Silicone wristbands reveal ubiquitous human exposure to ortho-phthalates and non-ortho-phthalate plasticizers in Southern California.

In the United States and abroad, ortho-phthalates and non-ortho-phthalate plasticizers continue to be used within a diverse array of consumer products. Prior California-specific biomonitoring programs for ortho-phthalates have focused on rural, agricultural communities and, to our knowledge, these programs have not measured the potential for exposure to non-ortho-phthalate plasticizers. Therefore, the potential for human exposure to ortho-phthalates and non-ortho-phthalate plasticizers have not been adequately addressed in regions of California that have higher population density. Since there are numerous sources of ortho-phthalates and non-ortho-phthalate plasticizers in population-dense, urban regions, the objective of this study was to leverage silicone wristbands to quantify aggregate ortho-phthalate and non-ortho-phthalate plasticizer exposure over a 5-day period across two different cohorts (2019 and 2020) of undergraduate students at the University of California, Riverside (UCR) that commute from all over Southern California. Based on 5 d of aggregate exposure across two different cohorts, total ortho-phthalate plus non-ortho-phthalate plasticizer concentrations ranged, on average, from ∼100,000-1,000,000 ng/g. Based on the distribution of individual ortho-phthalate and non-ortho-phthalate plasticizer concentrations, the concentrations of di-isononyl phthalate (DiNP, a high molecular weight ortho-phthalate), di (2-ethylhexyl) phthalate (DEHP, a high molecular weight ortho-phthalate), and di-2-ethylhexyl terephthalate (DEHT, a non-ortho-phthalate plasticizer) detected within wristbands were higher than the remaining seven ortho-phthalates and non-ortho-phthalate plasticizers measured, accounting for approximately 94-97% of the total mass depending on the cohort. Overall, our findings raise concerns about chronic DiNP, DEHP, and DEHT exposure in urban, population-dense regions throughout California.

Tris(1,3-dichloro-2-propyl) phosphate disrupts cellular metabolism within human embryonic kidney (HEK293) cells.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used, additive flame retardant that migrates from end-use products, leading to ubiquitous exposure of humans around the world. However, little is known about whether TDCIPP disrupts the physiology of human embryonic cells. Therefore, the objective of this study was to determine whether TDCIPP alters cell viability, cellular metabolism, cytosine methylation, and reactive oxygen species (ROS) levels within human embryonic kidney (HEK293) cells. Relative to vehicle controls, TDCIPP (0.015-0.1225 µM) resulted in a concentration-dependent increase in cell viability, a finding that was driven by an increase in relative ATP abundance. Interestingly, TDCIPP (0.061-0.98 µM) increased the rate of glycolysis - an adaptive mechanism consistent with the Warburg effect exhibited by tumorigenic cells. Moreover, relative to vehicle-treated cells, TDCIPP (0.245-15.63 µM) exposure for 48 h (but not 24 h) resulted in a significant, concentration-dependent decrease in ROS in situ, and TDCIPP (0.245 µM) exposure significantly increased carnosine within the histidine metabolism pathway. However, TDCIPP did not affect global 5-methylcytosine (5-mC) methylation (0.015-15.63 µM), cell membrane integrity (0.061-0.98 µM), nor the abundance of mitochondria (0.061-1.95 µM). Overall, our findings with TDCIPP point to a novel mechanism of action that may be relevant to human embryonic stem cells.

Triphenyl phosphate-induced pericardial edema in zebrafish embryos is reversible following depuration in clean water.

Triphenyl phosphate (TPHP) - a widely used organophosphate-based flame retardant - blocks cardiac looping during zebrafish development in a concentration-dependent manner, a phenotype that is dependent on disruption of embryonic osmoregulation and pericardial edema formation. However, it's currently unclear whether (1) TPHP-induced effects on osmoregulation are driven by direct TPHP-induced injury to the embryonic epidermis and (2) whether TPHP-induced pericardial edema is reversible or irreversible following cessation of exposure. Therefore, the objectives of this study were to determine whether TPHP-induced pericardial edema is reversible and whether TPHP causes injury to the embryonic epidermis by quantifying the number of DAPI-positive epidermal cells and analyzing the morphology of the yolk sac epithelium using scanning electron microscopy. First, we found that exposure to 5 µM TPHP from 24-72 h post-fertilization (hpf) did not increase prolactin - a hormone that regulates ions and water levels - in embryonic zebrafish, whereas high ionic strength exposure media was associated with elevated levels of prolactin. Second, we found that exposure to 5 µM TPHP from 24-72 hpf did not decrease DAPI-positive epidermal cells within the embryonic epithelium, and that co-exposure with 2.14 µM fenretinide - a synthetic retinoid that promotes epithelial wound repair - from 24-72 hpf did not mitigate the prevalence of TPHP-induced epidermal folds within the yolk sac epithelium when embryos were exposed within high ionic strength exposure media. Finally, we found that the pericardial area and body length of embryos exposed to 5 µM TPHP from 24-72 hpf were similar to vehicle-treated embryos at 120 hpf following transfer to clean water and depuration of TPHP from 72-120 hpf. Overall, our findings suggest that (1) the ionic strength of exposure media may influence the baseline physiology of zebrafish embryos; (2) TPHP does not cause direct injury to the embryonic epidermis; and (3) TPHP-induced effects on pericardial area and body length are reversible 48 h after transferring embryos to clean water.

Triphenyl phosphate-induced pericardial edema in zebrafish embryos is dependent on the ionic strength of exposure media.

Pericardial edema is commonly observed in zebrafish embryo-based chemical toxicity screens, and a mechanism underlying edema may be disruption of embryonic osmoregulation. Therefore, the objective of this study was to identify whether triphenyl phosphate (TPHP) - a widely used aryl phosphate ester-based flame retardant - induces pericardial edema via impacts on osmoregulation within embryonic zebrafish. In addition to an increase in TPHP-induced microridges in the embryonic yolk sac epithelium, an increase in ionic strength of exposure media exacerbated TPHP-induced pericardial edema when embryos were exposed from 24 to 72 h post-fertilization (hpf). However, there was no difference in embryonic sodium concentrations in situ within TPHP-exposed embryos relative to embryos exposed to vehicle (0.1% DMSO) from 24 to 72 hpf. Interestingly, increasing the osmolarity of exposure media with mannitol (an osmotic diuretic which mitigates TPHP-induced pericardial edema) and increasing the ionic strength of the exposure media (which exacerbates TPHP-induced pericardial edema) did not affect embryonic doses of TPHP, suggesting that TPHP uptake was not altered under these varying experimental conditions. Overall, our findings suggest that TPHP-induced pericardial edema within zebrafish embryos is dependent on the ionic strength of exposure media, underscoring the importance of further standardization of exposure media and embryo rearing protocols in zebrafish-based chemical toxicity screening assays.

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

Embryonic Exposure to Cigarette Smoke Extract Impedes Skeletal Development and Evokes Craniofacial Defects in Zebrafish.

Exposure to cigarette smoke represents the largest source of preventable death and disease in the United States. This may be in part due to the nature of the delayed harmful effects as well as the lack of awareness of the scope of harm presented by these products. The presence of "light" versions further clouds the harmful effects of tobacco products. While active smoking in expectant mothers may be reduced by educational and outreach campaigns, exposure to secondhand smoke is often involuntary yet may harm the developing embryo. In this study, we show that the main component of secondhand smoke, sidestream cigarette smoke, from several brands, including harm-reduction versions, triggered unsuccessful hatching at 3 dpf and reduced overall survival at 6 dpf in developing zebrafish. At non-lethal concentrations, craniofacial defects with different severity based on the cigarette smoke extract were noted by 6 dpf. All tested products, including harm-reduction products, significantly impacted cartilage formation and/or bone mineralization in zebrafish embryos, independent of whether the bones/cartilage formed from the mesoderm or neural crest. Together, these results in a model system often used to detect embryonic malformations imply that exposure of a woman to secondhand smoke while pregnant may lead to mineralization issues in the skeleton of her newborn, ultimately adding a direct in utero association to the increased fracture risk observed in children of mothers exposed to cigarette smoke.

Halogenated bisphenol a analogues induce PPARγ-independent toxicity within human hepatocellular carcinoma cells.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) - both halogenated bisphenol (BPA) analogues - are suspected ligands of peroxisome proliferator-activated receptor gamma (PPARγ). While previous studies have shown that TBBPA and TCBPA activate PPARγ within cell-free assays, the downstream effects of TBBPA- and TCBPA-induced PPARγ activation on cellular transcription and physiology have not been thoroughly investigated. Therefore, the objective of this study was to determine whether exposure to TBBPA or TCBPA (either alone or in combination) alters levels of neutral lipids and fatty acid synthase (FASN) - an enzyme that catalyzes synthesis of long-chain saturated fatty acids - within intact cells in a PPARγ-dependent manner. For this study, we relied on human hepatocellular carcinoma (HepG2) cells as a model since these liver cells express basal levels of PPARγ and have been used to study lipoprotein metabolism and regulation of drug metabolizing enzymes. Although exposure to TBBPA and TCBPA alone did not affect cell viability nor neutral lipid and FASN levels in a concentration-dependent manner, exposure to binary mixtures of TBBPA and TCBPA resulted in a concentration-dependent decrease in cell viability in the absence of concentration-dependent effects on neutral lipid and FASN levels. Interestingly, exposure to TBBPA or TCBPA alone or as a mixture enhanced the effects of a reference PPARγ agonist (ciglitazone) and antagonist (GW 9662) on cell viability (but not neutral lipid levels), suggesting that these two halogenated BPA analogues may interact synergistically with ciglitazone and GW 9662 to induce cytotoxicity. However, overexpression of PPARγ did not mitigate nor enhance the effects of TBBPA - a potent PPARγ ligand predicted by ToxCast's cell-free competitive binding assays - on cell viability, neutral lipid levels, nor the cellular transcriptome. Overall, our findings suggest that halogenated BPA analogues such as TCBPA and TBBPA induce toxicity within HepG2 cells in a PPARγ-independent manner.

Relationship between miR-203a inhibition and oil-induced toxicity in early life stage zebrafish (Danio rerio).

Dysregulation of microRNA (miRNA, miR) by environmental stressors influences the transcription of mRNA which may impair organism development and/or lead to adverse physiological outcomes. Early studies evaluating the effects of oil on developmental toxicity in early life stages of fish showed that reductions in expression of miR-203a were associated with enhanced expression of downstream mRNAs that predicted altered eye development, cardiovascular disease, and improper fin development. To better understand the effects of miR-203a inhibition as an outcome of oil-induced toxicity in early life stage (ELS) fish, embryonic zebrafish were injected with an miR-203a inhibitor or treated with 3.5 µM phenanthrene (Phe) as a positive control for morphological alterations of cardiovascular and eye development caused by oil. Embryos treated with Phe had diminished levels of miR-203a at 7 and 72 h after injection. Embryos treated with the miR-203a inhibitor and Phe exhibited a reduced heart rate by 48 h post fertilization (hpf), with an increased incidence of developmental deformities (including pericardial edema, altered eye development, and spinal deformities) and reduced caudal fin length by 72 hpf. There were significant reductions in lens and eye diameters in 120 hpf miR-203a-inhibitor and Phe-treated fish, as well as a significantly reduced number of eye saccades, determined by an optokinetic response (OKR) behavioral assay. The expression of vegfa, which is an important activator during neovascularization, was significantly upregulated in embryos receiving miR-203a inhibitor injections by 7 and 72 hpf with increased trends in vegfa expression in 72 hpf larvae treated with Phe. There were decreasing trends in crx, neurod1, and pde6h expression by 72 hpf in miR-203a inhibitor and Phe treatments, which are involved in photoreceptor function in developing eyes and regulated by miR-203a. These results suggest that an inhibition of miR-203a in ELS fish exhibits an oil-induced toxic response that is consistent with Phe treatment and specifically impacts retinal, cardiac, and fin development in ELS fish.

Tris(1,3-dichloro-2-propyl) phosphate disrupts the trajectory of cytosine methylation within developing zebrafish embryos.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate ester-based flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) reliably disrupts cytosine methylation from cleavage (2 hpf) through early-gastrulation (6 hpf). Therefore, the objective of this study was to determine whether TDCIPP-induced effects on cytosine methylation persist beyond 6 hpf. First, we exposed embryos to vehicle or TDCIPP from 0.75 hpf to 6, 24, or 48 hpf, and then conducted bisulfite amplicon sequencing of a target locus (lmo7b) using genomic DNA derived from whole embryos. Within both vehicle- and TDCIPP-treated embryos, CpG methylation was similar at 6 hpf and CHG/CHH methylation were similar at 24 and 48 hpf (relative to 6 hpf). However, relative to 6 hpf within the same treatment, CpG methylation was lower within vehicle-treated embryos at 48 hpf and TDCIPP-treated embryos at 24 and 48 hpf - an effect that was driven by acceleration of CpG hypomethylation. Similar to our previous findings with DNA methyltransferase, we found that, even at high µM concentrations, TDCIPP had no effect on zebrafish and human thymine DNA glycosylase activity (a key enzyme that decreases CpG methylation), suggesting that TDCIPP-induced effects on CpG methylation are not driven by direct interaction with thymine DNA glycosylase. Finally, using 5-methylcytosine (5-mC)-specific whole-mount immunochemistry and automated imaging, we found that exposure to TDCIPP increased 5-mC abundance within the yolk of blastula-stage embryos, suggesting that TDCIPP may impact cytosine methylation of maternally loaded mRNAs during the maternal-to-zygotic transition. Overall, our findings suggest that TDCIPP disrupts the trajectory of cytosine methylation during zebrafish embryogenesis, effects which do not appear to be driven by direct interaction of TDCIPP with key enzymes that regulate cytosine methylation.

Partial dust removal in vehicles does not mitigate human exposure to organophosphate esters.

Organophosphate esters (OPEs) have been detected within car interior dust, suggesting that the indoor microenvironment of vehicles may represent a potential route of human exposure to OPEs. We recently showed that people with longer commutes are exposed to higher concentrations of tris(1,3-dichloro-2-isopropyl)phosphate (TDCIPP) - a widely used OPE - and other studies have suggested that dust removal may lead to lower exposure to chemicals. Therefore, the overall objective of this study was to determine if a decrease in interior car dust results in mitigation of personal OPE exposure. Participants (N = 49) were asked to wear silicone wristbands, and a subset of them wiped interior parts at the front of their vehicles prior to one study week (N = 25) or both study weeks (N = 11). There were no significant differences in total OPE concentrations (77.79-13,660 ng/g) nor individual OPE concentrations (0.04-4852.81 ng/g) across the different wiping groups nor in relation to participant residence ZIP codes and AC/Heater usage. These findings suggest that higher exposure to TDCIPP for participants with longer commutes may be independent of dust located on interior parts at the front of the vehicle. Therefore, our study demonstrates that there is a need for research on the potential contribution of other sources of TDCIPP exposure within car interiors.

Triphenyl phosphate-induced pericardial edema is associated with elevated epidermal ionocytes within zebrafish embryos.

Triphenyl phosphate (TPHP) is an organophosphate ester-based plasticizer and flame retardant. The objective of this study was to identify the potential role of epidermal ionocytes in mediating TPHP-induced pericardial edema within zebrafish embryos. Exposure to TPHP from 24 to 72 h post fertilization (hpf) resulted in a significant increase in pericardial edema and the number of ionocytes at 72 hpf relative to time-matched embryos treated with vehicle. In addition, co-exposure of embryos to mannitol (an osmotic diuretic) blocked TPHP-induced pericardial edema and effects on ionocyte abundance. However, knockdown of ATPase1a1.4 - an abundant Na+/K+-ATPase localized to epidermal ionocytes - mitigated TPHP-induced effects on ionocyte abundance but not pericardial edema, whereas co-exposure of embryos to ouabain - a Na+/K+-ATPase inhibitor - enhanced TPHP-induced pericardial edema but not ionocyte abundance. Overall, our findings suggest that TPHP may have multiple mechanisms of toxicity leading to an increase in ionocyte abundance and pericardial edema within developing zebrafish embryos.

miR133b Microinjection during Early Development Targets Transcripts of Cardiomyocyte Ion Channels and Induces Oil-like Cardiotoxicity in Zebrafish (Danio rerio) Embryos.

Previous studies have shown that altered expression of a family of small noncoding RNAs (microRNAs, or miRs) regulates the expression of downstream mRNAs and is associated with diseases and developmental disorders. miR133b is highly expressed in mammalian cardiac and skeletal muscle, and aberrant expression is associated with cardiac disorders and electrophysiological changes in cardiomyocytes. Similarly, cardiac dysfunction has been observed in early life-stage mahi-mahi (Coryphaena hippurus) exposed to crude oil, a phenotype that has been associated with an upregulation of miR133b as well as subsequent downregulation of a delayed rectifier potassium channel (IKr) and calcium signaling genes that are important for proper heart development during embryogenesis. To examine the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish (Danio rerio) embryos were either (1) microinjected with ∼3 nL of negative control miR (75 µM) or miR133b (75 µM) or (2) exposed to a treatment solution containing 5 µM benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, as a positive control. At 72 h post fertilization (hpf), miR133b-injected fish exhibited BaP-like cardiovascular malformations, including a significantly increased pericardial area relative to negative control miR-injected embryos, as well as a significantly reduced eye area. qPCR revealed that miR133b microinjection decreased the abundance of cardiac-specific IKr kcnh6 at 5 hpf, which may contribute to action potential elongation in oil-exposed cardiomyocytes. Additionally, ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum, was also downregulated by miR133b. These results indicate that an oil-induced increase in miR133b may contribute to cardiac abnormalities in oil-exposed fish by targeting cardiac-specific genes essential for proper heart development.

Utilizing systems biology to reveal cellular responses to peroxisome proliferator-activated receptor γ ligand exposure.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that, upon activation by ligands, heterodimerizes with retinoid X receptor (RXR), binds to PPAR response elements (PPREs), and activates transcription of downstream genes. As PPARγ plays a central role in adipogenesis, fatty acid storage, and glucose metabolism, PPARγ-specific pharmaceuticals (e.g., thiazolidinediones) have been developed to treat Type II diabetes and obesity within human populations. However, to our knowledge, no prior studies have concurrently assessed the effects of PPARγ ligand exposure on genome-wide PPARγ binding as well as effects on the transcriptome and lipidome within human cells at biologically active, non-cytotoxic concentrations. In addition to quantifying concentration-dependent effects of ciglitazone (a reference PPARγ agonist) and GW 9662 (a reference PPARγ antagonist) on human hepatocarcinoma (HepG2) cell viability, PPARγ abundance in situ, and neutral lipids, HepG2 cells were exposed to either vehicle (0.1% DMSO), ciglitazone, or GW 9662 for up to 24 h, and then harvested for 1) chromatin immunoprecipitation-sequencing (ChIP-seq) to identify PPARγ-bound regions across the entire genome, 2) mRNA-sequencing (mRNA-seq) to identify potential impacts on the transcriptome, and 3) lipidomics to identify potential alterations in lipid profiles. Following exposure to ciglitazone and GW 9662, we found that PPARγ levels were not significantly different after 2-8 h of exposure. While ciglitazone and GW 9662 resulted in a concentration-dependent increase in neutral lipids, the magnitude and localization of PPARγ-bound regions across the genome (as identified by ChIP-seq) did not vary by treatment. However, mRNA-seq and lipidomics revealed that exposure of HepG2 cells to ciglitazone and GW 9662 resulted in significant, treatment-specific effects on the transcriptome and lipidome. Overall, our findings suggest that exposure of human cells to PPARγ ligands at biologically active, non-cytotoxic concentrations results in toxicity that may be driven by a combination of both PPARγ-dependent and PPARγ-independent mechanisms.

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning.

Dorsoventral (DV) patterning is a key landmark of embryonic development that is primarily regulated by bone morphogenetic protein (BMP) signaling. Disruption of DV patterning can result in downstream effects on cell specification and organogenesis. Zebrafish embryos have been extensively used to understand signaling pathways that regulate DV patterning because zebrafish embryos develop ex utero and, in contrast to mammalian embryos, which develop in utero, can be observed in real time using brightfield and fluorescence microscopy. Embryos with disrupted DV patterning are either dorsalized or ventralized, with lack of development of head or trunk/tail structures, respectively. Although these phenotypes are typically accompanied by effects on BMP signaling, exceptions exist where some drugs or environmental chemicals can disrupt DV patterning in the absence of effects on BMP signaling. Therefore, assessments of DV patterning should be accompanied by BMP signaling-specific readouts to confirm the role of BMP disruption. Here, we describe an exposure paradigm and steps for phenotyping zebrafish embryos for two types of DV defects, dorsalization and ventralization, with a range of severities. In addition, we describe a strategy for whole-mount immunohistochemistry of zebrafish embryos with an antibody specific for phospho-SMAD 1/5/9 (pSMAD 1/5/9), as disruption in pSMAD 1/5/9 localization is indicative of an effect on BMP signaling. Taken together, these protocols describe an initial strategy for evaluating DV patterning defects under various experimental conditions and confirming BMP-mediated DV patterning disruptions, which can be followed by additional studies that aim to uncover mechanisms leading to these adverse phenotypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Phenotyping for dorsalization and ventralization Basic Protocol 2: Whole-mount immunohistochemistry with antibody to phospho-SMAD 1/5/9.

Stage-dependent and regioselective toxicity of 2- and 6-hydroxychrysene during Japanese medaka embryogenesis.

Exposure to oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene being more potent than 6-hydroxychrysene. To better characterize this phenomenon, embryos of Japanese medaka (Oryzias latipes) were exposed to 2- or 6-hydroxychrysene (0.5, 2, or 5 µM) from 4 h-post-fertilization (hpf) to 7 d-post-fertilization. Following exposure, hemoglobin concentrations were quantified by staining fixed embryos with o-dianisidine (a hemoglobin-specific dye) and stained embryos were imaged using brightfield microscopy. Exposure to 2-hydroxychrysene resulted in a concentration-dependent decrease in hemoglobin relative to vehicle-exposed embryos, while only the highest concentration of 6-hydroxychrysene resulted in a significant decrease in hemoglobin. All tested concentrations of 2-hydroxychrysene also caused significant mortality (12.2 % ± 2.94, 38.9 % ± 14.4, 85.6 % ± 11.3), whereas mortality was not observed following exposure to 6-hydroxychrysene. Therefore, treatment of embryos with 2-hydroxychrysene at various developmental stages and durations was subsequently conducted to identify key developmental landmarks that may be targeted by 2-hydroxychrysene. A sensitive window of developmental toxicity to 2-hydroxychrysene was found between 52-100 hpf, with a 24 h exposure to 10 µM 2-hydroxychrysene resulting in significant anemia and mortality. Since exposure to 2-hydroxychrysene from 52 to 100 hpf, a window that includes liver morphogenesis in medaka, resulted in the highest magnitude of toxicity, liver development and function may have a role in 2-hydroxychrysene developmental toxicity.

Effects of Phenanthrene Exposure on Cholesterol Homeostasis and Cardiotoxicity in Zebrafish Embryos.

Polycyclic aromatic hydrocarbons (PAHs) are pervasive pollutants in aquatic ecosystems, and developing fish embryos are especially sensitive to PAH exposure. Exposure to crude oil or phenanthrene (a reference PAH found in oil) produces an array of gross morphological abnormalities in developing fish embryos, including cardiotoxicity. Recently, studies utilizing transcriptomic analyses in several oil-exposed fish embryos found significant changes in the abundance of transcripts involved in cholesterol biosynthesis. Given the vital role of cholesterol availability in embryonic heart development, we hypothesized that cholesterol dysregulation in early development contributes to phenanthrene-induced cardiotoxicity. We exposed zebrafish embryos to 12 or 15 µM phenanthrene from 6 to 72 h post fertilization (hpf) and demonstrated that, in conjunction with pericardial edema and bradycardia, several genes (fdft1 and hmgcra) in the cholesterol biosynthetic pathway were significantly altered. When embryos were pretreated with a cholesterol solution from 6 to 24 hpf followed by exposure to phenanthrene from 24 to 48 hpf, the effects of phenanthrene on heart rate were partially mitigated. Despite changes in gene expression, whole-mount in situ staining of cholesterol was not significantly affected in embryos exposed to phenanthrene ranging in stage from 24 to 72 hpf. However, the 2-dimensional yolk area was significantly increased with phenanthrene exposure at 72 hpf, suggesting that lipid transport from the yolk to the developing embryo was impaired. Environ Toxicol Chem 2021;40:1586-1595. © 2021 SETAC.

Inhalation of two Prop 65-listed chemicals within vehicles may be associated with increased cancer risk.

Chemicals are listed on California's Proposition 65 (Prop 65) for their potential to cause cancer, birth defects or other reproductive harm, and certain chemicals from this list are often detected within interior vehicle dust and air. Therefore, this study examined the potential risk associated with five Prop 65-listed chemicals detected within vehicle interiors: benzene, formaldehyde, di (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and tris(1,3-dichloro-2-propyl)phosphate (TDCIPP). Exposure estimates based on time spent within a vehicle were derived from a meta-analysis of estimated concentrations from the literature. Regulatory levels established by the California Office of Environmental Health Hazard Assessment (OEHHA) were then used to generate percent reference doses (%RfDs) for chemical-specific daily doses as well as determine the probability of risk (exceedance probability) as a function of %RfD for each chemical-specific daily dose. Based on our meta-analysis, benzene and formaldehyde were detected in vehicle interior air whereas DEHP, DBP and TDCIPP were detected in vehicle interior dust. Benzene and formaldehyde were the only two chemicals with an estimated %RfD > 100 across any of the commute times. For commute times of 20 min or longer, the %RfD was > 100 for maximum exposures based on the "maximum allowable daily level" for benzene, and for 95th-percentile exposures based on the "no significant risk level" for benzene and formaldehyde. Furthermore, the probability of exceeding 100% RfD was highest for cancer risks associated with benzene, followed by cancer risks associated with formaldehyde and the risk of reproductive and developmental toxicity associated with benzene. Lastly, within the entire state of California, the percent of commuters with a 10% probability of exceeding cancer risk associated with benzene or formaldehyde exposure was 78% and 63%, respectively. Overall, our study raises concerns about the potential risk associated with inhalation of benzene and formaldehyde for people who spend a significant amount of time in their vehicles, an issue that is especially pertinent to traffic-congested areas where people have longer commutes.

Environmentally relevant concentrations of boscalid exposure affects the neurobehavioral response of zebrafish by disrupting visual and nervous systems.

Boscalid is a persistent fungicide that is frequently detected in surface waters and may be neurotoxic to aquatic organisms. Herein, we evaluated the effects of environmentally relevant boscalid concentrations to zebrafish to explore its potentially neurotoxic mechanisms of effect. Behavioral responses (swimming, phototaxis, and predation), histopathology, transcriptomics, biochemical parameter analysis and gene expression of larval and adult zebrafish following boscalid treatment were assessed. We found that boscalid significantly inhibited the locomotor ability and phototactic response of larvae after an 8-d exposure, and altered the locomotor activity, predation trajectories and ability in adults after a 21-d exposure. It was noted that predation rates of zebrafish were significantly decreased by 30% and 100% after exposure to 0.1 and 1.0 mg/L boscalid, respectively. Adverse alterations in the cell differentiation of eyes and brain injury were also observed in both larvae and adults following boscalid exposure. The expression of genes related to neurodevelopment, neurotransmission, eye development, and visual function, in conjunction with RNA-Seq results, indicated that boscalid may impair visual phototransduction and nervous system processes in larval zebrafish. Conclusively, boscalid exposure may affect the neurobehavioral response of zebrafish by impairing proper visual and nervous system function.

Menthol in electronic cigarettes: A contributor to respiratory disease?

Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL® cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL® cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease.