Expanded Systematic Evidence Map for Hundreds of Per- and Polyfluoroalkyl Substances (PFAS) and Comprehensive PFAS Human Health Dashboard.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) encompass a class of chemically and structurally diverse compounds that are extensively used in industry and detected in the environment. The US Environmental Protection Agency (US EPA) 2021 PFAS Strategic Roadmap describes national research plans to address the challenge of PFAS. OBJECTIVES: Systematic Evidence Map (SEM) methods were used to survey and summarize available epidemiological and mammalian bioassay evidence that could inform human health hazard identification for a set of 345 PFAS that were identified by the US EPA's Center for Computational Toxicology and Exposure (CCTE) for in vitro toxicity and toxicokinetic assay testing and through interagency discussions on PFAS of interest. This work builds from the 2022 evidence map that collated evidence on a separate set of ∼150 PFAS. Like our previous work, this SEM does not include PFAS that are the subject of ongoing or completed assessments at the US EPA. METHODS: SEM methods were used to search, screen, and inventory mammalian bioassay and epidemiological literature from peer-reviewed and gray literature sources using manual review and machine-learning software. For each included study, study design details and health end points examined were summarized in interactive web-based literature inventories. Some included studies also underwent study evaluation and detailed extraction of health end point data. All underlying data is publicly available online as interactive visuals with downloadable metadata. RESULTS: More than 13,000 studies were identified from scientific databases. Screening processes identified 121 mammalian bioassay and 111 epidemiological studies that met screening criteria. Epidemiological evidence (available for 12 PFAS) mostly assessed the reproductive, endocrine, developmental, metabolic, cardiovascular, and immune systems. Mammalian bioassay evidence (available for 30 PFAS) commonly assessed effects in the reproductive, whole-body, nervous, and hepatic systems. Overall, 41 PFAS had evidence across mammalian bioassay and epidemiology data streams (roughly 11% of searched chemicals). DISCUSSION: No epidemiological and/or mammalian bioassay evidence were identified for most of the PFAS included in our search. Results from this SEM, our 2022 SEM on ∼150 PFAS, and other PFAS assessment products from the US EPA are compiled into a comprehensive PFAS dashboard that provides researchers and regulators an overview of the current PFAS human health landscape including data gaps and can serve as a scoping tool to facilitate prioritization of PFAS-related research and/or risk assessment activities. https://doi.org/10.1289/EHP13423.

Total Blood Mercury Predicts Methylmercury Exposure in Fish and Shellfish Consumers.

Many studies evaluating methylmercury (MeHg) toxicity rely on whole blood total mercury (THg) measurements to estimate MeHg exposure. However, whole blood THg includes other forms of mercury (Hg), such as inorganic Hg, which have different exposure sources and toxicological effects than MeHg. Therefore, estimating the whole blood MeHg/THg ratio is critical to predicting MeHg exposure and, subsequently, efforts to establish an exposure-response relationship for use in risk assessment. A large, representative dataset (National Health and Nutrition Examination Survey (NHANES) 2011-2016) was used to determine the whole blood MeHg/THg ratio among (a) self-reported fish and shellfish consumers, ≥ 15 years of age (the "full adult" population (N = 5268 training dataset; N = 2336 test dataset)) and (b) female fish and shellfish consumers, 15-44 years of age (the "women of reproductive age" population (N = 1285 training dataset; N = 560 test dataset)). Unadjusted and adjusted linear and spline models with direct measurements for both THg and MeHg were evaluated. The mean whole blood MeHg/THg ratio was 0.75 (95% confidence interval (CI): 0.74, 0.75). This ratio was significantly higher among those with higher THg concentrations. All models exhibited excellent fit (adjusted R2 from 0.957 to 0.982). Performance was slightly improved in spline versus linear models. For the full adult population and women of reproductive age, the unadjusted spline model predicted whole blood MeHg concentrations of 5.65 µg/L and 5.55 µg/L, respectively, when the THg concentration was 5.80 µg/L. These results suggest that whole blood THg is a good predictor of whole blood MeHg among fish and shellfish consumers.

An Effective Model of the Retinoic Acid Induced HL-60 Differentiation Program.

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.

Temporal Trends in Exposure to Organophosphate Flame Retardants in the United States.

During the past decade, use of organophosphate compounds as flame retardants and plasticizers has increased. Numerous studies investigating biomarkers (i.e., urinary metabolites) demonstrate ubiquitous human exposure and suggest that human exposure may be increasing. To formally assess temporal trends, we combined data from 14 U.S. epidemiologic studies for which our laboratory group previously assessed exposure to two commonly used organophosphate compounds, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPHP). Using individual-level data and samples collected between 2002 and 2015, we assessed temporal and seasonal trends in urinary bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) and diphenyl phosphate (DPHP), the metabolites of TDCIPP and TPHP, respectively. Data suggest that BDCIPP concentrations have increased dramatically since 2002. Samples collected in 2014 and 2015 had BDCIPP concentrations that were more than 15 times higher than those collected in 2002 and 2003 (10ß = 16.5; 95% confidence interval from 9.64 to 28.3). Our results also demonstrate significant increases in DPHP levels; however, increases were much smaller than for BDCIPP. Additionally, results suggest that exposure varies seasonally, with significantly higher levels of exposure in summer for both TDCIPP and TPHP. Given these increases, more research is needed to determine whether the levels of exposure experienced by the general population are related to adverse health outcomes.

Regional comparison of organophosphate flame retardant (PFR) urinary metabolites and tetrabromobenzoic acid (TBBA) in mother-toddler pairs from California and New Jersey.

The use of alternative chemical flame retardants in consumer products is increasing as the result of the phase-out of polybrominated diphenyl ethers. Today, the most commonly detected alternatives in residential furniture include the organophosphate flame retardants (PFRs) and the Firemaster (R) 550 mixture (FM550). Urinary levels of dialkyl and diaryl phosphate esters, and 2-ethylhexyl tetrabromobenzoate (EH-TBB) have been used as biomarkers of human exposure to PFRs and FM550, respectively. In a previous study, we demonstrated that toddlers had significantly higher levels of PFRs relative to their mothers in a cohort from New Jersey; however, it is unclear if there are regional differences in exposure. It is possible that exposure to PFRs may be higher in California relative to other US States due to the California flammability standard, as was seen previously observed for PBDEs. In the current study, we examined urinary levels of PFR metabolites and TBBA in 28 mother-child pairs from California, USA, collected in 2015, and compared them with levels measured in our previous study from New Jersey. Urine samples were extracted using solid-phase extraction and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Diphenyl phosphate (DPHP), isopropyl-phenyl phenyl phosphate (ip-PPP), bis(1,3-dichloro-2propyl) phosphate (BDCIPP) and BCIPHIPP conjugates were detected in 100% of mother and child urine samples, while bis(1-chloro-2-propyl) phosphate (BCIPP), tert-butyl-phenyl phenyl phosphate (tb-PPP) and TBBA were detected in < 50% of samples. Interestingly, BCIPHIPP conjugates were detected in 100% of the urine samples, suggesting ubiquitous exposure to the parent compound, tris(1-chloro-2-propyl) phosphate (TCIPP). The current study found significantly higher BDCIPP levels in California toddlers and higher and ip-PPP levels in mothers as compared to the New Jersey cohort, which may be reflective of California's furniture flammability standard. For example, BDCIPP levels in California children were 2.4 times higher than those in New Jersey children. Consistent with our previous work, the current study showed higher PFR and EH-TBB exposure in children, likely due to increased hand-mouth behavior. Children's DPHP and BDCIPP levels, on average, were 5.9 times and 15 times those of their mothers. Positive correlations between paired mothers and their children were shown for DPHP and BCIPHIPP conjugates but not BDCIPP or ip-PPP. In the children, several predictors of hand-mouth behavior were associated with BDCIPP, DPHP and ip-PPP urine levels, but no associations were observed with BCIPHIPP conjugates.

Nail polish as a source of exposure to triphenyl phosphate.

Triphenyl phosphate (TPHP) is primarily used as either a flame retardant or plasticizer, and is listed as an ingredient in nail polishes. However, the concentration of TPHP in nail polish and the extent of human exposure following applications have not been previously studied. We measured TPHP in ten different nail polish samples purchased from department stores and pharmacies in 2013-2014. Concentrations up to 1.68% TPHP by weight were detected in eight samples, including two that did not list TPHP as an ingredient. Two cohorts (n=26 participants) were recruited to assess fingernail painting as a pathway of TPHP exposure. Participants provided urine samples before and after applying one brand of polish containing 0.97% TPHP by weight. Diphenyl phosphate (DPHP), a TPHP metabolite, was then measured in urine samples (n=411) and found to increase nearly seven-fold 10-14h after fingernail painting (p<0.001). To determine relative contributions of inhalation and dermal exposure, ten participants also painted their nails and painted synthetic nails adhered to gloves on two separate occasions, and collected urine for 24h following applications. Urinary DPHP was significantly diminished when wearing gloves, suggesting that the primary exposure route is dermal. Our results indicate that nail polish may be a significant source of short-term TPHP exposure and a source of chronic exposure for frequent users or those occupationally exposed.

Metabolites of organophosphate flame retardants and 2-ethylhexyl tetrabromobenzoate in urine from paired mothers and toddlers.

As a result of the polybrominated diphenyl ether (PBDE) ban in the mid-2000s, the chemical flame retardant market has moved toward alterative compounds including chlorinated alkyl and nonchlorinated aryl organophosphate flame retardants (OPFRs) as well as aromatic brominated compounds such as Firemaster 550 (FM550). Recent studies have shown that the OPFRs and Firemaster 550 components are frequently detected in polyurethane foams and in indoor dust. Some OPFRs are considered carcinogenic and/or neurodevelopmental toxicants, and children's exposure to these compounds is a concern. OPFRs are readily metabolized and excreted in the urine as their dialkyl and diaryl compounds which function as biomarkers for OPFR exposure. Limited research has shown that adults are broadly exposed to OPFRs, but nothing is known about children's exposure. Similarly, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB), a FM550 component, is metabolized to tetrabromobenzoic acid (TBBA). The current study measured levels of bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), bis(1-chloro-2-propyl) phosphate (BCIPP), diphenyl phosphate (DPHP), 2 alkylated DPHPs, and TBBA in urine collected in 2013 from 21 US mother-toddler pairs. BDCIPP, DPHP, and ip-DPHP were detected in 100%, 98%, and 96% of all individuals, whereas BCIPP and tert-butyl-DPHP (tb-DPHP) were only detected in 8% and 13%. Further, TBBA was detected in 27% of adults but 70% of children. Overall, children had higher urinary levels of BDCIPP, DPHP, ip-DPHP, and TBBA as compared to their mothers, suggesting higher exposure. For example, on average, BDCIPP levels in children were 4.9 times those of mothers. BDCIPP and DPHP levels in mother's urine were also significantly correlated with levels in children's urine, suggesting similar exposure routes, likely in the home environment. Various potential predictors of OPFR exposure were assessed using a questionnaire. In children some predictors of hand-mouth exposure were associated with elevated BDCIPP and DPHP levels (e.g., less frequent hand washing for BDCIPP). Overall, these trends are consistent with higher flame retardant levels in children as a result of increased hand-mouth behavior and elevated dust exposure.

Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity.

The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38+ transfectants. In contrast, cells cultured for 48h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation.

The Src-family kinase inhibitor PP2 rescues inducible differentiation events in emergent retinoic acid-resistant myeloblastic leukemia cells.

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47(phox) expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid's biological effects in WT HL60 cells.

Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

ATRA-induced HL-60 myeloid leukemia cell differentiation depends on the CD38 cytosolic tail needed for membrane localization, but CD38 enzymatic activity is unnecessary.

Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11-20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11-20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11-20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function.

Mechanism-based small molecule probes for labeling CD38 on live cells.

CD38 is a type II transmembrane glycoprotein with multiple functions. It acts as an ecto-enzyme as well as a receptor. The enzymatic activity catalyzes the formation of two potent Ca(2+) releasing agents: cyclic adenosine diphosphate ribose (cADPR) from nicotinamide adenine dinucleotide (NAD) and nicotinic acid adenine dinucleotide phosphate (NAADP) from NAD phosphate (NADP). The receptor function of CD38 leads to the phosphorylation of intracellular signaling proteins and the up-regulation of cytokine production in immune cells. These two functions of CD38 underlie its involvement in various biological processes, such as hormone secretion, immune cell differentiation, and immune responses. Clinically, CD38 is used as a negative prognosis marker for chronic lymphatic leukemia (CLL). However, a clear molecular understanding of CD38's role in physiology and pathology is still lacking. To facilitate the study of CD38 at cellular and molecular levels, here we report a mechanism-based method for fluorescently labeling CD38 on live cells. This labeling method does not interfere with the receptor function of CD38 and the downstream signaling. The labeling method is thus a useful tool to study the receptor function of CD38 in live cells. In addition, since the mechanism-based labeling also inhibits the enzymatic activity of CD38, it should be useful for dissecting the receptor function of CD38 without interference from its enzyme function in complicated biological processes.