Separating Measurement Error and Signal in Environmental Data: Use of Replicates to Address Uncertainty.

Measurement uncertainty has long been a concern in the characterizing and interpreting environmental and toxicological measurements. We compared statistical analysis approaches when there are replicates: a Naïve approach that omits replicates, a Hybrid approach that inappropriately treats replicates as independent samples, and a Measurement Error Model (MEM) approach in a random effects analysis of variance (ANOVA) model that appropriately incorporates replicates. A simulation study assessed the effects of sample size and levels of replication, signal variance, and measurement error on estimates from the three statistical approaches. MEM results were superior overall with confidence intervals for the observed mean narrower on average than those from the Naïve approach, giving improved characterization. The MEM approach also featured an unparalleled advantage in estimating signal and measurement error variance separately, directly addressing measurement uncertainty. These MEM estimates were approximately unbiased on average with more replication and larger sample sizes. Case studies illustrated analyzing normally distributed arsenic and log-normally distributed chromium concentrations in tap water and calculating MEM confidence intervals for the true, latent signal mean and latent signal geometric mean (i.e., with measurement error removed). MEM estimates are valuable for study planning; we used simulation to compare various sample sizes and levels of replication.

Censoring Trace-Level Environmental Data: Statistical Analysis Considerations to Limit Bias.

Trace-level environmental data typically include values near or below detection and quantitation thresholds where health effects may result from low-concentration exposures to one chemical over time or to multiple chemicals. In a cook stove case study, bias in dibenzo[a,h]anthracene concentration means and standard deviations (SDs) was assessed following censoring at thresholds for selected analysis approaches: substituting threshold/2, maximum likelihood estimation, robust regression on order statistics, Kaplan-Meier, and omitting censored observations. Means and SDs for gas chromatography-mass spectrometry-determined concentrations were calculated after censoring at detection and calibration thresholds, 17% and 55% of the data, respectively. Threshold/2 substitution was the least biased. Measurement values were subsequently simulated from two log-normal distributions at two sample sizes. Means and SDs were calculated for 30%, 50%, and 80% censoring levels and compared to known distribution counterparts. Simulation results illustrated (1) threshold/2 substitution to be inferior to modern after-censoring statistical approaches and (2) all after-censoring approaches to be inferior to including all measurement data in analysis. Additionally, differences in stove-specific group means were tested for uncensored samples and after censoring. Group differences of means tests varied depending on censoring and distributional decisions. Investigators should guard against censoring-related bias from (explicit or implicit) distributional and analysis approach decisions.

Effects of Cold Temperature and Ethanol Content on VOC Emissions from Light-Duty Gasoline Vehicles.

Emissions of speciated volatile organic compounds (VOCs), including mobile source air toxics (MSATs), were measured in vehicle exhaust from three light-duty spark ignition vehicles operating on summer and winter grade gasoline (E0) and ethanol blended (E10 and E85) fuels. Vehicle testing was conducted using a three-phase LA92 driving cycle in a temperature-controlled chassis dynamometer at two ambient temperatures (-7 and 24 °C). The cold start driving phase and cold ambient temperature increased VOC and MSAT emissions up to several orders of magnitude compared to emissions during other vehicle operation phases and warm ambient temperature testing, respectively. As a result, calculated ozone formation potentials (OFPs) were 7 to 21 times greater for the cold starts during cold temperature tests than comparable warm temperature tests. The use of E85 fuel generally led to substantial reductions in hydrocarbons and increases in oxygenates such as ethanol and acetaldehyde compared to E0 and E10 fuels. However, at the same ambient temperature, the VOC emissions from the E0 and E10 fuels and OFPs from all fuels were not significantly different. Cold temperature effects on cold start MSAT emissions varied by individual MSAT compound, but were consistent over a range of modern spark ignition vehicles.

Cold temperature and biodiesel fuel effects on speciated emissions of volatile organic compounds from diesel trucks.

Speciated volatile organic compounds (VOCs) were measured in diesel exhaust from three heavy-duty trucks equipped with modern aftertreatment technologies. Emissions testing was conducted on a chassis dynamometer at two ambient temperatures (-7 and 22 °C) operating on two fuels (ultra low sulfur diesel and 20% soy biodiesel blend) over three driving cycles: cold start, warm start and heavy-duty urban dynamometer driving cycle. VOCs were measured separately for each drive cycle. Carbonyls such as formaldehyde and acetaldehyde dominated VOC emissions, making up ∼ 72% of the sum of the speciated VOC emissions (∑VOCs) overall. Biodiesel use led to minor reductions in aromatics and variable changes in carbonyls. Cold temperature and cold start conditions caused dramatic enhancements in VOC emissions, mostly carbonyls, compared to the warmer temperature and other drive cycles, respectively. Different 2007+ aftertreatment technologies involving catalyst regeneration led to significant modifications of VOC emissions that were compound-specific and highly dependent on test conditions. A comparison of this work with emission rates from different diesel engines under various test conditions showed that these newer technologies resulted in lower emission rates of aromatic compounds. However, emissions of other toxic partial combustion products such as carbonyls were not reduced in the modern diesel vehicles tested.

A new method for generating distributions of biomonitoring equivalents to support exposure assessment and prioritization.

Biomonitoring data are now available for hundreds of chemicals through state and national health surveys. Exposure guidance values also exist for many of these chemicals. Several methods are frequently used to evaluate biomarker data with respect to a guidance value. The "biomonitoring equivalent" (BE) approach estimates a single biomarker concentration (called the BE) that corresponds to a guidance value (e.g., Maximum Contaminant Level, Reference Dose, etc.), which can then be compared with measured biomarker data. The resulting "hazard quotient" estimates (HQ=biomarker concentration/BE) can then be used to prioritize chemicals for follow-up examinations. This approach is used exclusively for population-level assessments, and works best when the central tendency of measurement data is considered. Complementary approaches are therefore needed for assessing individual biomarker levels, particularly those that fall within the upper percentiles of measurement distributions. In this case study, probabilistic models were first used to generate distributions of BEs for perchlorate based on the point-of-departure (POD) of 7µg/kg/day. These distributions reflect possible biomarker concentrations in a hypothetical population where all individuals are exposed at the POD. A statistical analysis was then performed to evaluate urinary perchlorate measurements from adults in the 2001 to 2002 National Health and Nutrition Examination Survey (NHANES). Each NHANES adult was assumed to have experienced repeated exposure at the POD, and their biomarker concentration was interpreted probabilistically with respect to a BE distribution. The HQ based on the geometric mean (GM) urinary perchlorate concentration was estimated to be much lower than unity (HQ≈0.07). This result suggests that the average NHANES adult was exposed to perchlorate at a level well below the POD. Regarding individuals, at least a 99.8% probability was calculated for all but two NHANES adults that a higher biomarker concentration would have been observed compared to what was actually measured if the daily dietary exposure had been at the POD. This is strong evidence that individual perchlorate exposures in the 2001-2002 NHANES adult population were likely well below the POD. This case study demonstrates that the "stochastic BE approach" provides useful quantitative metrics, in addition to HQ estimates, for comparison across chemicals. This methodology should be considered when evaluating biomarker measurements against exposure guidance values, and when examining chemicals that have been identified as needing follow-up investigation based on existing HQ estimates.

Urinary cadmium in the 1999-2008 U.S. National Health and Nutrition Examination Survey (NHANES).

Chronic low-level cadmium (Cd) exposure is linked to kidney and cardiovascular disease, fractures, and cancer. Diet and smoking are primary sources of exposure in the general population. We analyzed urinary Cd in NHANES 1999-2008 to determine whether levels declined significantly over the decade for U.S. children, teens, and adults (nonsmokers and smokers) and, if so, factors influencing the decline(s). For each subpopulation, we modeled log urinary Cd using variable-threshold censored multiple regression. Models included individual-level covariates (age, gender, BMI, income, race/ethnicity/country of origin, education, survey period), smoking, housing (home age, water source, filter use), and diet (supplement use; 24-h calorie, fat, protein, micronutrient, and Cd-containing food intakes), creatinine, and survey year variables. Geometric mean urinary Cd (ng/mL) declined 20-25% in these subpopulations, and the regressions showed statistically significant declines in later years for teens and adults. While certain covariates were significantly associated with Cd by subpopulation (creatinine; age; BMI; race/ethnicity/origin; education; smokers in the home; serum cotinine; 24-h fat, Mg, Fe intakes; use of dietary supplements), they did not help explain the declines. Instead, unidentified time-related factors appeared responsible. Despite the declines, millions of Americans remain potentially at risk of adverse outcomes associated with low-level Cd exposure.

Carbonaceous aerosols emitted from light-duty vehicles operating on gasoline and ethanol fuel blends.

This study examines the chemical properties of carbonaceous aerosols emitted from three light-duty gasoline vehicles (LDVs) operating on gasoline (e0) and ethanol-gasoline fuel blends (e10 and e85). Vehicle road load simulations were performed on a chassis dynamometer using the three-phase LA-92 unified driving cycle (UDC). Effects of LDV operating conditions and ambient temperature (-7 and 24 °C) on particle-phase semivolatile organic compounds (SVOCs) and organic and elemental carbon (OC and EC) emissions were investigated. SVOC concentrations and OC and EC fractions were determined with thermal extraction-gas chromatography-mass spectrometry (TE-GC-MS) and thermal-optical analysis (TOA), respectively. LDV aerosol emissions were predominantly carbonaceous, and EC/PM (w/w) decreased linearly with increasing fuel ethanol content. TE-GC-MS analysis accounted for up to 4% of the fine particle (PM2.5) mass, showing the UDC phase-integrated sum of identified SVOC emissions ranging from 0.703 µg km(-1) to 18.8 µg km(-1). Generally, higher SVOC emissions were associated with low temperature (-7 °C) and engine ignition; mixed regression models suggest these emissions rate differences are significant. Use of e85 significantly reduced the emissions of lower molecular weight PAH. However, a reduction in higher molecular weight PAH entities in PM was not observed. Individual SVOC emissions from the Tier 2 LDVs and fuel technologies tested are substantially lower and distributed differently than those values populating the United States emissions inventories currently. Hence, this study is likely to influence future apportionment, climate, and air quality model predictions that rely on source combustion measurements of SVOCs in PM.

Assessing lead exposure in U.S. pregnant women using biological and residential measurements.

There is strong scientific evidence for multiple pathways of human exposure to lead (Pb) in residential settings, particularly for young children; however, less is known about maternal exposure during pregnancy and children's exposure during early lifestages. A robust, multi-faceted secondary analysis was conducted using data collected by the National Institute of Child Health and Human Development in the 2009-2014 National Children's Study Vanguard Studies. Descriptive statistics summarized Pb concentrations of maternal blood, maternal urine, and house dust vacuum samples collected during pregnancy and residence surface wipes collected both during pregnancy and six months post-partum. The maternal blood Pb level geometric mean was 0.44 µg/dL (n = 426), with no women having values ≥ 5 µg/dL; creatinine-adjusted maternal urinary Pb geometric mean was 0.43 µg/g (n = 366). These blood and urine concentrations are similar to those observed for females in the general U.S. population in the National Health and Nutrition Examination Survey 2010-2011 cycle. A modest correlation between maternal blood Pb and surface wipe measurements during pregnancy was observed (Spearman r = 0.35, p < 0.0001). Surface wipe Pb loadings obtained in mother's homes during pregnancy (n = 640) and from areas where children spent the most time at roughly 6 months of age (n = 99) ranged from 0.02 to 71.8 ng/cm2, with geometric means of 0.47 and 0.49 ng/cm2, respectively, which were relatively low compared to other national studies. Survey responses of demographic, lifestyle, and residence characteristics were assessed for associations with blood concentration and surface wipe loading. Demographic (e.g., race/ethnicity, income, education, marital status) and housing characteristics (e.g., year home built, paint condition, own or rent home, attached garage) were associated with both maternal blood and surface wipe loadings during pregnancy. The availability of residential environmental media and extensive survey data provided enhanced understanding of Pb exposure during pregnancy and early life.

Rehabilitation Using Mobile Health for Older Adults With Ischemic Heart Disease in the Home Setting (RESILIENT): Protocol for a Randomized Controlled Trial.

BACKGROUND: Participation in ambulatory cardiac rehabilitation remains low, especially among older adults. Although mobile health cardiac rehabilitation (mHealth-CR) provides a novel opportunity to deliver care, age-specific impairments may limit older adults' uptake, and efficacy data are currently lacking. OBJECTIVE: This study aims to describe the design of the rehabilitation using mobile health for older adults with ischemic heart disease in the home setting (RESILIENT) trial. METHODS: RESILIENT is a multicenter randomized clinical trial that is enrolling patients aged ≥65 years with ischemic heart disease in a 3:1 ratio to either an intervention (mHealth-CR) or control (usual care) arm, with a target sample size of 400 participants. mHealth-CR consists of a commercially available mobile health software platform coupled with weekly exercise therapist sessions to review progress and set new activity goals. The primary outcome is a change in functional mobility (6-minute walk distance), which is measured at baseline and 3 months. Secondary outcomes are health status, goal attainment, hospital readmission, and mortality. Among intervention participants, engagement with the mHealth-CR platform will be analyzed to understand the characteristics that determine different patterns of use (eg, persistent high engagement and declining engagement). RESULTS: As of December 2021, the RESILIENT trial had enrolled 116 participants. Enrollment is projected to continue until October 2023. The trial results are expected to be reported in 2024. CONCLUSIONS: The RESILIENT trial will generate important evidence about the efficacy of mHealth-CR among older adults in multiple domains and characteristics that determine the sustained use of mHealth-CR. These findings will help design future precision medicine approaches to mobile health implementation in older adults. This knowledge is especially important in light of the COVID-19 pandemic that has shifted much of health care to a remote, internet-based setting. TRIAL REGISTRATION: ClinicalTrials.gov NCT03978130; https://clinicaltrials.gov/ct2/show/NCT03978130. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/32163.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

Relationship between PM2.5 collected at residential outdoor locations and a central site.

Regression models are developed to describe the relationship between ambient PM2.5 (particulate matter [PM] < or = 2.5 microm in aerodynamic diameter) mass concentrations measured at a central-site monitor with those at residential outdoor monitors. Understanding the determinants and magnitude of variability and uncertainty in this relationship is critical for understanding personal exposures in the evaluation of epidemiological data. The repeated measures regression models presented here address temporal and spatial characteristics of data measured in the 2004-2007 Detroit Exposure and Aerosol Research Study, and they take into account missing data and other data features. The models incorporate turbulence kinetic energy and planetary boundary layer height, meteorological data that are not routinely considered in models that relate central-site concentrations to exposure to health effects. It was found that turbulence kinetic energy was highly statistically significant in explaining the relationship of PM2.5 measured at a particular stationary outdoor air monitoring site with PM2.5 measured outside nearby residences for the temporal coverage of the data.

Particle emissions from fused deposition modeling 3D printers: Evaluation and meta-analysis.

Fused deposition modeling (FDM) 3D printers, the most popular choice among home hobbyists, have been shown to release volatile organic chemicals (VOCs) and billions of airborne particles per minute, indicating the potential for consumer inhalation exposure and consequent health risks. Publications on FDM 3D printer emissions however, contain large heterogeneity of testing methods and analytical procedures making it difficult to reach overall conclusions for particle characteristics or particle number emission rates across the field. In this publication, data were collected over the printing time from 3D printer emission studies including particle count diameters (PCDs) (nanometers), particle number concentrations (PNCs) (particles/cm3), and particle number emission rates (PNERs) (particles min-1). Despite heterogeneity in methods, the majority of particles released were reported as ultrafine in size (i.e., <100 nm) indicating that using both acrylonitrile butadiene styrene (ABS) and poly-lactic acid (PLA) may present a risk of exposure to respirable particles. Mean PNC emitted in 3D printing tests ranged over several orders of magnitude across publications with overall means of 300,980 particles/cm3 for ABS and 65,482 particles/cm3 for PLA. Although mean PNC data were available from only 7 of the 16 papers reviewed, ABS resulted in greater particle numbers than PLA suggesting increased exposure to ultrafine particles. A linear mixed model was fitted for mean PNCs to further explore the impact of nozzle temperature and filament material. Finally, the PNER calculation method especially regarding losses, varied widely across studies, and directly impacted the PNERs reported. To strengthen direct comparability of results going forward, it is recommended that standard emissions testing protocols be developed for FDM 3D printers and particle influxes and losses be more uniformly calculated.

The Impact of Scaling Factor Variability on Risk-Relevant Pharmacokinetic Outcomes in Children: A Case Study Using Bromodichloromethane (BDCM).

Biotransformation rates extrapolated from in vitro data are used increasingly in human physiologically based pharmacokinetic (PBPK) models. This practice requires use of scaling factors, including microsomal content (mg of microsomal protein/g liver, MPPGL), enzyme specific content, and liver mass as a fraction of body weight (FVL). Previous analyses indicated that scaling factor variability impacts pharmacokinetic (PK) outcomes used in adult population dose-response studies. This analysis was extended to pediatric populations because large inter-individual differences in enzyme ontogeny likely would further contribute to scaling factor variability. An adult bromodichloromethane (BDCM) model (Kenyon, E. M., Eklund, C., Leavens, T. L., and Pegram, R. A. (2016a). Development and application of a human PBPK model for bromodichloromethane (BDCM) to investigate impacts of multi-route exposure. J. Appl. Toxicol. 36, 1095-1111) was re-parameterized for neonates, infants, and toddlers. Monte Carlo analysis was used to assess the impact of pediatric scaling factor variation on model-derived PK outcomes compared with adult findings. BDCM dose metrics were estimated following a single 0.05-liter drink of water or a 20-min bath, under typical (5 µg/l) and plausible higher (20 µg/l) BDCM concentrations. MPPGL, CYP2E1, and FVL values reflected the distribution of reported pediatric population values. The impact of scaling factor variability on PK outcome variation was different for each exposure scenario, but similar for each BDCM water concentration. The higher CYP2E1 expression variability during early childhood was reflected in greater variability in predicted PK outcomes in younger age groups, particularly for the oral exposure route. Sensitivity analysis confirmed the most influential parameter for this variability was CYP2E1, particularly in neonates. These findings demonstrate the importance of age-dependent scaling factor variation used for in vitro to in vivo extrapolation of biotransformation rates.

Moderate perinatal thyroid hormone insufficiency alters visual system function in adult rats.

Thyroid hormone (TH) is critical for many aspects of neurodevelopment and can be disrupted by a variety of environmental contaminants. Sensory systems, including audition and vision are vulnerable to TH insufficiencies, but little data are available on visual system development at less than severe levels of TH deprivation. The goal of the current experiments was to explore dose-response relations between graded levels of TH insufficiency during development and the visual function of adult offspring. Pregnant Long Evans rats received 0 or 3 ppm (Experiment 1), or 0, 1, 2, or 3 ppm (Experiment 2) of propylthiouracil (PTU), an inhibitor of thyroid hormone synthesis, in drinking water from gestation day (GD) 6 to postnatal day (PN) 21. Treatment with PTU caused dose-related reductions of serum T4, with recovery on termination of exposure, and euthyroidism by the time of visual function testing. Tests of retinal (electroretinograms; ERGs) and visual cortex (visual evoked potentials; VEPs) function were assessed in adult offspring. Dark-adapted ERG a-waves, reflecting rod photoreceptors, were increased in amplitude by PTU. Light-adapted green flicker ERGs, reflecting M-cone photoreceptors, were reduced by PTU exposure. UV-flicker ERGs, reflecting S-cones, were not altered. Pattern-elicited VEPs were significantly reduced by 2 and 3 ppm PTU across a range of stimulus contrast values. The slope of VEP amplitude-log contrast functions was reduced by PTU, suggesting impaired visual contrast gain. Visual contrast gain primarily reflects function of visual cortex, and is responsible for adjusting sensitivity of perceptual mechanisms in response to changing visual scenes. The results indicate that moderate levels of pre-and post-natal TH insufficiency led to alterations in visual function of adult rats, including both retinal and visual cortex sites of dysfunction.

Mutagenicity and Lung Toxicity of Smoldering vs. Flaming Emissions from Various Biomass Fuels: Implications for Health Effects from Wildland Fires.

BACKGROUND: The increasing size and frequency of wildland fires are leading to greater potential for cardiopulmonary disease and cancer in exposed populations; however, little is known about how the types of fuel and combustion phases affect these adverse outcomes. OBJECTIVES: We evaluated the mutagenicity and lung toxicity of particulate matter (PM) from flaming vs. smoldering phases of five biomass fuels, and compared results by equal mass or emission factors (EFs) derived from amount of fuel consumed. METHODS: A quartz-tube furnace coupled to a multistage cryotrap was employed to collect smoke condensate from flaming and smoldering combustion of red oak, peat, pine needles, pine, and eucalyptus. Samples were analyzed chemically and assessed for acute lung toxicity in mice and mutagenicity in Salmonella. RESULTS: The average combustion efficiency was 73 and 98% for the smoldering and flaming phases, respectively. On an equal mass basis, PM from eucalyptus and peat burned under flaming conditions induced significant lung toxicity potencies (neutrophil/mass of PM) compared to smoldering PM, whereas high levels of mutagenicity potencies were observed for flaming pine and peat PM compared to smoldering PM. When effects were adjusted for EF, the smoldering eucalyptus PM had the highest lung toxicity EF (neutrophil/mass of fuel burned), whereas smoldering pine and pine needles had the highest mutagenicity EF. These latter values were approximately 5, 10, and 30 times greater than those reported for open burning of agricultural plastic, woodburning cookstoves, and some municipal waste combustors, respectively. CONCLUSIONS: PM from different fuels and combustion phases have appreciable differences in lung toxic and mutagenic potency, and on a mass basis, flaming samples are more active, whereas smoldering samples have greater effect when EFs are taken into account. Knowledge of the differential toxicity of biomass emissions will contribute to more accurate hazard assessment of biomass smoke exposures. https://doi.org/10.1289/EHP2200.

Chemical-agnostic hazard prediction: statistical inference of in vitro toxicity pathways from proteomics responses to chemical mixtures.

Toxicity pathways have been defined as normal cellular pathways that, when sufficiently perturbed as a consequence of chemical exposure, lead to an adverse outcome. If an exposure alters one or more normal biological pathways to an extent that leads to an adverse toxicity outcome, a significant correlation must exist between the exposure, the extent of pathway alteration, and the degree of adverse outcome. Biological pathways are regulated at multiple levels, including transcriptional, post-transcriptional, post-translational, and targeted degradation, each of which can affect the levels and extents of modification of proteins involved in the pathways. Significant alterations of toxicity pathways resulting from changes in regulation at any of these levels therefore are likely to be detectable as alterations in the proteome. We hypothesize that significant correlations between exposures, adverse outcomes, and changes in the proteome have the potential to identify putative toxicity pathways, facilitating selection of candidate targets for high throughput screening, even in the absence of a priori knowledge of either the specific pathways involved or the specific agents inducing the pathway alterations. We explored this hypothesis in vitro in BEAS-2B human airway epithelial cells exposed to different concentrations of Ni2+, Cd2+, and Cr6+, alone and in defined mixtures. Levels and phosphorylation status of a variety of signaling pathway proteins and cytokines were measured after 48 hours exposure, together with cytotoxicity. Least Absolute Shrinkage and Selection Operator (LASSO) multiple regression was used to identify a subset of these proteins that constitute a putative toxicity pathway capable of predicting cytotoxicity. The putative toxicity pathway for cytotoxicity of these metals and metal mixtures identified by LASSO is composed of phospho-RPS6KB1, phospho-p53, cleaved CASP3, phospho-MAPK8, IL-10, and Hif-1α. As this approach does not depend on knowledge of the chemical composition of the mixtures, it may be generally useful for identifying sets of proteins predictive of adverse effects for a variety of mixtures, including complex environmental mixtures of unknown composition.

Temperature and Driving Cycle Significantly Affect Carbonaceous Gas and Particle Matter Emissions from Diesel Trucks.

The present study examines the effects of fuel [an ultralow sulfur diesel (ULSD) versus a 20% v/v soy-based biodiesel-80% v/v petroleum blend (B20)], temperature, load, vehicle, driving cycle, and active regeneration technology on gas- and particle-phase carbon emissions from light and medium heavy-duty diesel vehicles (L/MHDDV). The study is performed using chassis dynamometer facilities that support low-temperature operation (-6.7 °C versus 21.7 °C) and heavy loads up to 12 000 kg. Organic and elemental carbon (OC-EC) composition of aerosol particles is determined using a thermal-optical technique. Gas- and particle-phase semivolatile organic compound (SVOC) emissions collected using traditional filter and polyurethane foam sampling media are analyzed using advanced gas chromatograpy/mass spectrometry methods. Study-wide OC and EC emissions are 0.735 and 0.733 mg/km, on average. The emissions factors for diesel vehicles vary widely, and use of a catalyzed diesel particle filter (CDPF) device generally mutes the carbon particle emissions in the exhaust, which contains ~90% w/w gas-phase matter. Interestingly, replacing ULSD with B20 did not significantly influence SVOC emissions, for which sums range from 0.030 to 9.4 mg/km for the L/MHDDVs. However, both low temperature and vehicle cold-starts significantly increase SVOCs in the exhaust. Real-time particle measurements indicate vehicle regeneration technology did influence emissions, although regeneration effects went unresolved using bulk chemistry techniques. A multistudy comparison of the toxic particle-phase polycyclic aromatic hydrocarbons (PAHs; molecular weight (MW) ≥ 252 amu) in diesel exhaust indicates emission factors that span up to 8 orders of magnitude over the past several decades. This study observes conditions under which PAH compounds with MW ≥ 252 amu appear in diesel particles downstream of the CDPF and can even reach low-end concentrations reported earlier for much larger HDDVs with poorly controlled exhaust streams. This rare observation suggests that analysis of PAHs in particles emitted from modern L/MHDDVs may be more complex than recognized previously.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.