Distinct developmental trajectories of autonomous behaviors and agency in rodents.

Behavioral interactions within the nuclear family may play a pivotal role in the emergence of autonomy and agency in mammals. While the emergence of a behavior may arise over weeks in line with nervous system maturation, individual events occur on sub-second time scales. This makes it uniquely challenging to track development in the lab where observations are made over minutes to hours or in ecological studies which lack individual specificity and sub-second precision. Here we study families of gerbils, a highly social rodent, raised in enlarged home-cage environments over weeks of development, using continuous video recordings to capture tens of millions of time points per family. Focusing on postnatal day 15 (when pups leave the nest) to day 30 (around the time when pups would disperse) we identify distinct developmental trajectories for both autonomous behaviors (exploration, food and water foraging), and social behaviors (huddling, approach, time spent together). Most of these behaviors emerge in concert with clear diurnal and crepuscular patterns and we find sex differences in both autonomous and social behaviors. Our work supports the emergence of distinct autonomous and social behavior phenotypes as the behavioral correlates of critical developmental periods of maturation of the rodent brain and can form the basis of future research on development from both neuroscience and behavioral biology perspectives.

Robust symptom networks in recurrent major depression across different levels of genetic and environmental risk.

BACKGROUND: Genetic risk and environmental adversity-both important risk factors for major depression (MD)-are thought to differentially impact on depressive symptom types and associations. Does heterogeneity in these risk factors result in different depressive symptom networks in patients with MD? METHODS: A clinical sample of 5784 Han Chinese women with recurrent MD were interviewed about their depressive symptoms during their lifetime worst episode of MD. The cases were classified into subgroups based on their genetic risk for MD (family history, polygenic risk score, early age at onset) and severe adversity (childhood sexual abuse, stressful life events). Differences in MD symptom network structure were statistically examined for these subgroups using permutation-based network comparison tests. RESULTS: Although significant differences in symptom endorsement rates were seen in 18.8% of group comparisons, associations between depressive symptoms were similar across the different subgroups of genetic and environmental risk. Network comparison tests showed no significant differences in network strength, structure, or specific edges (P-value > 0.05) and correlations between edges were strong (0.60-0.71). LIMITATIONS: This study analyzed depressive symptoms retrospectively reported by severely depressed women using novel statistical methods. Future studies are warranted to investigate whether similar findings hold in prospective longitudinal data, less severely depressed patients, and men. CONCLUSIONS: Similar depressive symptom networks for MD patients with a higher or lower genetic or environmental risk suggest that differences in these etiological influences may produce similar symptom networks downstream for severely depressed women.

Polygenic risk for severe psychopathology among Europeans is associated with major depressive disorder in Han Chinese women.

BACKGROUND: Previous studies have demonstrated that several major psychiatric disorders are influenced by shared genetic factors. This shared liability may influence clinical features of a given disorder (e.g. severity, age at onset). However, findings have largely been limited to European samples; little is known about the consistency of shared genetic liability across ethnicities. METHOD: The relationship between polygenic risk for several major psychiatric diagnoses and major depressive disorder (MDD) was examined in a sample of unrelated Han Chinese women. Polygenic risk scores (PRSs) were generated using European discovery samples and tested in the China, Oxford, and VCU Experimental Research on Genetic Epidemiology [CONVERGE (maximum N = 10 502)], a sample ascertained for recurrent MDD. Genetic correlations between discovery phenotypes and MDD were also assessed. In addition, within-case characteristics were examined. RESULTS: European-based polygenic risk for several major psychiatric disorder phenotypes was significantly associated with the MDD case status in CONVERGE. Risk for clinically significant indicators (neuroticism and subjective well-being) was also associated with case-control status. The variance accounted for by PRS for both psychopathology and for well-being was similar to estimates reported for within-ethnicity comparisons in European samples. However, European-based PRS were largely unassociated with CONVERGE family history, clinical characteristics, or comorbidity. CONCLUSIONS: The shared genetic liability across severe forms of psychopathology is largely consistent across European and Han Chinese ethnicities, with little attenuation of genetic signal relative to within-ethnicity analyses. The overall absence of associations between PRS for other disorders and within-MDD variation suggests that clinical characteristics of MDD may arise due to contributions from ethnicity-specific factors and/or pathoplasticity.

Genetic effects influencing risk for major depressive disorder in China and Europe.

Major depressive disorder (MDD) is a common, complex psychiatric disorder and a leading cause of disability worldwide. Despite twin studies indicating its modest heritability (~30-40%), extensive heterogeneity and a complex genetic architecture have complicated efforts to detect associated genetic risk variants. We combined single-nucleotide polymorphism (SNP) summary statistics from the CONVERGE and PGC studies of MDD, representing 10 502 Chinese (5282 cases and 5220 controls) and 18 663 European (9447 cases and 9215 controls) subjects. We determined the fraction of SNPs displaying consistent directions of effect, assessed the significance of polygenic risk scores and estimated the genetic correlation of MDD across ancestries. Subsequent trans-ancestry meta-analyses combined SNP-level evidence of association. Sign tests and polygenic score profiling weakly support an overlap of SNP effects between East Asian and European populations. We estimated the trans-ancestry genetic correlation of lifetime MDD as 0.33; female-only and recurrent MDD yielded estimates of 0.40 and 0.41, respectively. Common variants downstream of GPHN achieved genome-wide significance by Bayesian trans-ancestry meta-analysis (rs9323497; log10 Bayes Factor=8.08) but failed to replicate in an independent European sample (P=0.911). Gene-set enrichment analyses indicate enrichment of genes involved in neuronal development and axonal trafficking. We successfully demonstrate a partially shared polygenic basis of MDD in East Asian and European populations. Taken together, these findings support a complex etiology for MDD and possible population differences in predisposing genetic factors, with important implications for future genetic studies.

Using citrate-functionalized TiO2 nanoparticles to study the effect of particle size on zebrafish embryo toxicity.

TiO2 nanoparticles (NPs) are photoactive, potentially producing toxicity in vivo in the presence of sunlight. We have previously demonstrated photodependent toxicity in zebrafish embryos exposed to TiO2 NPs. Here we investigate the effect of particle size on developing zebrafish exposed to 6, 12 and 15 nm citrate-functionalized anatase TiO2 NPs under either simulated sunlight illumination or in the dark. All three sizes of TiO2 NPs caused photo-dependent toxicity. Under simulated sunlight illumination, the acute toxicity of the 6 nm citrate-TiO2 NPs (120 h LC50 of 23.4 mg L(-1)) exceeded that of the 12 and 15 nm citrate-TiO2 NPs. Exposure to 6 nm particles under illumination also caused a higher incidence of developmental defects than the larger particles. These abnormalities included pericardial edema, yolk-sac edema, craniofacial malformation, and opaque yolk. To gain insight into the mechanisms of toxicity, we measured hydroxyl radicals (˙OH) generated by NPs in vitro and reactive oxygen species (ROS) produced in vivo. We found that on a mass basis, smaller particles generated higher levels of ROS both in vitro and in vivo, and the 6 nm citrate-TiO2 NPs induced more oxidative stress than larger particles in the zebrafish embryo. We examined oxidative DNA damage by measuring 8-hydroxydeoxyguanosine in zebrafish exposed to different-sized citrate-TiO2 NPs and found that 6 nm particles caused more DNA damage than did larger particles (12 and 15 nm) under illumination. Our results indicate a photo-dependent toxicity of citrate-TiO2 NPs to zebrafish embryos, with an inverse relationship between particle size and toxicity. Production of more ROS, resulting in more oxidative stress and more DNA damage, represents one possible mechanism of the higher toxicity of smaller citrate-TiO2 NPs. These results highlight the relationship between citrate-TiO2 NP size and toxicity/oxidative stress in developing zebrafish embryos.

Effects of wheat or corn distillers dried grains with solubles on feedlot performance, fecal shedding, and persistence of Escherichia coli O157:H7.

Distillers dried grains with solubles (DDGS) are a coproduct of the ethanol industry and are often used as a replacement for grain in livestock production. Feeding corn DDGS to cattle has been linked to increased fecal shedding of Escherichia coli O157:H7, although in Canada, DDGS are often produced from wheat. This study assessed the effects of including 22.5% wheat or corn DDGS (DM basis) into barley-based diets on performance, carcass characteristics, animal health, and fecal E. coli O157:H7 shedding of commercial feedlot cattle. Cattle (n = 6,817) were randomly allocated to 10 pens per treatment group: WDDGS (diets including 22.5% wheat DDGS), CDDGS (diets including 22.5% corn DDGS), or CTRL (barley substituted for DDGS). Freshly voided fecal pats (n = 588) were collected and pooled monthly for fecal pH measurement and screened for naturally occurring E. coli O157:H7 by immunomagnetic separation (IMS) and direct plating (DP). Hide swabs (n = 367) were collected from randomly selected cattle from each pen before slaughter. Pen-floor fecal samples (n = 18) were collected from treatment groups at entry to the feedlot (<14 d on the finishing diet) and after adapting to the finishing diet for ≥ 14 d, inoculated (10(9) cfu of a 5 strain naldixic acid-resistant E. coli O157:H7 mixture), incubated (20°C) and evaluated weekly (IMS and DP) to assess fecal E. coli O157:H7 persistence. The WDDGS group had 3.0% poorer ADG (P = 0.007), 5.3% poorer G:F (P < 0.001), and a decreased proportion of Canada Quality Grade AAA carcasses (P = 0.022) compared with CTRL cattle. The CDDGS group had a similar ADG (P = 0.06), a decreased proportion of Canada Yield Grade (YG) 1 (P < 0.001), and greater proportions of Canada YG 2 (P = 0.003) and YG 3 (P < 0.001) carcasses compared with the CTRL group. There were no differences among groups in any of the animal health parameters assessed. Inclusion of DDGS in cattle finishing diets had no effect on fecal shedding (P = 0.650) or persistence (P = 0.953) of E. coli O157:H7. However, feces from cattle on starter diets <14 d had longer persistence of E. coli O157:H7 (week) than cattle on finishing diets ≥ 14 d (P < 0.003). Inclusion of DDGS in feedlot diets depends on commodity pricing relative to that of barley and for WDDGS must also include the risk of feedlot performance and carcass grading disadvantages. Feeding cattle barley based-diets with 22.5% corn or wheat DDGS did not affect fecal shedding of E. coli O157:H7.

A putative 'pre-nervous' endocannabinoid system in early echinoderm development.

Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences.

Efficacy of dose regimen and observation of herd immunity from a vaccine against Escherichia coli O157:H7 for feedlot cattle.

A clinical trial was conducted to test the effect of a vaccine product containing type III secreted proteins of Escherichia coli O157:H7 on the probability that feedlot steers shed E. coli O157:H7 in feces. Six hundred eight same-source steers were utilized. Of these, 480 steers were assigned randomly to 60 pens (eight head per pen) and to one of four vaccination treatments (120 cattle per treatment, two head per treatment per pen). The four treatments were (i) no vaccination; (ii) one dose, vaccinated once at reimplant (day 42); (iii) two doses, vaccinated on arrival (day 0) and again at reimplant (day 42); and (iv) three doses, vaccinated on arrival (day 0), on day 21, and again at reimplant (day 42). The remaining 128 steers were assigned randomly to 12 pens within the same feedlot to serve as unvaccinated external controls. The probability of detecting E. coli O157:H7 among cattle receiving different doses of vaccine was compared with that of unvaccinated external control cattle, accounting for clustering by repeated measures, block, and pen and fixed effects of vaccine, corn product, and test period. Vaccine efficacy of receiving one, two, and three doses of vaccine was 68, 66, and 73%, respectively, compared with cattle in pens not receiving vaccine. Cattle receiving three doses of vaccine were significantly less likely to shed E. coli O157:H7 than unvaccinated cattle within the same pen. Unvaccinated cattle housed with vaccinated cattle were 59% less likely to shed E. coli O157:H7 than cattle in pens not receiving vaccine, likely because they benefited from herd immunity. This study supports the hypothesis that vaccination with this vaccine product effectively reduces the probability for cattle to shed E. coli O157:H7. There was no indication that the vaccine affected performance or carcass quality. In addition, we found that vaccinating a majority of cattle within a pen offered a significant protective effect (herd immunity) to unvaccinated cattle within the same pen.

Effect of a vaccine product containing type III secreted proteins on the probability of Escherichia coli O157:H7 fecal shedding and mucosal colonization in feedlot cattle.

Preharvest intervention strategies to reduce Escherichia coli O157:H7 in cattle have been sought as a means to reduce human foodborne illness. A blinded clinical trial was conducted to test the effect of a vaccine product on the probability that feedlot steers, under conditions of natural exposure, shed E. coli O157:H7 in feces, are colonized by this organism in the terminal rectum, or develop a humoral response to the respective antigens. Steers (n = 288) were assigned randomly to 36 pens (eight head per pen), and pens were randomized to vaccination treatment in a balanced fashion within six dietary treatments of an unrelated nutrition study. Treatments included vaccination or placebo (three doses at 3-week intervals). Fecal samples for culture (n = 1,410) were collected from the rectum of each steer on pretreatment day 0 and posttreatment days 14, 28, 42, and 56. Terminal rectum mucosal (TRM) cells were aseptically collected for culture at harvest (day 57 posttreatment) by scraping the mucosa 3.0 to 5.5 cm proximal to the rectoanal junction. E. coli O157:H7 was isolated and identified with selective enrichment, immunomagnetic separation, and PCR confirmation. Vaccinated cattle were 98.3% less likely to be colonized by E. coli O157:H7 in TRM cells (odds ratio = 0.014, P < 0.0001). Diet was also associated with the probability of cattle being colonized (P = 0.04). Vaccinated cattle demonstrated significant humoral responses to Tir and O157 lipopolysaccharide. These results provide evidence that this vaccine product reduces E. coli O157:H7 colonization of the terminal rectum of feedlot beef cattle under conditions of natural exposure, a first step in its evaluation as an effective intervention for food and environmental safety.

Past radioactive particle contamination in the Columbia river at the Hanford site, USA.

One closed-loop and eight single-pass plutonium production reactors originally operated on the Columbia river. During the 26 years of single-pass reactor operations, small amounts of radioactive particles were released in liquid discharges to the Columbia river and were deposited in sediment and cobble along the shoreline and on islands in the river. Islands located adjacent to D island and immediately downstream of D island had the greatest density of particles. In 1979, the small particles contained between 63 and 890 kBq of cobalt-60 activity. Dose rates emanating from those particles ranged from 1 to 14 microGy h(-1). Because of the short half-life of cobalt-60 (5.3 y), the hot-particle problem at Hanford has taken care of itself through radiological decay. Some scientists have proposed that it is economically and environmentally advantageous to manage isolated low-level contaminated sites with institutional controls until the activity decays and the sites can be released rather than to pursue expensive clean-up options.

Effect of Lactobacillus acidophilus strain NP51 on Escherichia coil O157:H7 fecal shedding and finishing performance in beef feedlot cattle.

A 2-year study was conducted during the summer months (May to September) to test the effectiveness of feeding Lactobacillus acidophilus strain NP51 on the proportion of cattle shedding Escherichia coli O157:H7 in the feces and evaluate the effect of the treatment on finishing performance. Steers (n = 448) were assigned randomly to pens, and pens of cattle were assigned randomly to NP51 supplementation or no supplementation (control). NP51 products were mixed with water and applied as the feed was mixed daily in treatment-designated trucks at the rate of 10(9) CFU per steer. Fecal samples were collected (n = 3,360) from the rectum from each animal every 3 weeks, and E. coli O157:H7 was isolated by standard procedures, using selective enrichment, immunomagnetic separation, and PCR confirmation. The outcome variable was the recovery of E. coli O157:H7 from feces, and was modeled using logistic regression accounting for year, repeated measures of pens of cattle, and block. No significant differences were detected for gain, intakes, or feed efficiency of control or NP51-fed steers. The probability for cattle to shed E. coli O157:H7 varied significantly between 2002 and 2003 (P = 0.004). In 2002 and 2003, the probability for NP51-treated steers to shed E. coli O157:H7 over the test periods was 13 and 21%, respectively, compared with 21 and 28% among controls. Over the 2 years, NP51-treated steers were 35% less likely to shed E. coli O157: H7 than were steers in untreated pens (odds ratio = 0.58, P = 0.008). This study is consistent with previous reports that feeding NP51 is effective in reducing E. coli O157:H7 fecal shedding in feedlot cattle.

Growth, morphogenesis, and differentiation during mouse prostate development in situ, in renal grafts, and in vitro.

BACKGROUND: In vitro organ culture and renal grafting of the urogenital sinus (UGS) have both been used as models of prostate development. However, neither has been rigorously examined for its fidelity to replicate the canonical process of prostate differentiation in situ. METHODS: We assessed size, morphology, histology, and the mRNA expression of differentiation marker genes of the E14 male mouse UGS grown for 0-28 days as sub-renal capsule allografts in nude mice or in culture containing androgen and compared these to UGS development in situ. RESULTS: Development of grafted tissues was morphologically and histologically similar to development in situ but differentiation occurred more rapidly. UGS growth in organ culture resulted in bud formation, but did not trigger cellular differentiation. However, the potential for differentiation was maintained and could be rescued by grafting tissues into nude mice. CONCLUSIONS: In vitro organ culture and renal grafting of UGS tissues may be appropriate models for studying prostatic bud formation, but only grafting is an appropriate model for prostatic differentiation.

Tetrachlorodibenzo-p-dioxin exposure alters radial arm maze performance and hippocampal morphology in female AhR mice.

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been reported to alter spatial learning in rats tested on a radial arm maze (RAM). TCDD is believed to exert most of its effects through binding to the aryl hydrocarbon receptor (AhR). To determine whether the AhR mediates TCDD-induced alterations in spatial learning, we tested male and female AhR-knockout (AhR-/-), heterozygous (AhR+/-) and wild-type (AhR+/+) mice on the RAM. AhR+/- male and female mice were time mated, and treated dams were dosed with 5 microg TCDD/kg body weight on day 13 of gestation. When offspring reached adulthood, male and female AhR+/+, AhR+/- and AhR-/- mice from TCDD-exposed and unexposed litters were tested on the eight-arm RAM. After testing, we examined hippocampal morphology as visualized by the Timm's silver sulfide stain. TCDD-exposed female AhR+/- mice made more errors than their respective controls on the RAM and exhibited a decrease in the size of the intra- and infrapyramidal mossy fiber (IIP-MF) field of the hippocampus. None of the other TCDD-exposed groups differed from their respective control groups with regard to maze performance or hippocampal morphology. The reduction of IIP-MF field indicates a possible morphological basis for the learning deficit that was observed in the female AhR+/- mice. It is hypothesized that the effect of TCDD exposure is AhR dependent and that TCDD may alter GABAergic activity in the hippocampus of female mice during development.

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal vein in developing zebrafish.

A role for the aryl hydrocarbon receptor (AHR) pathway in vascular maturation has been implicated by studies in Ahr-null mice. In this study the hypothesis that activation of AHR signaling by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters common cardinal vein (CCV) development in the zebrafish embryo was investigated. The CCV is a paired vessel that grows across the yolk, connecting to the heart. It is extensively remodeled and regresses as the heart migrates dorsally within the pericardium. TCDD significantly reduced CCV growth as early as 44 h post fertilization (hpf), and CCV area was reduced to 63% of control at 62 hpf. This vascular response to TCDD was at least as sensitive as previously defined endpoints of TCDD developmental toxicity in zebrafish. TCDD also blocked regression of the CCV (by 80 hpf), possibly contributing to the "string-like" heart phenotype seen in TCDD-exposed zebrafish larvae. Dependence of the block in CCV regression on zebrafish (zf) AHR2 was investigated using a zfahr2 specific morpholino to knock down expression of AHR2. The zfahr2 morpholino had no effect on CCV regression in the absence of TCDD, but did protect against the TCDD-induced block of CCV regression. This demonstrates that the TCDD-induced block in CCV regression is AHR2 dependent. It is significant that decreased CCV growth occurs before and inhibition of CCV regression occurs concurrent with overt signs of TCDD developmental toxicity. This suggests that alterations of vascular growth and remodeling may play a role in TCDD developmental toxicity in zebrafish.

Disruption of erythropoiesis by dioxin in the zebrafish.

2,3,7,8-Tetrachlorodibenzo-p- dioxin (TCDD, or dioxin) causes early life stage mortality in a variety of fish species. We have used the zebrafish (Danio rerio) to study the cardiovascular effects of TCDD treatment over the time course of zebrafish development. Early TCDD exposure (6 ng/ml) starting at 4 hr postfertilization (hpf) produced reductions in blood flow and in the number of circulating erythrocytes. These defects were consistently observable by 72 hpf. However, these responses were not observed when TCDD exposure was delayed until 96 hpf or later. These results suggest a model in which TCDD interferes with cardiovascular and erythropoietic developmental processes that are normally completed by 96 hpf. This model is strengthened by the finding that TCDD exposure blocks the step in hematopoiesis in which developing zebrafish switch from the primitive phase to the definitive phase of erythropoiesis. We observed no effect of TCDD on the levels of circulating primitive erythrocytes before 72 hpf and the expression of markers for early hematopoiesis, GATA-1 and GATA-2. However, early TCDD exposure prevented the appearance of definitive phase erythrocytes. TCDD produced a small delay in the migration of blood cells expressing SCL from the intermediate cell mass to the dorsal mesentery and dorsal aorta. Despite the decrease in blood flow produced by TCDD, confocal microscopy of the trunk vasculature by using a Tie2/green fluorescence protein endothelial marker at 48, 60, 72, and 96 hpf of TCDD-exposed (4 hpf) revealed no apparent defects in blood vessel structure.

Investigation of Prx1 protein expression provides evidence for conservation of cardiac-specific posttranscriptional regulation in vertebrates.

Gene targeting experiments have defined that the homeobox gene Prx1 is essential for normal craniofacial, limb, and vascular development. Although its RNA expression pattern is well established, Prx1 protein expression in the developing embryo has not been examined. A novel Prx1 antibody was produced to define the normal Prx1 protein expression pattern in the developing mouse embryo. In craniofacial and limb mesenchyme, Prx1 protein expression is consistent with previously published data on RNA localization. However, a remarkable discrepancy was found in cardiac tissue. Prx1 protein is undetectable in the murine embryonic and adult heart, despite the presence of Prx1 transcripts. These data demonstrate that Prx1 expression is posttranscriptionally regulated. This discrepancy between the presence of Prx1 transcript and the absence of detectable protein was also observed in embryonic chick heart, suggesting conservation of the regulatory mechanism in vertebrates. This observation provides a new explanation of why the Prx null mice lack cardiac malformations.

Role of the aryl hydrocarbon receptor in the development of control and 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed male mice.

Experiments were conducted to determine the role of the aryl hydrocarbon receptor (AhR) in the development of control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 male mice. Male and female mice heterozygous for the AhR (Ahr+/-) were mated, and pregnant females were dosed orally on gestation day 13 with corn oil vehicle or TCDD (5 microg/kg). Pups were necropsied on postnatal day (PND) 21, 35, and 90. Comparison of vehicle-exposed wild-type (Ahr+/+) pups with vehicle-exposed AhR knockout (AhRKO; Ahr-/-) pups confirmed and extended previous reports that development of the liver, heart, spleen, thymus, and kidney is affected by absence of the AhR. Lung, submandibular gland, testis, and epididymis weights were also affected, indicating that the AhR plays a role in normal development of these organs as well. The presence or absence of the AhR had no effect on the incidence of hydronephrosis, daily sperm production, or cauda epididymal sperm numbers in vehicle-exposed mice. TCDD caused numerous effects in wild-type mice that were absent in AhRKO mice; specifically, hydronephrosis, increases in relative liver and heart weight, decreases in absolute heart and lung weight, and decreases in absolute and relative thymus, submandibular gland, epididymis, and testis weight. In several cases, TCDD produced one effect in wild-type mice (reductions in body weight and absolute thymus, submandibular gland, and epididymis weight on PND 21; and reductions in absolute and relative submandibular gland and absolute testis weight on PND 35) but caused the opposite effect in AhRKO mice. In yet other cases (reduced relative spleen weight on PND 21 and reductions in absolute and relative thymus weight on PND 35), TCDD produced similar effects in wildtype and AhRKO mice. There were also cases in which TCDD significantly affected AhRKO mice without significantly altering the same endpoint in wild-type mice; absolute liver, lung, and kidney weight were increased and relative submandibular gland weight was decreased on PND 21; relative heart weight was reduced and absolute lung weight increased on PND 35; and relative liver weight was decreased on PND 90. Although many effects of TCDD required the presence of the AhR, these results provide evidence either for multiple forms of the AhR in mice (one or more of which are still present in AhRKO mice), or for AhR-independent effects of low-level TCDD exposure.

In utero and lactational treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs mammary gland differentiation but does not block the response to exogenous estrogen in the postpubertal female rat.

These experiments tested whether in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters mammary gland differentiation, estrogen receptor alpha (ERalpha) expression levels, or the response to estrogen in the female postpubertal rat mammary gland. Pregnant Holtzman rats were administered a single oral dose of 1 microg/kg TCDD or vehicle on gestation-day 15. Exposed and non-exposed female offspring were weaned on postnatal day 21 and ovariectomized at 9 weeks of age. Two weeks later, both TCDD and control animals were divided into 3 groups, receiving treatment with placebo, 0.025, or 0.1 mg 17beta-estradiol pellet implants. After 48 h, mammary tissue was removed for analysis following euthanasia. TCDD-exposed mammary glands demonstrated impaired differentiation as measured by the distribution of terminal ductal structures and increased expression levels of ERalpha. The response to exogenous estrogen was tested in TCDD-exposed animals and compared to control non-exposed animals. Estrogen stimulation of the TCDD-exposed glands induced progesterone receptor expression and mammary gland differentiation as measured by a shift in distribution from terminal end buds and terminal ducts to Types I and II lobules. Control glands were better differentiated at baseline and did not exhibit any significant changes in the distribution of terminal ductal structures following estrogen stimulation. The increase in progesterone receptor-expression levels by exogenous estrogen in control glands was similar to the TCDD-exposed glands. These experiments demonstrate that in utero and lactational exposures to TCDD impair mammary gland differentiation but that TCDD-exposed mammary glands retain the ability to differentiate in response to estrogen.

Relative potencies of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners to induce cytochrome P4501A mRNA in a zebrafish liver cell line.

Induction of cytochrome P4501A (CYP1A) mRNA by polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF), and polychlorinated biphenyl (PCB) congeners was measured in a zebrafish liver (ZF-L) cell line. The ZF-L cells were far less sensitive to PCDD, PCDF, and PCB congeners than were other fish cell lines. The 2,3,7,8-PCDDs, 2,3,7,8-PCDFs, and PCB 126 caused dose-related induction. All other PCBs tested, including other coplanar as well as ortho-substituted congeners, were ineffective at inducing CYP1A. The potency of each congener that gave a response, relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin, was determined. The ZF-L cell-derived relative potency values (REPs) are similar to other in vitro REPs in that the ZF-L cell-derived REPs are generally higher than those derived from in vivo models. Furthermore, the ZF-L cell-derived REPs are generally within fivefold of REPs determined in a variety of rainbow trout systems when the same endpoint in the same tissue are compared. Analysis of these data indicates that REPs based on molecular and biochemical responses in sensitive and insensitive species are similar, but overestimate relative in vivo toxicity in the rainbow trout. The ZF-L cell-derived REPs expand the database of REPs, providing additional information that will be useful in quantifying the uncertainty associated with applying consensus fish-specific toxic equivalency factors in ecological risk assessment.

Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer Di(2-ethylhexyl) phthalate.

Several members of the phthalate ester family have antiandrogenic properties, yet little is known about how exposure to these ubiquitous environmental contaminants early in development may affect sexual development. We conducted experiments to determine effects of in utero and lactational exposure to the most prevalent phthalate ester, di(2-ethylhexyl) phthalate (DEHP), on male reproductive system development and sexual behavior. Sprague-Dawley rats were dosed with corn oil or DEHP (0, 375, 750, or 1,500 mg/kg/day, per os) from gestation day 3 through postnatal day (PND) 21. Dose-related effects on male offspring included reduced anogenital distance, areola and nipple retention, undescended testes, and permanently incomplete preputial separation. Testis, epididymis, glans penis, ventral prostate, dorsolateral prostate, anterior prostate, and seminal vesicle weights were reduced at PND 21, 63, and/or 105-112. Additional dose-related effects included a high incidence of anterior prostate agenesis, a lower incidence of partial or complete ventral prostate agenesis, occasional dorsolateral prostate and seminal vesicle agenesis, reduced sperm counts, and testicular, epididymal, and penile malformations. Many DEHP-exposed males were sexually inactive in the presence of receptive control females, but sexual inactivity did not correlate with abnormal male reproductive organs. These results suggest that in utero and lactational DEHP exposure also inhibited sexually dimorphic central nervous system development. No major abnormalities were found in any of eight control litters, but DEHP caused severe male reproductive system toxicity in five of eight litters at 375 mg/kg/day, seven of eight litters at 750 mg/kg/day, and five of five litters at 1,500 mg/kg/day. These results demonstrate that the male reproductive system is far more sensitive to DEHP early in development than when animals are exposed as juveniles or adults. The effects of DEHP on male reproductive organs and sexual behaviors and the lack of significant effects on time to vaginal opening and first estrus in their littermates demonstrate that DEHP (and/or its metabolites) affects development of the male reproductive system primarily by acting as an antiandrogen. The pattern of effects of in utero and lactational DEHP exposure differed from patterns caused by other phthalate esters, and the preponderance of anterior prostate agenesis appears to be unique among all chemicals. These results suggest that DEHP acts partly by mechanisms distinct from those of other antiandrogens.