Mice with conditional inactivation of fibroblast growth factor receptor-2 signaling in oligodendrocytes have normal myelin but display dramatic hyperactivity when combined with Cnp1 inactivation.

Fibroblast growth factor receptors (Fgfr) comprise a widely expressed family of developmental regulators implicated in oligodendrocyte (OL) maturation of the CNS. Fgfr2 is expressed by OLs in myelinated fiber tracks. In vitro, Fgfr2 is highly upregulated during OL terminal differentiation, and its activation leads to enhanced growth of OL processes and the formation of myelin-like membranes. To investigate the in vivo function of Fgfr2 signaling by myelinating glial cells, we inactivated the floxed Fgfr2 gene in mice that coexpress Cre recombinase (cre) as a knock-in gene into the OL-specific 2',3'-cyclic nucleotide phosphodiesterase (Cnp1) locus. Surprisingly, no obvious defects were detected in brain development of these conditional mutants, including the number of OLs, the onset and extent of myelination, the ultrastructure of myelin, and the expression level of myelin proteins. However, unexpectedly, a subset of these conditional Fgfr2 knock-out mice that are homozygous for cre and therefore are also Cnp1 null, displayed a dramatic hyperactive behavior starting at approximately 2 weeks of age. This hyperactivity was abolished by treatment with dopamine receptor antagonists or catecholamine biosynthesis inhibitors, suggesting that the symptoms involve a dysregulation of the dopaminergic system. Although the molecular mechanisms are presently unknown, this novel mouse model of hyperactivity demonstrates the potential involvement of OLs in neuropsychiatric disorders, as well as the nonpredictable role of genetic interactions in the behavioral phenotype of mice.

In utero alcohol exposure increases mammary tumorigenesis in rats.

Findings in humans and animal models suggest that in utero hormonal and dietary exposures increase later breast cancer risk. Since alcohol intake by adult women consistently increases their breast cancer risk, we wondered whether maternal alcohol consumption during pregnancy increases female offspring's mammary tumorigenesis. In our study, pregnant female rats were pair-fed isocaloric diets containing either 0 (control), 16 or 25 g alcohol kg(-1) feed between days 7 and 19 of gestation. These alcohol exposures generate blood alcohol levels that correspond to low and moderate alcohol consumption and are lower than those that induce foetal alcohol syndrome. Serum oestradiol levels were elevated in pregnant rats exposed to alcohol (P<0.003). When adult, female offspring of alcohol-exposed dams developed significantly more 7,12-dimethylbenz[a]anthracene -induced mammary tumours, compared to the controls (tumour multiplicity; mean+/-s.e.m., controls: 2.0+/-0.3, 16 g alcohol: 2.7+/-0.4 and 25 g alcohol: 3.7+/-0.4; P<0.006). In addition, the mammary epithelial tree of the alcohol-exposed offspring was denser (P<0.004) and contained more structures that are susceptible for the initiation of breast cancer (P<0.001). Immunohistochemical assessment indicated that the mammary glands of 22-week-old in utero alcohol-exposed rats contained elevated levels of oestrogen receptor-alpha (P<0.04) that is consistent with the changes in mammary gland morphology. In summary, maternal alcohol intake during pregnancy increases female offspring's mammary tumorigenesis, perhaps by programming the foetal mammary gland to exhibit persistent alterations in morphology and gene expression. It remains to be determined whether an increase in pregnancy oestradiol levels mediated alcohol's effects on offspring's mammary tumorigenesis.

Plasma concentrations of endogenous benzodiazepine-receptor ligands in patients with hepatic encephalopathy: a comparative study.

OBJECTIVE: Hepatic encephalopathy (HE) is a complex neuropsychiatric disorder secondary to acute or chronic liver failure. Although the exact causes of HE have not been clarified, enhanced central nervous system inhibition at the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex, mediated by increased levels of endogenous benzodiazepine-receptor ligands (BZRL), has been proposed. Research exploring this hypothesis has yielded contradictory findings. This study evaluated the presence and levels of BZRL in plasma from patients with HE and 3 comparison groups. DESIGN: Cross-sectional study. PATIENTS: Twenty-four patients with HE, 10 patients with liver cirrhosis without encephalopathy (LC), 4 patients with uremic encephalopathy (UE), and 9 healthy subjects. INTERVENTIONS: Radio-receptor assay of plasma samples from patients and controls. MAIN OUTCOME MEASURES: Plasma levels of BZRL. RESULTS: The patients in the HE group had significantly higher plasma BZRL levels than the patients with UE and the healthy subjects, but not than those with LC, in whom these compounds were also detected in significant concentrations. When patients were classified according to the severity of HE, plasma of BZRL showed a modest correlation with stage of severity (r = 0.37). Interestingly, approximately one-third of the patients with HE did not have detectable levels of BZRL. CONCLUSION: Endogenous BZRL may play a role in the pathogenesis of HE, although neuropsychiatric symptoms in HE are difficult to explain in terms of these compounds alone.

Adult rats stressed as neonates show exaggerated behavioral responses to both pharmacological and environmental challenges.

Adult rats that were isolated from the mother and nest for 1 hr per day from Postnatal Day 2 to 9 were studied. Controls consisted of handled littermates as well as separate litters that were never handled. As adults, animals were given either a pharmacological challenge (1.0 or 2.0 mg/kg amphetamine) or an environmental challenge (restraint). Previously isolated animals demonstrated increased activity compared to controls at both drug doses. Similarly, isolated animals manifested exaggerated inhibition of activity after restraint. Previously isolated animals usually did not show differences compared to controls under baseline conditions (saline injection or no restraint). The neuroplastic changes that result from the neonatal experience are long lasting and appear when the system is challenged.

Repeated isolation stress in the neonatal rat: relation to brain dopamine systems in the 10-day-old rat.

Isolation of the rat pup from the nest and dam for one hour per day from PN 2-9 is a useful paradigm for producing stress in the neonate. These previously isolated rats respond to an amphetamine challenge with alterations in activity at the juvenile stage or as adults. Furthermore, when dopamine release is measured in the nucleus accumbens, juveniles release 3 times more dopamine after amphetamine than do controls. This study describes changes in behavior and brain dopamine systems at PN 10. Experiment 1 determined an appropriate amphetamine dose that could be used for behavioral activation at PN 10. Experiment 2 produced significant evidence of enhanced behavioral activation after the isolation paradigm and indicated that brain regions innervated by the mesolimbic dopamine system, septum, and hypothalamus display increased dopamine turnover and that the nigrostriatal pathway is less active. Likewise, in Experiment 3, in vivo microdialysis of the nucleus accumbens indicated that previously isolated pups respond to an amphetamine challenge with a several-fold increase in dopamine release over a 4-hour session.

Ethanol attenuation of morphine dependence: comparison to dizocilpine.

Recent studies indicate that morphine dependence, assessed as the severity of naloxone-precipitated opiate withdrawal in rats, is attenuated by dizocipline, a non-competitive, excitatory amino acid antagonist. Because ethanol is a putative excitatory amino acid antagonist, the present study compared the effects of co-administration of ethanol to that of dizocilpine on morphine dependence. Rats were administered morphine (10 mg/kg) twice daily for 9 days. One group received ethanol (1 g/kg) co-administration, another received dizocilpine (0.05 mg/kg) co-administration, and a third served as vehicle controls. On day 10, all rats received naloxone (4 mg/kg) injections and ratings of several classic signs of opiate withdrawal were made. Both ethanol- and dizocilpine-treated rats showed significantly less severe precipitated opiate withdrawal overall, with the ethanol group showing reduced ratings of some specific signs. These results demonstrate that ethanol, like dizocilpine, attenuates the development of morphine dependence. The results are consistent with the action of ethanol at glutamate receptors.

Effects of 5,7-dihydroxytryptamine lesions of the prefrontal cortex on consumption of sucrose-ethanol solutions: relationship to prefrontal monoamines.

Thirty adult male Wistar rats received 8 micrograms bilaterally of 5,7-dihydroxytryptamine into the medial prefrontal cortex (mPFC). Rats were then trained, via a sucrose, fading paradigm, to consume increasing concentrations of alcohol. After death, dopamine (DA), norepinephrine (NE), serotonin (5-HT), and their metabolites were measured in the mPFC, nucleus accumbens (NA), and raphe nucleus. The lesioned group demonstrated a reduction in 5-hydroxyindoleacetic acid (5-HIAA), DA, and NE in the mPFC (p < 0.05), and a trend toward reduction of 5-HT in the NA. In comparison with controls, lesioned animals consumed less of all solutions containing sucrose and alcohol. On regression analyses, monoamines in the mPFC (i.e., 5-HIAA, dihydrophenylacetic acid and NE) predicted consumption of the 5% ethanol solution (p = 0.009), 10% ethanol solution (p = 0.0006), and the 5% sucrose solutions (p = 0.0006), but not the 20% sucrose solutions. In each case, monoamine levels were positively correlated with consumption. No relationships were seen between monoamine levels in the NA and raphe, and in consummatory behavior.

Repeated isolation in the neonatal rat produces alterations in behavior and ventral striatal dopamine release in the juvenile after amphetamine challenge.

Rat pups were isolated from the mother and nest for 1 hr per day from Postnatal Day (PN) 2 to 9 At PN 27, rats were tested for behavioral responsiveness to 2.0 or 7.5 mg/kg amphetamine. Only isolated rats receiving the 7.5 mg/kg dose displayed increased activity scores, compared with nonisolated and nonhandled controls. Their increased activity is attributed to a slower latency to enter into stereotypy. In a second experiment, similarly treated groups were challenged by the 7.5 mg/kg dose during a session in which a microdialysis probe implanted in the ventral striatum was being perfused. The challenge drug elicited a much greater increase in dialysate dopamine in isolated vs. nonisolated groups. Results are discussed with regard to dissociation between sensitized and subsensitized responses.

Dorsal lesions of the prefrontal cortex: effects on alcohol consumption and subcortical monoaminergic systems.

Male Wistar rats were subjected to either bilateral aspiration lesions of the dorsal regions of the prefrontal cortex (PFC) or sham lesions and placed on a 6-week, modified sucrose-fading procedure. At the time of sacrifice, the size of the lesion, both in anterior-posterior and medial-lateral dimensions, was measured. Following sacrifice, levels of dopamine (DA), serotonin (5-HT), norepinephrine (NE), and their metabolites were measured in the midbrain (raphe) and nucleus accumbens (NA). Lesioned animals had reductions in 5-HT in the NA, and DA and NE in the raphe. The lesioned group drank more of a solution of 5% alcohol than controls early in the sucrose fading, and less during the later stages. In the lesioned group, the size of the left- and right-hemisphere lesions predicted 5-HIAA levels in the NA, and 5-HT and 5-HIAA levels in the raphe. A laterality effect was noted, such that the size of left-hemisphere lesions were positively associated with raphe 5-HT and 5-HIAA levels, and negatively associated with 5-HT levels in the NA, while right-hemisphere lesions showed the opposite relationships. In addition, the width of the left-hemisphere lesion predicted some measures of alcohol intake. These results suggest that, in the rat, the dorsal PFC is involved in the regulation of monoamines in subcortical regions known to be important in the regulation of reinforced behaviors, and that this regulation differs between hemispheres and shows a laterality effect. In addition, the dorsal PFC appears to have a subtle involvement in the regulation of alcohol intake.

Brain dopamine response in isolated 10-day-old rats: assessment using D2 binding and dopamine turnover.

A single 5-min isolation from the nest, dam, and siblings in 10-day-old rat pups was investigated for its effect on brain dopamine systems. The release of dopamine in innervated brain regions was measured in separate studies using in vivo ligand binding of 3H-raclopride, ex vivo binding using 3H-raclopride, and neurochemical measurement of the dopamine turnover using levels of DOPAC and dopamine. In addition, in vitro homogenate binding was performed to determine baseline Bmax and Kd values for 3H-raclopride binding sites across treatments. Isolation for 5 min in a "novel" environment resulted in decreased 3H-raclopride binding in striatum and septum as determined by both in vivo and ex vivo binding, as well as increased dopamine turnover. There was no difference in Bmax and Kd values for 3H-raclopride in these brain regions after the 5-min isolation, indicating that the binding decreases were due to an increase of available dopamine, presumably from terminal release. The convergence of results from three different techniques supports the interpretation that dopamine is released during the 5-min isolation in both brain regions.

Prefrontal levels of 5-HIAA, but not dopamine, predict alcohol consumption in male Wistar rats following 6-OHDA lesions.

To examine the effects of dopamine (DA) on alcohol consumption, male Wistar rats were subjected either to 6-OHDA lesions of the frontal cortex (MPFC) or to a sham lesion/no lesion. Following surgery, rats were trained to drink alcohol on a sucrose-fading paradigm over the course of 6 weeks, at the completion of which they consumed a solution of 3% sucrose/10% alcohol. Daily consumption of alcohol was computed for each rat. Animals were sacrificed and the MPFC, nucleus accumbens (NA), and ventral tegmentum (VTA) were removed. Levels of DA and its metabolites (i.e., HVA and DOPAC), norepinephrine (NE), and serotonin (5-HT) and its metabolite (i.e., 5-HIAA) were measured for each brain region using HPLC with electrochemical detection. Post hoc analyses were run examining the relationship of DA and its metabolites, 5-HT and its metabolite (5-HIAA), and norepinephrine (NE) in the MPFC, NA, and VTA with alcohol consumption. The 6-OHDA lesions depleted DA to 74.5% of control levels in the MPFC, but did not significantly affect alcohol consumption. Post hoc analyses found that the "high" alcohol consumption group had significantly reduced levels of MPFC 5-HIAA in comparison to the "low" consumption group, but that there was no relationship of 5-HIAA levels in the VTA or NA to alcohol consumption. These findings suggest that MPFC DA is not critically involved in the regulation of alcohol consumption. They further suggest that MPFC serotonergic systems may play an important role in the regulation of alcohol consumption, although future experimentation directly manipulating 5-HT systems in the MPFC will be required to fully assess these findings.

Effect of prenatal ethanol and stress on levels of beta-endorphin in different brain regions of the rat.

The combination of prenatal ethanol exposure and footshock stress was investigated for its effects on brain beta-endorphin levels. Subjects were offspring of rats that received 1 of 3 prenatal dietary treatments: an ethanol-containing liquid diet, a identical liquid diet with ethanol substituted isocalorically with maltose-dextrin (pair-fed group), and standard laboratory rat chow (chow-fed group). Two different stress paradigms were used: a short (30-sec) footshock stress paradigm and a prolonged (180-sec) footshock stress paradigm. Levels of beta-endorphin were measured with radioimmunoassay in eight brain regions of unstressed (baseline) rats, and of stressed rats at 3 and 30 min following termination of the stress. Seven brain regions containing high densities of beta-endorphin axons and terminals were chosen, as well as the arcuate region of the hypothalamus, the only brain region where both beta-endorphin perikarya and terminals are located. Following the short footshock stress paradigm, there were no changes in beta-endorphin levels, except for a trend toward increased levels in the pair-fed group. After the prolonged stress paradigm, levels of beta-endorphin in both the pair-fed and chow-fed groups tended to be decreased in several brain regions, including the arcuate region, at 3 min after termination of the stress. In contrast, for the prenatally ethanol-exposed group, beta-endorphin levels were increased significantly in the arcuate region, and moderately increased in the septal/preoptic region and medulla/pons at 3 min after the prolonged stress paradigm.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of isolation conditions on brain regional enkephalin and beta-endorphin levels and vocalizations in 10-day-old rat pups.

Young rat pups were isolated from their dams under different conditions. The endogenous opioid peptides were measured in brain regions after isolation. Because there is no uptake mechanism for peptides released at the synapse and because released peptide is rapidly degraded enzymatically, decreases in peptide levels over this time course can be interpreted as release from terminals. No change was observed in either peptide in the hypothalamus, septum, or amygdala after isolation compared with controls. Significant decreases were seen in the midbrain after isolation. A comparison of peptide levels and ultrasonic vocalizations in the pups isolated in familiar, novel, or control conditions was also performed. Enkephalin levels in the midbrain were decreased in familiar and novel conditions, but in the brainstem opioid peptides were decreased only in the familiar condition. The greater involvement of the opioid peptides in the pups isolated in familiar conditions may contribute to the ability of naltrexone to block vocalization.

X-ray diffraction analysis of brain lipid membrane structure in Alzheimer's disease and beta-amyloid peptide interactions.

Small angle x-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes reconstituted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 A reduction in the lipid bilayer width and marked changes in membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of six AD and six age-matched controls showed that the phospholipid:protein mass ratio was unchanged, but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. The C:PL mole ratio was not significantly different for samples prepared from cerebellum of AD versus control patients. X-ray diffraction analysis of a cholesterol-enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Addition of beta-amyloid peptide to bovine brain phospholipid membranes significantly changed the electron density associated with the hydrocarbon core. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the beta-amyloid precursor protein.

The distribution of catecholamines and beta-endorphin in the brains of three behaviorally distinct breeds of dogs and their F1 hybrids.

This study examines neurochemical and behavioral differences among three types of domestic dogs and F1 hybrids derived from them. Purebred dogs included Border Collies, representing herding dogs, Shar Plaininetz, representing livestock protecting dogs, and Siberian Huskies, representing Northern dogs. Composite behavioral scores were derived from frequency measures of various components of predatory behavior observed when the dogs were tested with mice. Catecholamine levels, including norepinephrine (NE), dopamine (DA), and epineprine (EPI), were determined in various brain regions by high-performance liquid chromatography (HPLC) with electrochemical detection. beta-endorphin levels were determined in the same regions by RIA. Collies showed the highest levels of non-consummatory behaviors and Huskies the highest levels of consummatory behaviors. Shars were found to have lower levels of NE and DA than Collies and Huskies in several brain regions, including those comprising the nigrostriatal DA system. Positive correlations between neurochemical and behavioral characteristics could be made between Shars and Collies. Comparisons of F1 hybrids with their respective parental breeds revealed no clear pattern of inheritance for these characteristics but suggested that multiple factors, both independent and epistatic, are involved. Based on previous studies on nigrostriatal DA and behavior, the levels of DA in this system may be causally related to the levels of predatory behavior expressed by Collies and Shars.

Evidence for changes in the Alzheimer's disease brain cortical membrane structure mediated by cholesterol.

Small angle X-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes extracted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 A reduction in the lipid bilayer width and significant changes in the membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of 6 AD and 6 age-matched controls showed that the phospholipid:protein mass ratio was unchanged but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. By contrast, the C:PL mole ratio in the cerebellum did not change significantly. X-ray diffraction analysis of a cholesterol enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the beta-amyloid precursor protein.

beta-Endorphin-immunoreactive neurons in the hypothalamus and medulla of the rat brain: Effect of prenatal ethanol.

The number and distribution of beta-endorphin neurons in the hypothalamus and medulla of rats is compared among three experimental treatment groups: prenatally ethanol-exposed rats, pair-fed control rats, and normally fed control rats. Quantitative analysis of beta-endorphin-immunoreactive neurons in 12 sections through the rostral-caudal extent of the mediobasal hypothalamus revealed no significant difference in the total number or distribution of beta-endorphin neurons among any of the three treatment groups. The mean number of beta-endorphin neurons was consistently less in each of the 12 sections of pair-fed rats compared to normally fed rats, but there was no consistent difference between the mean number of neurons/section in prenatally ethanol-exposed rats compared to the normally fed controls. The distribution and time of appearance of a second population of beta-en-dorphin neurons was analyzed in the caudal medulla. beta-endorphin-immunoreactive neurons were easily visualized in the caudal medulla of rats from each treatment group on Postnatal Days 9-11. However, this population could not be identified with antisera against beta-endorphin on Postnatal Days 19-21 in any treatment group, even though neurons in the hypothalamus of the same rats were easily visualized histochemically at this age. These data suggest that the well-characterized prenatally ethanol-induced alterations in central opioid systems cannot be attributed to changes in the number, distribution, or time of appearance of beta-endorphin neurons in the brain.

Effect of ethanol on maternal and offspring characteristics: comparison of three liquid diet formulations fed during gestation.

Maternal blood alcohol levels, weight gain during pregnancy, parturition time, perinatal mortality, and postnatal growth of offspring were compared in groups of pregnant rats fed one of three ethanol-containing liquid diets (Kahn's formula = BSA diet, Revised Wiener's = RA6 diet, and Lieber-DeCarli's high protein 82C diet = LDA diet). The three ethanol diets all contained the same amount of ethanol-derived energy (36% of total energy), but differed in the amount of energy contributed by protein (17, 30, and 25%), fat (36, 24, and 13%), and carbohydrate (12, 10, and 27%), respectively. The experimental design also included dams that were pair-fed isocaloric ethanol-free versions of the three ethanol diets (designated BSP, RP6, and LDA, respectively) and a group of dams fed a pelleted casein-based solid diet (PC diet). All experimental diets were fed ad libitum from gestational day 7 to delivery. The effect of ethanol exposure in utero was most severe in mothers and offspring fed the BSA diet. The feed efficiency ratio (maternal weight gain/total dietary energy consumed) of this low-protein ethanol diet was less than that of RA6 or LDA diets. The feed efficiency ratio calculated for RA6 and LDA diets was not different from that of PC diet. Compared with rats fed RA6 and LDA diets, the rats that were fed BSA diet exhibited deficient maternal weight gain, greater parturition delay, impaired fetal growth, and increased perinatal mortality among the offspring. BSA dams had the highest blood ethanol levels of all groups fed ethanol diets, and exhibited the least difference in blood ethanol concentrations between the day (2 PM) and night (9 PM) periods of the diurnal cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroanatomic and neurochemical abnormalities in nonhuman primate infants exposed to weekly doses of ethanol during gestation.

Ethanol was orally administered once per week to 54 gravid pigtailed macaques (Macaca nemestrina) in doses of 0.0, 0.3, 0.6, 1.2, 1.8, 2.5 or 4.1 gm/kg from the 1st week in gestation or in doses of 2.5, 3.3 or 4.1 gm/kg from the 5th week. Mean maternal peak plasma ethanol concentrations (MPPEC's) ranged from 24 +/- 6 mg/dl at the 0.3 g/kg dose to 549 +/- 71 mg/dl at the 4.1 g/kg dose. Thirty-three live born infants were assessed for abnormalities of physical and behavioral development. Ocular pathology, neuropathologic and neurochemical assessements were done on 31 animals at 6 months postnatal age. Microphthalmia was noted in three of the 26 animals exposed to ethanol. Retinal ganglion cell loss was significantly associated with intra-uterine ethanol exposure. Microphthalmia and retinal ganglion cell loss was observed in both the delayed and full-gestational exposed animals. No structural anomalies were found in the brains via gross inspection or light microscopy. Chemical abnormalities in the striatal nuclei were identified. Striatal dopamine concentrations increased with increasing MPPEC exposure (0-249 mg/dl) among animals exposed weekly to ethanol throughout gestation. Striatal dopamine concentrations decreased with increasing MPPEC exposure (260-540 mg/dl) among animals whose weekly exposure to ethanol was delayed until the 5th week of gestation. The same pattern of association was also noted between MPPEC and ultrastructural alterations in the caudate nucleus. The extent of ultrastructural alterations increased with increasing MPPEC among the full-gestational exposed animals and decreased with increasing MPPEC among the delayed-dose animals.