. The findings revealed post-implantation loss in the highest dose group, indicating the risk of abortion. Soft tissue abnormalities and skeletal alterations were observed in the highest dose group. Humoral immunity was assessed by estimating hemagglutination titer and immunoglobulin production. Cell mediated immunity was assessed by Delayed Type Hypersensitivity, whereas, non-specific immunity was assessed by phagocytic index, and other phenotypic parameters. These data revealed that imidacloprid caused age-dependent adverse effects on the developing immunity which was aggravated when exposure continued throughout development, leading to a compromised immune system.     

Behavioral evaluation of male and female mice pups exposed to fluoxetine during pregnancy and lactation

BACKGROUND/AIMS: Fluoxetine (FLX) has been widely prescribed for depression during pregnancy and/or lactation. Since serotonin is a neurotrophic factor, the use of FLX by mothers could disrupt brain development resulting in behavioral alterations in their progeny. This study evaluated the effects of developmental FLX exposure on anxiety, depression, aggressivity and pain sensitivity of male and female mice pups. METHODS: Swiss dams were treated daily, by gavage, with 7.5 mg/kg of FLX during pregnancy and lactation. Pups were submitted to open-field, forced swimming, elevated plus-maze, intruder-resident and hot plate tests at adolescence and adulthood. RESULTS AND CONCLUSION: In male pups, exposure to FLX decreased ambulation at postnatal day (PND) 40 and tended (p=0.07) to increase the latency to the first attack in the intruder-resident test at PND 70, suggesting decreased impulsivity. In female pups, FLX exposure increased immobility time in the forced swimming test at both PND 30 and 70, which is interpreted as depressive-like behavior. In conclusion, our results suggest that maternal exposure to FLX during pregnancy and lactation results in enduring behavioral alterations in male and female pups throughout life.     

Gestational PFOA exposure of mice is associated with altered mammary gland development in dams and female offspring.

Perfluorooctanoic acid (PFOA), with diverse and widespread commercial and industrial applications, has been detected in human and wildlife sera. Previous mouse studies linked prenatal PFOA exposure to decreased neonatal body weights (BWs) and survival in a dose-dependent manner. To determine whether effects were linked to gestational time of exposure or to subsequent lactational changes, timed-pregnant CD-1 mice were orally dosed with 5 mg PFOA/kg on gestation days (GD) 1-17, 8-17, 12-17, or vehicle on GD 1-17. PFOA exposure had no effect on maternal weight gain or number of live pups born. Mean pup BWs on postnatal day (PND) 1 in all PFOA-exposed groups were significantly reduced and decrements persisted until weaning. Mammary glands from lactating dams and female pups on PND 10 and 20 were scored based on differentiation or developmental stages. A significant reduction in mammary differentiation among dams exposed GD 1-17 or 8-17 was evident on PND 10. On PND 20, delays in normal epithelial involution and alterations in milk protein gene expression were observed. All exposed female pups displayed stunted mammary epithelial branching and growth at PND 10 and 20. While control litters at PND 10 and 20 had average scores of 3.1 and 3.3, respectively, all treated litters had scores of 1.7 or less, with no progression of duct epithelial growth evident over time. BW was an insignificant covariate for these effects. These findings suggest that in addition to gestational exposure, abnormal lactational development of dams may play a role in early growth retardation of developmentally exposed offspring.     

Transplacental and early life exposure to inorganic arsenic affected development and behavior in offspring rats

To evaluate the developmental neurotoxicity of arsenic in offspring rats by transplacental and early life exposure to sodium arsenite in drinking water, the pregnant rats or lactating dams, and weaned pups were given free access to drinking water, which contained arsenic at concentrations of 0, 10, 50, 100 mg/L from GD 6 until PND 42. A battery of physical and behavioral tests was applied to evaluate the functional outcome of pups. Pups in arsenic exposed groups weighed less than controls throughout lactation and weaning. Body weight of 10, 50 and 100 mg/L arsenic exposed groups decreased significantly on PND 42, 16 and 12, respectively. Physical development (pinna unfolding, fur appearance, incisor eruption, or eye opening) in pups displayed no significant differences between control and arsenic treated groups. The number of incidences within the 100 mg/L arsenic treated group, in tail hung, auditory startle and visual placing showed significant decrease compared to the control group (p < 0.05). In square water maze test, the trained numbers to finish the trials successfully in 50 and 100 mg/L arsenic exposed groups increased remarkably compared to control group, and there was a dose-related increase (p < 0.01) observed. Taken together, these data show that exposure of inorganic arsenite to pregnant dams and offspring pups at levels up to 100 mg/L in drinking water may affect their learning and memory functions and neuromotor reflex.     

Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic–pituitary–ovarian axis development and function in Wistar rats

We studied delayed effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether (PEG-b-PLA) on the endpoints related to pubertal development and reproductive function in female Wistar rats from postnatal day 4 (PND4) to PND 176. Female pups were injected intraperitoneally, daily, from PND4 to PND7 with PEG-b-PLA (20 or 40 mg/kg b.w.). Both doses of PEG-b-PLA accelerated the onset of vaginal opening compared with the control group. In the low-dose PEG-b-PLA-treated group, a significantly reduced number of regular estrous cycles, increased pituitary weight due to hyperemia, vascular dilatation and congestion, altered course of hypothalamic gonadotropin-releasing hormone-stimulated luteinizing hormone secretion, and increased progesterone serum levels were observed. The obtained data indicate that neonatal exposure to PEG-b-PLA might affect the development and function of hypothalamic–pituitary–ovarian axis (HPO), and thereby alter functions of the reproductive system in adult female rats. Our study indicates a possible neuroendocrine disrupting effect of PEG-b-PLA nanoparticles.     

Hepatotoxicity studies in the progeny of pregnant dams treated with methimazole, monocrotophos and lead acetate

An experimental study was conducted to evaluate the hepatotoxic effects in the progeny of dams treated with methimazole, monocrotophos (MCP) and lead acetate. Female pregnant albino rats of Wistar kyoto strain were divided into five groups and treated as follows, from day 3 of pregnancy till weaning of pups on postnatal day (PND) 21. Group 1 served as sham control, group 2 received methimazole 0.02% in drinking water, group 3 received MCP (0.3 mg/kg orally), group 4 received lead acetate at 0.2% in drinking water and group 5 received MCP + lead acetate. Thyroid hormone profile was recorded on 14 (th)day of gestation in dams. Eight pups from each group were euthanized on PND 21 and 90, and liver tissues were collected for analysis. Thiobarbituric acid reactive substances (TBARS), protein carbonyls and reduced glutathione (GSH) of liver were studied on PND 21 and 90, while the activities of Na (+)/K(+)ATPase and Mg (2+)ATPase in the liver were studied on PND 90. T(3), T(4), GSH, Na(+)/K(+)ATPase and Mg(2+)ATPase were significantly (P<0.05) decreased, while TBARS and protein carbonyls were significantly (P < 0.05) increased in all the test groups as compared to group 1. From this study, it is concluded that both MCP and lead acetate have a possible influence on thyroid gland of dams as the thyroid profile was altered significantly and the hepatotoxic effects were comparable to those induced by methimazole.     

Increased high-affinity nicotinic receptor-binding in rats exposed to lead during development

Receptor autoradiography and membrane radioligand-binding assays were used to determine the expression of nicotinic cholinergic receptors in the brains of weanling rats exposed to low-levels of lead (Pb) during development. Nicotinic receptors were identified with the frog toxin epibatidine (EB) that binds with high affinity to a variety of receptors containing and β subunits. Rat pups were exposed to Pb from their mothers given 750-ppm Pb in the diet beginning on gestational day 0 through postnatal day (PN) 21. Blood Pb levels ranged from 36.5 to 46.5 μg/dl in the PN21 pups, and this exposure did not alter their body weight when compared to control rats. Several brain regions identified by autoradiographic studies as having significant binding of EB were dissected from control and Pb-treated pups and used in saturation-binding experiments with membrane preparations to determine the affinity constant (K_d) and maximal-binding capacity (B_max) of [³H]EB. Results indicate that the B_max of [³H]EB was increased in several brain regions in Pb-treated rat pups, without a significant effect on K_d estimates. [³H]EB-binding to membranes from untreated rats was not affected by in vitro exposure to 20-μM Pb, indicating that the effect of Pb on [³H]EB-binding in vivo was not likely due to direct influence of free Pb remaining in the tissue at the time of assay. The data therefore suggest that expression of nicotinic receptors that bind [³H]EB were increased by developmental exposure to Pb. Several possible mechanisms for these effects and the potential toxicological significance are discussed.     

Towards a Pragmatic Alternative Testing Strategy for the Detection of Reproductive Toxicants

Exposure of rodents in utero to perfluorooctane sulfonate (PFOS) impairs perinatal development and survival. Following intravenous or gavage exposure of C57Bl/6 mouse dams on gestational day (GD) 16 to ³⁵S-PFOS (12.5 mg/kg), we determined the distribution in dams, fetuses (GD18 and GD20) and pups (postnatal day 1, PND1) employing whole-body autoradiography and liquid scintillation counting. In dams, levels were highest in liver and lungs. After placental transfer, ³⁵S-PFOS was present on GD18 at 2–3 times higher levels in lungs, liver and kidneys than in maternal blood. In PND1 pups, levels in lungs were significantly higher than in GD18 fetuses. A heterogeneous distribution of ³⁵S-PFOS was observed in brains of fetuses and pups, with levels higher than in maternal brain. This first demonstration of substantial localization of PFOS to both perinatal and adult lungs is consistent with evidence describing the lung as a target for the toxicity of PFOS at these ages.     

Disruption of thyroid hormone homeostasis at weaning of Holtzman rats by lactational but not in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The purpose of this study is to clarify whether lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is entirely responsible for the perturbation in thyroid hormone homeostasis during the neonatal period. Pregnant Holtzman rats were given a single oral dose of 1.0 mug TCDD/kg body weight on gestational day 15. Half of the litters were cross-fostered with the half of the dams treated with vehicle on postnatal day (PND) 1 to make four groups of rats, control (C/C), prenatal TCDD exposure only (T/C), postnatal TCDD exposure only (C/T), and both prenatal and postnatal TCDD exposure (T/T). On PND 21, the C/T and T/T groups, but not the T/C and C/C groups, showed a significant decrease in serum total thyroxin (TT4) and free thyroxin (FT4) concentrations in both sexes and a significant increase in serum thyroid-stimulating hormone (TSH) levels, particularly male pups. These two groups of male and female pups had significantly higher concentrations of TCDD in the liver, with marked induction of cytochrome P450 (CYP) 1A1 mRNA and intense immunostaining of CYP1A1 in the liver. UDP glycosyltransferase 1 family, polypeptide A6 (UGT1A6) and UGT1A7 mRNAs were induced in their livers, with marked immunostaining of UGT1A6. The transfer of TCDD from dams to the pups was confirmed by the detection of TCDD in mother's milk remaining in the stomachs of lactationally exposed pups on PND 1. The present results demonstrate that lactational, but not in utero, exposure to TCDD was responsible for the disruption of thyroid hormone homeostasis.     

DEVELOPMENT OF AN INHALATION SYSTEM FOR THE SIMULTANEOUS EXPOSURE OF RAT DAMS AND PUPS DURING DEVELOPMENTAL NEUROTOXICITY STUDIES

Due to mass flux constraints, the exposure of pups along with dams to particles cannot be easily done in traditional whole-body inhalation systems. Typical inhalation developmental neurotoxicity studies rely on maternal separation during exposure, resulting in pup stress that may alter behavioral development, thereby confounding study results. We developed an individual-rat, whole-body inhalation exposure cylinder that allows for the simultaneous exposure of rat dams and their pups to vapors, aerosols, and particles. We evaluated pup growth and development following gestational and lactational 6-h/ day exposures to HEPA-filtered air using the novel exposure cylinder, in which dams and pups were maintained together, versus rat pups for which only the dams were exposed or control animals that remained in the domiciliary cage with the dams. Rat pups that were separated from their dams during neonatal exposure to HEPA-filtered air demonstrated significant delays in several developmental landmarks such as surface righting, negative geotaxis, and eye opening, as well as decreased terminal body and brain weights. Increased spontaneous motor activity was observed on postnatal day (PND) 13, 17, and 60 in pups that were separated from their dams. Malnutrition is the likely cause for the observed hyperactivity and delayed development. We then compared maternal blood levels of tertiary amyl methyl ether (TAME) and its metabolite tertiary amyl alcohol (TAA) in the 2 exposure systems following exposure to 500 ppm TAME for 6 h. Dams exposed in the novel exposure cylinder had higher blood levels of TAME compared with those exposed in stainless steel caging. We also compared levels of particulate manganese phosphate within the novel exposure cylinder to determine if levels achieved within the exposure cylinder were similar to levels generated within a 1-m3 chamber. The novel exposure cylinder is an ideal system for exposure of dams and pups to particles, due to the high horizontal flow rates, which result in an adequate mass flux flowing past the breathing zone of the animals with retained normal growth and development.