Despite epidemiological links between rare earth elements (REEs) exposure and thyroid dysfunction, health risks of chronic, low-dose mixture exposure in populations remain unknown. We exposed human thyroid follicular epithelial cells Nthy-ori 3-1 to a biomimetic 14-REE mixture, encompassing light (La, Ce, Pr, Nd, Sm, and Eu) and heavy (Gd, Tb, Dy, Ho, Yb, Lu, Sc, and Y) elements, at concentrations reflecting human internal exposure from 1× to 1000× serum background concentration (SBC). REEs significantly inhibited triiodothyronine (T3) secretion without affecting thyroxine (T4). This disruption was associated with dysregulation of the dual oxidase 1 (DUOX1)/catalase (CAT)-mediated H₂O₂ redox system involved in thyroid hormone synthesis, together with impaired deiodinase-mediated T4-to-T3 conversion. Upregulation of DUOX1 and thyroid peroxidase (TPO), alongside NRF2-mediated defense activation, was significantly initiated at the 1× SBC level, indicating high sensitivity to background environmental concentrations. REE-induced oxidative stress triggered mitochondrial damage and ATP depletion, which may be mechanistically linked to the impaired deiodinase-mediated T4-to-T3 conversion. Antioxidant intervention effectively restored mitochondrial integrity, ATP levels, and deiodinase activities, thereby rescuing T3 secretion. This study revealed a thyrotoxic mechanism of REEs under realistic exposure scenarios, providing critical evidence for assessing their environmental health risks.

Obesity, a significant global health issue, heightens the risk of non-alcoholic fatty liver disease (NAFLD). Its interaction with environmental pollutants might exacerbate NAFLD's severity. Haloacetamides (HAcAms), a group of emerging nitrogenous disinfection byproducts (DBPs) and potent oxidative stressors, are found in chlorinated drinking water. Since oxidative stress is associated with HAcAms-DBP cytotoxicity and a key factor in NAFLD pathogenesis, we hypothesize that HAcAms-DBPs could exacerbate liver injury and NAFLD, particularly with high-fat diets. This study examined HAcAms-DBPs' impact on liver lipid metabolism in mice treated with 1 to 100 times the background drinking water level (13.05 µg/L) for up to 16 weeks of oral administration. Compared to a high-fat-only group, mice co-exposed to a high-fat diet and HAcAms-DBPs for 16 weeks had elevated serum alanine transaminase, aspartate transaminase, triglyceride, hepatic lipid aggregation, and inflammation response. Under high-fat conditions, background drinking water levels of HAcAms significantly upregulated liver Acetyl-CoA carboxylase 1, fatty acid synthase, peroxisome proliferator-activated receptor gamma (PPARγ), PPARγ coactivator-1α, glucose transporter 1 and 4 protein expression in C57BL/6J mice; 10 times background significantly increased expression of inflammatory marker tumor necrosis factor and liver fibrosis marker protein alpha-smooth muscle actin; 100 times further increased both liver damage and markers of early non-alcoholic steatohepatitis phenotypes like steatosis and lobular inflammation. HAcAms-DBPs plus high-fat conditions worsened liver damage. The possible health risks of NAFLD induced by HAcAms in obese individuals deserve further study.

Unhealthy lifestyles, obesity, and environmental pollutants are strongly correlated with the development of nonalcoholic fatty liver disease (NAFLD). Haloacetaldehyde-associated disinfection byproducts (HAL-DBPs) at various multiples of concentrations found in finished drinking water together with high-fat (HF) were examined to gauge their mixed effects on hepatic lipid metabolism. Using new alternative methods (NAMs), studying effects in human cells in vitro for risk assessment, we investigated the combined effects of HF and HAL-DBPs on hepatic lipid metabolism and lipotoxicity in immortalized LO-2 human hepatocytes. Coexposure of HAL-DBPs at various multiples of environmental exposure levels with HF increased the levels of triglycerides, interfered with de novo lipogenesis, enhanced fatty acid oxidation, and inhibited the secretion of very low-density lipoproteins. Lipid accumulation caused by the coexposure of HAL-DBPs and HF also resulted in more severe lipotoxicity in these cells. Our results using an in vitro NAM-based method provide novel insights into metabolic reprogramming in hepatocytes due to coexposure of HF and HAL-DBPs and strongly suggest that the risk of NAFLD in sensitive populations due to HAL-DBPs and poor lifestyle deserves further investigation both with laboratory and epidemiological tools. We also discuss how results from our studies could be used in health risk assessments for HAL-DBPs.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by abnormal lipid deposition, with oxidative stress being a risk factor in its onset and progression. Haloacetamides (HAcAms), as unregulated disinfection by-products in drinking water, may alter the incidence and severity of NAFLD through the production of oxidative stress. We explored whether HAcAms at 1, 10, and 100-fold concentrations in Shanghai drinking water perturbed lipid metabolism in normal human liver LO-2 cells. CRISPR/Cas9 was used to construct a LO-2 line with stable NRF2 knock-down (NRF2-KD) to investigate the mechanism underlying abnormal lipid accumulation and hepatocyte damage caused by mixed exposure to HAcAms. At 100-fold real-world concentration, HAcAms caused lipid deposition and increased triglyceride accumulation in LO-2 cells, consistent with altered de novo lipogenesis. Differences in responses to HAcAms in normal and NRF2-KD LO-2 cells indicated that HAcAms caused hepatocyte lipid deposition and triglyceride accumulation by activation of the NRF2/PPARγ pathway and aggravated liver cell toxicity by inducing ferroptosis. These results indicate that HAcAms are important risk factors for NAFLD. Further observations and verifications of the effect of HAcAms on NAFLD in the population are warranted in the future.

To identify pathway perturbations and examine biological modes of action (MOAs) for various perfluoroalkyl substances, we re-analyzed published in vitro gene expression studies from human primary liver spheroids. With treatment times ranging from 10 to 14 days, shorter-chain PFAS (those with 6 or fewer fluorinated carbon atoms in the alkyl chain) showed enrichment for pathways of fatty acid metabolism and fatty acid beta-oxidation with upregulated genes. Longer-chain PFAS compounds, specifically PFOS (perfluorooctane sulfonate), PFDS (perfluorodecane sulfonate), and higher doses of PFOA (perfluorooctanoic acid), had enrichment for pathways involved in steroid metabolism, fatty acid metabolism, and biological oxidation for downregulated genes. Although PFNA (perfluorononanoic acid), PFDA (perfluorodecanoic acid), and PFUnDA (perfluoroundecanoic acid) were more toxic and could only be examined after a 1-day treatment, all three had enrichment patterns similar to those observed with PFOS. With PFOA there were dose-dependent changes in pathway enrichment, shifting from upregulation of fatty acid metabolism and downregulation of steroid metabolism to downregulation of both at higher doses. The response to PFHpS (perfluoroheptanesulfonic acid) was similar to the PFOA pattern at the lower treatment dose. Based on results of transcription factor binding sites analyses, we propose that downregulation of pathways of lipid metabolism by longer chain PFAS may be due to inhibitory interactions of PPARD on genes controlled by PPARA and PPARG. In conclusion, our transcriptomic analysis indicates that the biological MOAs of PFAS compounds differ according to chain length and dose, and that risk assessments for PFAS should consider these differences in biological MOAs when evaluating mixtures of these compounds.

Single, high doses of TCDD in rats caused wasting, a progressive loss of 30 to 50% body weight and death within several weeks. To identify pathway perturbations at or near doses causing wasting, we examined differentially gene expression (DGE) and pathway enrichment in centrilobular (CL) and periportal (PP) regions of female rat livers following 6 dose levels of TCDD – 0, 3, 22, 100, 300, and 1000 ng/kg/day, 5 days/week for 4 weeks. At the higher doses, rats lost weight, had increased liver/body weight ratios and nearly complete cessation of liver cell proliferation, signs consistent with wasting. DGE curves were left shifted for the CL versus the PP regions. Canonical Phase I and Phase II genes were maximally increased at lower doses and remained elevated at all doses. At lower doses, < 22 ng/kg/day in the CL and < 100 ng/kg/day, upregulated genes showed transcription factor (TF) enrichment for AHR and ARNT. At the mid- and hi-dose doses, there was a large number of downregulated genes and pathway enrichment for DEGs showed downregulation of many cellular metabolism processes including those for steroids, fatty acid metabolism, pyruvate metabolism and citric acid cycle. There was significant TF enrichment of the hi-dose downregulated genes for RXR, ESR1, LXR, PPARalpha. At the highest dose, there was also pathway enrichment with upregulated genes for extracellular matrix organization, collagen formation, hemostasis and innate immune system. TCDD demonstrates most of its effects through binding the aryl hydrocarbon receptor (AHR) while the downregulation of metabolism genes at higher TCDD doses is known to be independent of AHR binding to DREs. Based on our results with DEG, we provide a hypothesis for wasting in which high doses of TCDD shifts circadian processes away from the resting state leading to greatly reduced synthesis of steroids and complex lipids needed for cell growth and producing gene expression signals consistent with an epithelial-to-mesenchymal transition in hepatocytes.### Competing Interest StatementDuring the course of this study, M.E.A., A.R.B. and M.B.B. has been employed by ScitoVation LLC, and J.A.H. has been employed by the EPA.

With the use of in vitro new approach methodologies (NAMs) for the assessment of non-combustible next-generation nicotine delivery products, new extrapolation methods will also be required to interpret and contextualize the physiological relevance of these results. Quantitative in vitro to in vivo extrapolation (QIVIVE) can translate in vitro concentrations into in-life exposures with physiologically-based pharmacokinetic (PBPK) modelling and provide estimates of the likelihood of harmful effects from expected exposures. A major challenge for evaluating inhalation toxicology is an accurate assessment of the delivered dose to the surface of the cells and the internalized dose. To estimate this, we ran the multiple-path particle dosimetry (MPPD) model to characterize particle deposition in the respiratory tract and developed a PBPK model for nicotine that was validated with human clinical trial data for cigarettes. Finally, we estimated a Human Equivalent Concentration (HEC) and predicted plasma concentrations based on the minimum effective concentration (MEC) derived after acute exposure of BEAS-2B cells to cigarette smoke (1R6F), or heated tobacco product (HTP) aerosol at the air liquid interface (ALI). The MPPD-PBPK model predicted the in vivo data from clinical studies within a factor of two, indicating good agreement as noted by WHO International Programme on Chemical Safety (2010) guidance. We then used QIVIVE to derive the exposure concentration (HEC) that matched the estimated in vitro deposition point of departure (POD) (MEC cigarette = 0.38 puffs or 11.6 µg nicotine, HTP = 22.9 puffs or 125.6 µg nicotine) and subsequently derived the equivalent human plasma concentrations. Results indicate that for the 1R6F cigarette, inhaling 1/6th of a stick would be required to induce the same effects observed in vitro, in vivo. Whereas, for HTP it would be necessary to consume 3 sticks simultaneously to induce in vivo the effects observed in vitro. This data further demonstrates the reduced physiological potency potential of HTP aerosol compared to cigarette smoke. The QIVIVE approach demonstrates great promise in assisting human health risk assessments, however, further optimization and standardization are required for the substantiation of a meaningful contribution to tobacco harm reduction by alternative nicotine delivery products.

Single, high doses of TCDD in rats are known to cause wasting, a progressive loss of 30 to 50% body weight and death within several weeks. To identify pathway perturbations at or near doses causing wasting, we examined differentially gene expression (DGE) and pathway enrichment in centrilobular (CL) and periportal (PP) regions of female rat livers following 6 dose levels of TCDD - 0, 3, 22, 100, 300, and 1000 ng/kg/day, 5 days/week for 4 weeks. At the higher doses, rats lost weight, had increased liver/body weight ratios and nearly complete cessation of liver cell proliferation, signs consistent with wasting. DGE curves were left shifted for the CL versus the PP regions. Canonical Phase I and Phase II genes were maximally increased at lower doses and remained elevated at all doses. At lower doses, ≤ 22 ng/kg/day in the CL and ≤ 100 ng/kg/day, upregulated genes showed transcription factor (TF) enrichment for AHR and ARNT. At the mid- and high-dose doses, there was a large number of downregulated genes and pathway enrichment for DEGs which showed downregulation of many cellular metabolism processes including those for steroids, fatty acid metabolism, pyruvate metabolism and citric acid cycle. There was significant TF enrichment of the hi-dose downregulated genes for RXR, ESR1, LXR, PPARalpha. At the highest dose, there was also pathway enrichment with upregulated genes for extracellular matrix organization, collagen formation, hemostasis and innate immune system. TCDD demonstrates most of its effects through binding the aryl hydrocarbon receptor (AHR) while the downregulation of metabolism genes at higher TCDD doses is known to be independent of AHR binding to DREs. Based on our results with DEG, we provide a hypothesis for wasting in which high doses of TCDD shift circadian processes away from the resting state, leading to greatly reduced synthesis of steroids and complex lipids needed for cell growth, and producing gene expression signals consistent with an epithelial-to-mesenchymal transition in hepatocytes.

Single doses of perfluoro-n-decanoic acid (PFDA) cause wasting, a progressive loss of 30 to 50% body weight, increasing liver/body weight ratios, and death within several weeks ([Olson and Andersen, 1983][1]). Repeat high doses of perfluorooctane sulfonate (PFOS) produce a subset of these responses in rats and monkeys. The mode of action (MOA) of these wasting-like syndromes is not clear, nor is it understood if these responses are limited to a subset of perfluoroacid substances (PFAS) or a common response to high dose exposure with a larger number of PFAS. To identify pathway perturbations in liver caused by PFAS, we analyzed published in vitro gene expression studies from human primary liver spheroids treated with various PFAS for treatment times up to 14 days ([Rowan-Carroll et al ., 2021][2]). With treatment times of 10 to 14 days, longer-chain PFAS compounds, specifically PFOS, perfluorodecane sulfonate (PFDS) and higher doses of perfluorooctanoic acid (PFOA), downregulated large numbers of genes in pathways for steroid metabolism, fatty acid metabolism and biological oxidations. Shorter chain PFAS compounds upregulated genes in pathways for fatty acid metabolism. Although PFDA was more toxic and could only be examined at 1-day of treatment, it also downregulated genes for lipid metabolism, steroid metabolism, and biological oxidations. Shorter chain PFAS, both carboxylic and sulfonic acids, did not lead to downregulation of pathways for fatty acid or steroid metabolism. TCDD is also known to cause wasting responses in rodents and humans. In intact rats, high dose responses of longer chain PFAS produce downregulation of batteries of genes associated with fatty acid oxidation and lipogenesis similar to those seen with TCDD. Based on our results, when combined with other literature, we propose that the longer-chain PFAS impair lipogenic pathways through inhibitory interactions between PPARβ, PPARα and PPARγ. ### Competing Interest Statement During the course of this study, A.R.B., M.B. and M.E.A. has been employed by ScitoVation, LLC. [1]: #ref-25 [2]: #ref-30

Human exposures to rare earth elements are increasing with expanded use in aerospace, precision instruments, and new energy batteries, materials, and fertilizers. Individually these elements have low toxicity, although few investigations have examined the health effects of longer-term mixture exposures. We used the LO-2 cell line to examine the effects of graded exposures to lanthanum, cerium, and yttrium (LCY) mixtures at 1-, 100-, and 1000-fold their human background levels (0.31 mu g/L La, 0.25 mu g/L Ce, and 0.12 mu g/L Y) on cell cycle, oxidative stress, and nuclear factor erythroid-2-related factor (NRF2) pathway biomarkers, assessing responses every 10 passages up to 100 passages. Cell migration, concanavalin A, malignant transformation, and tumorigenesis in nude mice were also examined. Mixed LCY exposures activated oxidative stress and the NRF2 pathway by the 30th passage and increased the proportion of cells in the S phase and cell cycle-specific biomarkers by the 40th passage. LCY exposures did not cause malignant transformation of hepatocytes or induced tumorigenesis in nude mice but enhanced cell proliferation, migration, and agglutination. Importantly, LCY mixtures with longer-term exposure activated the NRF2 pathway and altered the hepatocyte cell cycle at doses far below those used in previous toxicological studies. The consequences of LCY mixtures for public health merit further study.

Abstract The aryl hydrocarbon receptor (AhR) is an inducible transcription factor whose ligands include the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand-activated AhR binds to DNA at dioxin response elements (DREs) containing the core motif 5′-GCGTG-3′. However, AhR binding is highly tissue specific. Most DREs in accessible chromatin are not bound by TCDD-activated AhR, and DREs accessible in multiple tissues can be bound in some and unbound in others. As such, AhR functions similarly to many nuclear receptors. Given that AhR possesses a strong core motif, it is suited for a motif-centered analysis of its binding. We developed interpretable machine learning models predicting the AhR binding status of DREs in MCF-7, GM17212, and HepG2 cells, as well as primary human hepatocytes. Cross-tissue models predicting transcription factor (TF)-DNA binding generally perform poorly. However, reasons for the low performance remain unexplored. By interpreting the results of individual within-tissue models and by examining the features leading to low cross-tissue performance, we identified sequence and chromatin context patterns correlated with AhR binding. We conclude that AhR binding is driven by a complex interplay of tissue-agnostic DRE flanking DNA sequence and tissue-specific local chromatin context. Additionally, we demonstrate that interpretable machine learning models can provide novel and experimentally testable mechanistic insights into DNA binding by inducible TFs.

Chronic inhalation of formaldehyde by F344 rats causes nasal squamous cell carcinoma (SCC). This outcome is well-characterized: including dose-response and time course data for SCC, mechanistic endpoints, and nasal dosimetry. Conolly et al. (Toxicol. Sci. 75, 432-447, 2003) used these resources to develop a biologically based dose-response (BBDR) model for SCC in F344 rats. This model, scaled up to humans, has informed dose-response conclusions reached by several international regulatory agencies. However, USEPA concluded that uncertainties precluded its use for cancer risk assessment. Here, we describe an updated BBDR model that addresses uncertainties through refined dosimetry modeling, revised analysis of labeling index data, and an extended dataset where both inhaled (exogenous) and endogenous formaldehyde (exogF, endoF) form DNA adducts. Further, since Conolly et al. (ibid) was published, it has become clear that, when controls from all F344 inhalation bioassays are considered, accounting for over 4000 rats, at most one nasal SCC occurred. This low spontaneous incidence constrains possible contribution of endoF to the formation of nasal SCC via DNA reactivity. Further, since both exogF and endoF form DNA adducts, this constraint also applies to exogF. The revised BBDR model therefore drives SCC formation through the cytotoxicity of high concentration exogF. An option for direct mutagenicity associated with DNA adducts is retained to allow estimation of an upper bound on adduct mutagenicity consistent with the lack of a spontaneous SCC incidence. These updates represent an iterative refinement of the 2003 model, incorporating new data and insights to reduce identified model uncertainties.

In earlier physiologically based pharmacokinetic (PBPK) models for manganese (Mn), the kinetics of transport of Mn into and out of tissues were primarily driven by slow rates of association and dissociation of Mn with tissue binding sites. However, Mn is known to show rapidly reversible binding in tissues. An updated Mn model for primates, following similar work with rats, was developed that included rapid association/dissociation processes with tissue Mn-binding sites, accumulation of free Mn in tissues after saturation of these Mn-binding sites and rapid rates of entry into tissues. This alternative structure successfully described Mn kinetics in tissues in monkeys exposed to Mn via various routes including oral, inhalation, and intraperitoneal, subcutaneous, or intravenous injection and whole-body kinetics and tissue levels in humans. An important contribution of this effort is showing that the extension of the rate constants for binding and cellular uptake established in the monkey were also able to describe kinetic data from humans. With a consistent model structure for monkeys and humans, there is less need to rely on cadaver data and whole-body tracer studies alone to calibrate a human model. The increased biological relevance of the Mn model structure and parameters provides greater confidence in applying the Mn PBPK models to risk assessment. This model is also well-suited to explicitly incorporate emerging information on the role of transporters in tissue disposition, intestinal uptake, and hepatobiliary excretion of Mn.

The most recent version of the octamethylcyclotetrasiloxane (D4) physiologically based pharmacokinetic (model) was developed using the available kinetic studies in male and female F344 rats. Additional data, which had not been included in the D4 model development, allowed for a more detailed assessment of the loss of D4 following long-term exposure in both SD and F344 rats. This new data demonstrated a deficiency in the published PBPK model predictions of terminal concentrations of D4 in plasma and fat 14 days after the end of exposures for 28 -days, 6 h/day, where the model predictions were an order of magnitude lower than the data. To capture this time-point without altering the end-of-exposure peak concentrations in blood and fat required conversion of the one-way (liver to fat) mobile lipoprotein pool (MLP) into a bi-directional pool between liver and fat. Simulation of the D4 pharmacokinetics in the SD rat, as opposed to the F344 rat, also required a reduction of both fold induction of liver metabolism (KMAX: 5-to 2-fold) and maximal rate of metabolism (VMAXC: 5.0-1.54 mg/ kg0.75). The revised MLP description was extended to the human D4 model using a parallelogram approach between rat and human MLP parameters to establish the parameters for the current model in the absence of similar long-term clearance data in the human. The revised human D4 model provided good fits to the human inhalation and dermal exposure studies while not appreciably altering cross-species dose metrics based on the free concentration of D4 in blood.

Introduction: ß-chloroprene (2-chloro-1,3-butadiene; CP) causes lung tumors after inhalation exposures in rats and mice. Mice develop these tumors at lower exposures than rats. In rats CP exposures cause depletion of lung glutathione (GSH). Methods: PBPK models developed to relate the appearance of mouse lung tumors with rates of CP metabolism to reactive metabolites or total amounts metabolized during exposures have been expanded to include production of reactive metabolites from CP. The extended PBPK model describes both the unstable oxirane metabolite, 2-CEO, and metabolism of the more stable oxirane, 1-CEO, to reactive metabolites via microsomal oxidation to a diepoxide, and linked production of these metabolites to a PK model predicting GSH depletion with increasing CP exposure. Key information required to develop the model were available from literature studies identifying: 1) microsomal metabolites of CP, and 2) in vitro rates of clearance of CP and 1-CEO from active microsomal preparations from mice, rats, hamsters and humans. Results: Model simulation of concentration dependence of disproportionate increases in reactive metabolite concentrations as exposures increases and decreases in tissue GSH are consistent with the dose-dependence of tumor formation. At the middle bioassay concentrations with a lung tumor incidence, the predicted tissue GSH is less than 50% background. These simulations of reduction in GSH are also consistent with the gene expression results showing the most sensitive pathways are Nrf2-regulation of oxidative stress and GSH metabolism. Discussion: The PBPK model is used to correlate predicted tissue exposure to reactive metabolites with toxicity and carcinogenicity of CP.

Liver responses are the most common endpoints used as the basis for setting exposure standards. Liver hepatocytes play a vital role in biotransformation of xenobiotics, but non-parenchymal cells (NPCs) in the liver are also involved in certain liver responses. Development of in vitro systems that more faithfully capture liver responses to reduce reliance on animals is a major focus of New Approach Methodology (NAMs). Since rodent regulatory studies are frequently the sole source safety assessment data, mode-of-action data, and used for risk assessments, in vitro rodent models that reflect in vivo responses need to be developed to reduce reliance on animal models. In the work presented in this paper, we developed a 2-D hepatocyte monoculture and 2-D liver cell co-culture system using rat liver cells. These models were assessed for conditions for short-term stability of the cultures and phenotypic and transcriptomic responses of 2 prototypic hepatotoxicants compounds - acetaminophen and phenobarbital. The optimized multi-cellular 2-D culture required use of freshly prepared hepatocytes and NPCs from a single rat, a 3:1 ratio of hepatocytes to NPCs and growth medium using 50% Complete Williams E medium (WEM) and 50% Endothelial Cell Medium (ECM). The transcriptomic responses of the 2 model systems to PB were compared to previous studies from TG-Gates on the gene expression changes in intact rats and the co-culture model responses were more representative of the in vivo responses. Transcriptomic read-outs promise to move beyond conventional phenotypic evaluations with these in vitro NAMs and provide insights about modes of action.

Comprehensive enrichment of contaminants in drinking water is an essential step for accurately determining exposure levels of contaminants and testing their biological effects. Traditional methods using a single absorbent for enriching contaminants in water might not be adequate for complicated matrices with different physical-chemical profiles. To examine this hypothesis, we used an integrated enrichment system that had three sequential stages-XAD-2 resin, poly (styrene-divinylbenzene) and activated charcoal to capture organic pollutants and disinfection by-products (DBPs) from drinking water in Shanghai. Un-adsorbed Organic Compounds in Eluates (UOCEs) named UOCEs-A, -B, and-C following each adsorption stage were determined by gas chromatography-mass spectrometry to evaluate adsorption efficiency of the enrichment system. Meanwhile, biological effects such as cytotoxicity, effects on reactive oxygen species (ROS) generation and glutathione (GSH) depletion were determined in human LO2 cells to identify potential adverse effects on exposure to low dose contaminants. We found that poly-styrene-divinylbenzene (PS-DVB) and activated charcoal (AC) could still partly collect UOCEs-A and-B that the upper adsorption column incompletely captured, and that potential carcinogens like 2-naphthamine were present in all eluates. UOCEs-A at (1-4000), UOCEs-B at (1000-4000), and UOCEs-C at (2400-4000) folds of the actual concentrations had significant cytotoxicity to LO2 cells. Additionally, ROS and GSH change in cells treated with UOCEs indicated the potential for long-term effects of exposure to some mixtures of contaminants such as DBPs at low doses. These results suggested that an enriching system with a single adsorbent would underestimate the exposure level of pollutants and the biological effects of organic pollutants from drinking water. Effective methods for pollutants' enrichment and capture of drinking water should be given priority in future studies on accurate evaluation of biological effects exposed to mixed pollutants via drinking water.

Current computational technologies hold promise for prioritizing the testing of the thousands of chemicals in commerce. Here, a case study is presented demonstrating comparative risk-prioritization approaches based on the ratio of surrogate hazard and exposure data, called margins of exposure (MoEs). Exposures were estimated using a U.S. EPA's ExpoCast predictive model (SEEM3) results and estimates of bioactivity were predicted using: 1) Oral equivalent doses (OEDs) derived from U.S. EPA's ToxCast high-throughput screening program, together with in vitro to in vivo extrapolation and 2) thresholds of toxicological concern (TTCs) determined using a structure-based decision-tree using the Toxtree open source software. To ground-truth these computational approaches, we compared the MoEs based on predicted noncancer TTC and OED values to those derived using the traditional method of deriving points of departure from no-observed adverse effect levels (NOAELs) from in vivo oral exposures in rodents. TTC-based MoEs were lower than NOAEL-based MoEs for 520 out of 522 (99.6%) compounds in this smaller overlapping dataset, but were relatively well correlated with the same (r2 = 0.59). TTC-based MoEs were also lower than OED-based MoEs for 590 (83.2%) of the 709 evaluated chemicals, indicating that TTCs may serve as a conservative surrogate in the absence of chemical-specific experimental data. The TTC-based MoE prioritization process was then applied to over 45,000 curated environmental chemical structures as a proof-of-concept for high-throughput prioritization using TTC-based MoEs. This study demonstrates the utility of exploiting existing computational methods at the pre-assessment phase of a tiered risk-based approach to quickly, and conservatively, prioritize thousands of untested chemicals for further study.

Octamethylcyclotetrasiloxane (D4), a highly lipophilic, volatile compound with low water solubility, is metabolized to lower molecular weight, linear silanols. Toxicity has been documented in several tissues in animals following mixed vapor/aerosol exposures by inhalation at near saturating vapor concentrations or with gavage dosing in vegetable oil vehicles. These results, together with more mechanism-based studies and detailed pharmacokinetic information, were used to assess likely modes of action (MOAs) and the tissue dose measures of D4 and metabolites that would serve as key events leading to these biological responses. This MOA analysis indicates that pulmonary effects arise from direct epithelial contact with mixed vapor/aerosol atmospheres of D4; liver hypertrophy and hepatocyte proliferation arise from adaptive, rodent-specific actions of D4 with nuclear receptor signaling pathways; and, nephropathy results from a combination of chronic progresive nephropathy and silanol metabolites binding with alpha-2u globulin (a male rat specific protein). At this time, the MOAs of other liver effects – pigment accumulation and bile duct hyperplasia (BDH) preferentially observed in Sprague-Dawley (SD) rats- are not known. Hypothalamic actions of D4 delaying the rat mid-cycle gonadotrophin releasing hormone (GnRH) surge that result in reproductive effects and subsequent vaginal/uterine/ovarian tissue responses, including small increases in incidence of benign endometrial adenomas, are associated with prolongation of endogenous estrogen exposures due to delays in ovulation. Human reproduction is not controlled by a mid-cycle GnRH surge. Since the rodent-specific reproductive and the vaginal/uterine/ovarian tissue responses are not relevant for risk assessments in human populations, D4 should neither be classified as a CMR (i.e., carcinogenic, mutagenic, or toxic for reproduction) substance nor be regarded as an endocrine disruptor. Bile duct hyperplasia (BDH) and pigment accumulation in liver seen in SD rats are endpoints that could serve to define a Benchmark Dose (BMD) or No-Observed-Effect-Level (NOEL) for D4 although their human relevance remains uncertain.