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When analyzing fetal defect incidence in laboratory animal studies, correlation in responses within litters (i.e., litter effects) can lead to increased false-positive rates if litter effects are not incorporated into the analysis. Studies of fetal defects require analysis methods that are robust across a broad range of defect types, including those with zero or near-zero incidence rates in control groups.A simulation study compared power and false-positive rates for six approaches across a range of background defect rates and litter size distributions. Statistical methods evaluated included ignoring the litter effect as well as parametric and nonparametric approaches based on litter proportions, generalized linear mixed models (GLMMs), the Rao-Scott Cochran-Armitage (RSCA) trend test, and a modification to the RSCA (mRSCA) introduced here to improve estimation at low background rates. These methods were also applied to a common and a rare defect from two prenatal developmental toxicology studies conducted by the National Toxicology Program (NTP).At background defect rates of 1%, the mRSCA and parametric litter proportion methods provided gains in power over the nonparametric litter proportion method, the GLMM method, and the RSCA method. Simulations involving litter loss in high-dose groups showed loss of power for both litter proportion methods.The mRSCA test developed here compares favorably with other litter-based approaches and is robust across a range of background defect rates and litter size distributions, making it a practical choice for prenatal developmental toxicology studies involving both common and rare fetal defects.

The general population, including children and adolescents, is exposed to 4-methylimidazole (4-MI) in the diet. 4-MI is a by-product of caramel color manufacturing. It has been previously classified as a possible human carcinogen and displays potential reproductive toxicity. A follow up assessment of reproductive toxicity was conducted in rats utilizing the reproductive assessment by continuous breeding paradigm, in which multiple generations were exposed to 4-MI in diet at 750, 2500, and 5000 ppm. 4-MI exposure was associated with delays in preputial separation and vaginal opening, impairment in reproductive performance, and concomitant histopathological findings in the prostate, testis, and epididymis at 2500 and 5000 ppm. The Lowest Observed Adverse Effect Level for reproductive (based on prostate atrophy) and developmental toxicity (based on delays in preputial separation and vaginal opening) was 750 ppm, equivalent to approximately 50-60 mg/kg bw/day.

Due to structural similarity to bisphenol A and lack of safety data, the National Toxicology Program (NTP) is evaluating the potential toxicity of bisphenol AF (BPAF) in rodent models. The current investigation reports the internal exposure data for free (unconjugated BPAF) and total (free and conjugated forms) BPAF during critical stages of development following perinatal dietary exposure in Hsd:Sprague Dawley®SD® rats to 0 (vehicle control), 338, 1125, and 3750 ppm BPAF from gestation day (GD) 6 to postnatal day (PND) 28. Free and total BPAF concentrations in maternal plasma at GD 18, PND 4, and PND 28 increased with the exposure concentration; free BPAF concentrations were ≤ 1.61% those of total BPAF demonstrating extensive first pass metabolism of BPAF following dietary exposure in adults. Free and total BPAF were quantified in GD 18 fetuses and PND 4 pups with free concentrations 11.7–53.4% that of corresponding total concentrations. In addition, free concentrations were higher (130–571%) and total concentrations were lower (1.71–7.23%) than corresponding concentrations in dams, demonstrating either preferential transfer of free BPAF and/or inability of fetuses and pups to conjugate BPAF. Free and total concentrations in PND 28 pups were similar to maternal concentrations demonstrating direct exposure of pups via feed and that conjugating enzymes are developed in PND 28 pups. In conclusion, these data demonstrate considerable gestational and lactational transfer of parent aglycone from the mother to offspring. Since the ontogeny of conjugating enzymes in humans is similar to that of rodents, the data from rodent BPAF studies may be useful in predicting human risk from exposure to BPAF.

In recent years, several genome-wide association studies have identified candidate regions for genetic susceptibility in major mood disorders. Most notable are regions in a locus in chromosome 3p21, encompassing the genes NEK4-ITIH1-ITIH3-ITIH4. Three of these genes represent heavy chains of the composite protein inter-α-inhibitor (IαI). In order to further establish associations of these genes with mood disorders, we evaluated behavioral phenotypes in mice deficient in either Ambp/bikunin, which is necessary for functional ITIH1 and ITIH3 complexes, or in Itih4, the gene encoding the heavy chain Itih4. We found that loss of Itih4 had no effect on the behaviors tested, but loss of Ambp/bikunin led to increased anxiety-like behavior in the light/dark and open field tests and reduced exploratory activity in the elevated plus maze, light/dark preference and open field tests. Ambp/bikunin knockout mice also exhibited a sex-dependent exaggeration of acoustic startle responses, alterations in social approach during a three-chamber choice test, and an elevated fear conditioning response. These results provide experimental support for the role of ITIH1/ITIH3 in the development of mood disorders.

Perfluoroalkyl and polyfluoroalkyl substances are used in commercial applications and developmental exposure has been implicated in alterations in neurobehavioral functioning. While associations between developmental perfluorooctanoic acid (PFOA) exposure and human outcomes have been inconsistent, studies in experimental animals suggest alterations in motor related behaviors. To examine a dose-response pattern of neurobehavioral effects following gestational exposure to PFOA, pregnant CD-1 mice received PFOA (0, 0.1, 0.3, 1.0 mg/kg/day) via oral gavage from gestational day 1–17 and the male offspring examined. Motor activity assessments on postnatal day (PND)18, 19, and 20 indicated a shift in the developmental pattern with an elevated activity level observed in the 1.0 mg/kg/day dose group on PND18. In the adult, no alterations were observed in body weights, activity levels, diurnal pattern of running wheel activity, startle response, or pre-pulse startle inhibition. In response to a subcutaneous injection of saline or nicotine (80 µg/kg), all animals displayed a transient increase in activity likely associated with handling with no differences observed across dose groups. Inhibition of motor activity over 18 days of 400µg/kg nicotine injection was not significantly different across dose groups. Hyperactivity induced by 2mg/kg (+)-methamphetamine hydrochloride intraperitoneal injection was significantly lower in the 1.0 mg/kg/day PFOA dose group as compared to controls. Taken together, these data suggest that the effects on motor-related behaviors with gestational PFOA exposure do not mimic those reported for acute postnatal exposure. Changes were not observed at dose level under 1.0 mg/kg/day PFOA. Further examination of pathways associated with methamphetamine-induced activity is warranted.

In the US EPA's 1998 Baltimore Epidemiology-Exposure Panel Study, a group of 16 residents of a single building retirement community wore personal monitors recording personal fine particulate air pollution concentrations (PM2.5) for 27 days, while other monitors recorded concurrent apartment, central indoor, outdoor and ambient site PM2.5 concentrations. Using the Baltimore panel study data, we develop a Bayesian hierarchical model to characterize the relationship between personal exposure and concentrations of PM2.5 indoors and outdoors. Personal exposure is expressed as a linear combination of time spent in microenvironments and associated microenvironmental concentrations. The model incorporates all available monitoring data and accounts for missing data and sources of uncertainty such as measurement error and individual differences in exposure. We discuss the implications of using personal versus ambient PM2.5 measurements in characterization of personal exposure to PM2.5.

In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an "antimicrobial fingerprint," which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways.

Thyroid hormones (TH) regulate biological processes implicated in neurodevelopmental disorders and can be altered with environmental exposures. Developmental exposure to the dioxin-like compound, 3,3',4,4'-tetrachloroazobenzene (TCAB), induced a dose response deficit in serum T4 levels with no change in 3,5,3'-triiodothyronine or thyroid stimulating hormone. Female Sprague-Dawley rats were orally gavaged (corn oil, 0.1, 1.0, or 10 mg TCAB/kg/day) two weeks prior to cohabitation until post-partum day 3 and male offspring from post-natal day (PND) 4–21. At PND21, the high dose showed a deficit in body weight gain. Conventional neuropathology detected no neuronal death, myelin disruption, or gliosis. Astrocytes displayed thinner and less complex processes at 1.0 and 10 mg/kg/day. At 10 mg/kg/day, microglia showed less complex processes, unbiased stereology detected fewer hippocampal CA1 pyramidal neurons and dentate granule neurons (GC) and Golgi staining of the cerebellum showed diminished Purkinje cell dendritic arbor. At PND150, normal maturation of GC number and Purkinje cell branching area was not observed in the 1.0 mg/kg/day dose group with a diminished number and branching suggestive of effects initiated during developmental exposure. No effects were observed on post-weaning behavioral assessments in control, 0.1 and 1.0 mg/kg/day dose groups. The demonstrated sensitivity of hippocampal neurons and glial cells to TCAB and T4 deficit raises support for considering additional anatomical features of brain development in future DNT evaluations.

In indoor air quality studies, discrepancies between personal and stationary indoor air quality monitors arise because of the source proximity effect, in which pollutant sources near the respondent cause elevated and highly variable exposures. In a set of experiments in a home, concentrations of a continuously emitting tracer gas were simultaneously monitored at different distances from the tracer gas source. Concentration time series are modeled at collinear monitoring sites as the sum of a slowly varying baseline time series and the superposition of transient elevated concentrations, or “microplumes.” Microplume arrivals appear as pulses in the time series, with pulse magnitudes and duration varying by location relative to the source. A nonparametric method is developed to estimate the time-varying parameters of the baseline time series. Parameters of superposed microplumes are estimated using the method of moments. Bias and sampling error of estimates are investigated using a simulation study. Estimate...

More than a dozen indoor air quality studies have reported a large discrepancy between concentrations measured by stationary indoor monitors (SIMs) and personal exposure monitors (PEMs). One possible cause of this discrepancy is a source proximity effect, in which pollutant sources close to the respondent cause elevated and highly variable exposures. This paper describes three sets of experiments in a home using real-time measurements to characterize and quantify the proximity effect relative to a fixed distant location analogous to a SIM. In the first set of experiments, using sulfur hexafluoride (SF6) as a continuously emitting tracer pollutant from a point source, measurements of pollutant concentrations were made at different distances from the source under different air exchange rates and source strengths. A second set of experiments used a continuous point source of carbon monoxide (CO) tracer pollutant and an array of high time resolution monitors to collect simultaneous concentration readings at different locations in the room. A third set of experiments measured particle count density and particle-bound polycyclic aromatic hydrocarbon (PAH) concentrations emitted from a continuous particle point source (an incense stick) using two particle counters and two PAH monitors, and included human activity periods both before and during the source emission period. Results from the SF6 and CO experiments show that while the source is emitting, a source proximity effect can be seen in the increases in the mean and median and in the variability of concentrations closest to the source, even at a distance of 2.0 m from the source under certain settings of air exchange rate and source strength. CO concentrations at locations near the source were found to be higher and more variable than the predictions of the mass balance model. For particles emitted from the incense source, a source proximity effect was evident for the fine particle sizes (0.3 to 2.5 microm) and particle-bound PAH up to at least 1.0 m from the source. Analysis of spatial and temporal patterns in the data for the three tracer pollutants reveal marked transient elevations of concentrations as seen by the monitor, referred to as "microplumes," particularly at locations close to the source. Mixing patterns in the room show complex patterns and directional effects, as evidenced by the variable intensity of the microplume activity at different locations. By characterizing the spatial and temporal variability of pollutant concentrations in the home, the proximity effect can be quantified, leading to improved indoor monitoring designs and models of human exposure to air pollutants.

Studies have linked increased levels of particulate air pollution to decreased autonomic control, as measured by heart rate variability (HRV), particularly in susceptible populations such as the elderly. In this study, we use data obtained from the 1998 USEPA epidemiology-exposure longitudinal panel study of elderly adults in a Baltimore retirement home to examine the relationship between HRV and PM2.5 personal exposure. We consider PM2.5 personal exposure in the aggregate and personal exposure to the components of PM2.5 as estimated in two ways using receptor models. We develop a Bayesian hierarchical model for HRV as a function of personal exposure to PM2.5, which integrates HRV measurements and data obtained from personal, indoor and outdoor PM2.5 monitoring and meteorological data. We found a strong relationship between decreased HRV (HF, LF, r-MSSD and SDNN) and total personal exposure to PM2.5 at a lag of 1 day. Using personal exposure monitoring (PEM) apportionment results, we examined the relative importance of ambient and non-ambient personal PM2.5 exposure to HRV and found the effect of internal non-ambient sources of PM2.5 on HRV to be minimal. Using the PEM apportionment data, a consistent effect of soil at short time scales (lag 0) was found across all five HRV measures, and an effect of sulfate on HRV was seen for HF and r-MSSD at the moving average of lags 0 and 1 days. Modeling of ambient site apportionment data indicated effects of nitrate on HRV at lags of 1 day, and moving averages of days 0 and 1 and days 0–2 for all but the ratio LF/HF. Sulfate had an effect on HRV at a lag of 1 day for four HRV measures (HF, LF, r-MSSD, SDNN) and for LF/HF at a moving average of days 0–2.

The National Research Council has outlined the need for non-mammalian toxicological models to test the potential health effects of a large number of chemicals while also reducing the use of traditional animal models. The nematode Caenorhabditis elegans is an attractive alternative model because of its well-characterized and evolutionarily conserved biology, low cost, and ability to be used in high-throughput screening. A high-throughput method is described for quantifying the reproductive capacity of C. elegans exposed to chemicals for 48 h from the last larval stage (L4) to adulthood using a COPAS Biosort. Initially, the effects of exposure conditions that could influence reproduction were defined. Concentrations of DMSO vehicle

Background Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative. Methodology/Principal Findings Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC50 of 2 µM. Conclusions/Significance The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.