Smoking Behaviors Among Indigenous Pregnant People Compared to a Matched Regional Cohort.

INTRODUCTION: Smoking commercial tobacco products is highly prevalent in American Indian and Alaska Native (Indigenous) pregnancies. This disparity directly contributes to maternal and fetal mortality. Our objective was to describe cigarette smoking prevalence, cessation intervention uptake, and cessation behaviors of pregnant Indigenous people compared to sex and age-matched regional cohort. AIMS AND METHODS: Pregnancies from an Indigenous cohort in Olmsted County, Minnesota, identified in the Rochester Epidemiology Project, were compared to pregnancies identified in a sex and age-matched non-Indigenous cohort from 2006 to 2019. Smoking status was defined as current, former, or never. All pregnancies were reviewed to identify cessation interventions and cessation events. The primary outcome was smoking prevalence during pregnancy, with secondary outcomes measuring uptake of smoking cessation interventions and cessation. RESULTS: The Indigenous cohort included 57 people with 81 pregnancies, compared to 226 non-Indigenous people with 358 pregnancies. Smoking was identified during 45.7% of Indigenous pregnancies versus 11.2% of non-Indigenous pregnancies (RR: 3.25, 95% CI = 1.98-5.31, p ≤ .0001). Although there was no difference in uptake of cessation interventions between cohorts, smoking cessation was significantly less likely during Indigenous pregnancies compared to non-Indigenous pregnancies (OR: 0.23, 95% CI = 0.07-0.72, p = .012). CONCLUSIONS: Indigenous pregnant people in Olmsted County, Minnesota were more than three times as likely to smoke cigarettes during pregnancy compared to the non-indigenous cohort. Despite equivalent uptake of cessation interventions, Indigenous people were less likely to quit than non-Indigenous people. Understanding why conventional smoking cessation interventions were ineffective at promoting cessation during pregnancy among Indigenous women warrants further study. IMPLICATIONS: Indigenous pregnant people in Olmsted County, Minnesota, were greater than three times more likely to smoke during pregnancy compared to a regional age matched non-Indigenous cohort. Although Indigenous and non-Indigenous pregnant people had equivalent uptake of cessation interventions offered during pregnancy, Indigenous people were significantly less likely to quit smoking before fetal delivery. This disparity in the effectiveness of standard of care interventions highlights the need for further study to understand barriers to cessation in pregnant Indigenous people.

Indigenous Smoking Behaviors in Olmsted County, Minnesota: A Longitudinal Population-Based Study.

OBJECTIVE: To describe smoking behaviors and pharmaceutical cessation aid uptake in a population-based Indigenous cohort compared with an age- and sex-matched non-Indigenous cohort. PATIENTS AND METHODS: Using the health record-linkage system of the Rochester Epidemiology Project (January 1, 2006, to December 31, 2019), smoking data of Indigenous residents of Olmsted County in Minnesota were abstracted to define the smoking prevalence, incidence, cessation, relapse after cessation, and pharmaceutical smoking cessation aid uptake compared with a matched non-Indigenous cohort. Prevalence was analyzed with a modified Poisson regression; cessation and relapse were evaluated with generalized estimating equations. Incidence was evaluated with a Cox proportional hazards model. RESULTS: Smoking prevalence was higher in the Indigenous cohort (39.0% to 47.0%; n=898) than the matched cohort (25.6% to 30.3%; n=1780). Pharmaceutical uptake was higher among the Indigenous cohort (35.8% of n=584 ever smokers vs 16.3% of n=778 ever smokers; P<.001). Smoking cessation events occurred more frequently in the Indigenous cohort (relative risk, 1.10; 95% CI, 1.06 to 1.13; P<.001). Indigenous former smokers were more likely to resume smoking (relative risk, 3.03; 95% CI, 2.93 to 3.14; P<.001) compared with the matched cohort. These findings were independent of socioeconomic status, age, and sex. CONCLUSION: Smoking in this Indigenous cohort was more prevalent compared with a sex- and age-matched non-Indigenous cohort despite more smoking cessation events and higher use of smoking cessation aids in the Indigenous cohort. The relapse rate after achieving cessation in the Indigenous cohort was more than three times higher than the non-Indigenous cohort. This finding has not been previously described and represents a potential target for relapse prevention efforts in US Indigenous populations.

Characterizing cleft palate toxicants using ToxCast data, chemical structure, and the biomedical literature.

Cleft palate has been linked to both genetic and environmental factors that perturb key events during palatal morphogenesis. As a developmental outcome, it presents a challenging, mechanistically complex endpoint for predictive modeling. A data set of 500 chemicals evaluated for their ability to induce cleft palate in animal prenatal developmental studies was compiled from Toxicity Reference Database and the biomedical literature, which included 63 cleft palate active and 437 inactive chemicals. To characterize the potential molecular targets for chemical-induced cleft palate, we mined the ToxCast high-throughput screening database for patterns and linkages in bioactivity profiles and chemical structural descriptors. ToxCast assay results were filtered for cytotoxicity and grouped by target gene activity to produce a "gene score." Following unsuccessful attempts to derive a global prediction model using structural and gene score descriptors, hierarchical clustering was applied to the set of 63 cleft palate positives to extract local structure-bioactivity clusters for follow-up study. Patterns of enrichment were confirmed on the complete data set, that is, including cleft palate inactives, and putative molecular initiating events identified. The clusters corresponded to ToxCast assays for cytochrome P450s, G-protein coupled receptors, retinoic acid receptors, the glucocorticoid receptor, and tyrosine kinases/phosphatases. These patterns and linkages were organized into preliminary decision trees and the resulting inferences were mapped to a putative adverse outcome pathway framework for cleft palate supported by literature evidence of current mechanistic understanding. This general data-driven approach offers a promising avenue for mining chemical-bioassay drivers of complex developmental endpoints where data are often limited.

TNF-Alpha Inhibition and Other Immunosuppressants in the Development of Uveal and Cutaneous Melanoma.

OBJECTIVE: To investigate an association between tumor necrosis factors-alpha (TNFα) inhibitors or other immunosuppressants and the development of uveal and cutaneous melanoma. PATIENTS AND METHODS: We performed a retrospective incidence and case-control analysis of patients in Olmsted County, MN, who were diagnosed with uveal or cutaneous melanoma from January 1, 2000, to December 31, 2014. Incidence was adjusted by age and gender to the 2010 US white population. Controls were matched by sex and age to cases at time of diagnosis of melanoma. RESULTS: There were 1221 cases of melanoma (33 uveal, 1188 cutaneous). Combined incidence of uveal and cutaneous melanoma per 100,000 person-years varied by gender (male > female), age (older > younger), and time period: 2010 to 2014 (77.9, 95% confidence interval [CI], 71.1-84.7) ≈ 2005 to 2009 (78.0, 95% CI, 70.9-85.0) > 2000 to 2004 (42.5, 95% CI, 36.9-48.1, P<.001). TNFa inhibitor prescription was not associated with significantly increased risk of melanoma vs controls (1.06% vs 0.41%, P=.06). Immunosuppressive agents, high-dose corticosteroids, and topical immunosuppressants were associated with melanoma (odds ratio [OR] 1.42 CI, 1.03-1.95, 3.30 CI, 2.44-4.48, and 1.87 CI, 1.06-3.28, respectively). An increased number of patients with uveal melanoma received immune modulating agents vs controls, but this was not statistically significant (P=.36). Autoimmune disease itself was not correlated with melanoma (P=.73). CONCLUSION: Exposure to immunosuppressive agents is associated with melanoma. TNFa inhibition and autoimmune disease alone do not significantly increase risk of melanoma. In patients receiving immunosuppressive treatments, physicians should consider monitoring with dilated ophthalmic and full-body skin examinations. Further studies are needed to assess the impact of TNFa inhibitors on development of melanoma, particularly in uveal melanoma.

Embryonic Midfacial Palatal Organ Culture Methods in Developmental Toxicology.

The morphogenesis of the secondary palate provides an interesting model for many of the processes involved in embryonic development. A number of in vitro models have been used to study craniofacial development, including whole embryo culture, palatal mesenchymal and micromass cell cultures, and Trowell-like palatal cultures in which dissected palates are cultured individually or as pairs in contact on a support above medium. This chapter presents a detailed protocol for the culture of maxillary midfacial tissues, including the palatal shelves, in suspension culture. This method involves isolation of the midfacial tissues (maxillary arch and palatal shelves) and suspension of the tissues in medium in flasks. On a rocker in an incubator, the palatal shelves elevate, grow, make contact, and fuse in a time span analogous to that occurring in the intact embryo in utero.

A Three-Dimensional Organoid Culture Model to Assess the Influence of Chemicals on Morphogenetic Fusion.

Embryologic development involves cell differentiation and organization events that are unique to each tissue and organ and are susceptible to developmental toxicants. Animal models are the gold standard for identifying putative teratogens, but the limited throughput of developmental toxicological studies in animals coupled with the limited concordance between animal and human teratogenicity motivates a different approach. In vitro organoid models can mimic the three-dimensional (3D) morphogenesis of developing tissues and can thus be useful tools for studying developmental toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro organoid models that capture key morphogenesis events that occur in the embryo. We previously described organoids composed of human stem and progenitor cells that recapitulated the cellular features of palate fusion, and here we further characterized the model by examining pharmacological inhibitors targeting known palatogenesis and epithelial morphogenesis pathways as well as 12 cleft palate teratogens identified from rodent models. Organoid survival was dependent on signaling through EGF, IGF, HGF, and FGF pathways, and organoid fusion was disrupted by inhibition of BMP signaling. We observed concordance between the effects of EGF, FGF, and BMP inhibitors on organoid fusion and epithelial cell migration in vitro, suggesting that organoid fusion is dependent on epithelial morphogenesis. Three of the 12 putative cleft palate teratogens studied here (theophylline, triamcinolone, and valproic acid) significantly disrupted in vitro organoid fusion, while tributyltin chloride and all-trans retinoic acid were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid morphogenesis in a scalable format amenable to toxicology screening.

Development of an organotypic stem cell model for the study of human embryonic palatal fusion.

BACKGROUND: Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1,000 births worldwide. The secondary palate is formed by paired palatal shelves, consisting of a mesenchymal core with an outer layer of epithelial cells that grow toward each other, attach, and fuse. One of the mechanisms that can cause CP is failure of fusion, that is, failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme confluence. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. METHODS: We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical-derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEKs). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and time to fusion. RESULTS: Osteogenic differentiation in hMSC spheroids was highest by Day 13. hEGF delayed fusion of organoids after 12 and 18 hr of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone. CONCLUSION: Our results show that this model of human palatal fusion appropriately mimics the morphology of the developing human palate and responds to hEGF as expected.

Engineering human cell spheroids to model embryonic tissue fusion in vitro.

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton's Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.

Parkinson Disease and Melanoma: Confirming and Reexamining an Association.

OBJECTIVE: To examine an association between melanoma and Parkinson disease (PD). PATIENTS AND METHODS: Phase I: Rochester Epidemiology Project records were used to identify (between January 1, 1976, and December 31, 2013) patients with PD in Olmsted County, Minnesota, with 3 matched controls per case. After review, JMP statistical software with logistic regression analysis was used to assess the risk of preexisting melanoma in patients with PD vs controls. Phase II: All Rochester Epidemiology Project cases of melanoma were identified (between January 1, 1976, and December 31, 2014), with 1 control per case. A Cox proportional hazards model was used to assess the risk of developing PD after the index date in cases vs controls, and Kaplan-Meier analysis was performed to determine the 35-year cumulative risk of PD. A Cox proportional hazards model was used to assess the risk of death from metastatic melanoma in patients with melanoma without PD compared with those with PD. RESULTS: Phase I: Patients with PD had a 3.8-fold increased likelihood of having preexisting melanoma as compared with controls (95% CI, 2.1-6.8; P<.001). Phase II: Patients with melanoma had a 4.2-fold increased risk of developing PD (95% CI, 2.0-8.8; P<.001). Kaplan-Meier analysis revealed an increased 35-year cumulative risk of PD in patients with melanoma (11.8%) compared with controls (2.6%) (P<.001). Patients with melanoma without PD had a 10.5-fold increased relative risk of death from metastatic melanoma compared with patients with melanoma with PD (95% CI, 1.5-72.2) (P=.02). CONCLUSION: There appears to be an association between melanoma and PD. Further study is warranted; but on the basis of these results, physicians may consider counseling patients with melanoma about PD risk and implementing cutaneous and ocular melanoma surveillance in patients with PD.

PPARα-independent transcriptional targets of perfluoroalkyl acids revealed by transcript profiling.

Perfluoroalkyl acids (PFAAs) are ubiquitous and persistent environmental contaminants. Compounds such as perfluoroocanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonate (PFHxS) are readily found in the tissues of humans and wildlife. While PFOA and PFOS have been the subject of numerous studies since they were first described over a decade ago, less is known about the biological activity of PFHxS and PFNA. Most PFAAs are activators of peroxisome proliferator-activated receptor α (PPARα), although the biological effects of these compounds are likely mediated by other factors in addition to PPARα. To evaluate the effects of PFHxS and PFNA, male wild-type and Pparα-null mice were dosed by oral gavage with PFHxS (3 or 10mg/kg/day), PFNA (1 or 3mg/kg/day), or vehicle for 7days, and liver gene expression was evaluated by full-genome microarrays. Gene expression patterns were then compared to historical in-house data for PFOA and PFOS in addition to the experimental hypolipidemic agent, WY-14,643. While WY-14,643 altered most genes in a PPARα-dependent manner, approximately 11-24% of regulated genes in PFAA-treated mice were independent of PPARα. The possibility that PFAAs regulate gene expression through other molecular pathways was evaluated. Using data available through a microarray database, PFAA gene expression profiles were found to exhibit significant similarity to profiles from mouse tissues exposed to agonists of the constitutive activated receptor (CAR), estrogen receptor α (ERα), and PPARγ. Human PPARγ and ERα were activated by all four PFAAs in trans-activation assays from the ToxCast screening program. Predictive gene expression biomarkers showed that PFAAs activate CAR in both genotypes and cause feminization of the liver transcriptome through suppression of signal transducer and activator of transcription 5B (STAT5B). These results indicate that, in addition to activating PPARα as a primary target, PFAAs also have the potential to activate CAR, PPARγ, and ERα as well as suppress STAT5B.

Engineering epithelial-stromal interactions in vitro for toxicology assessment.

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues.

Perfluoroalkyl acids-induced liver steatosis: Effects on genes controlling lipid homeostasis.

Persistent presence of perfluoroalkyl acids (PFAAs) in the environment is due to their extensive use in industrial and consumer products, and their slow decay. Biochemical tests in rodent demonstrated that these chemicals are potent modifiers of lipid metabolism and cause hepatocellular steatosis. However, the molecular mechanism of PFAAs interference with lipid metabolism remains to be elucidated. Currently, two major hypotheses are that PFAAs interfere with mitochondrial beta-oxidation of fatty acids and/or they affect the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) in liver. To determine the ability of structurally-diverse PFAAs to cause steatosis, as well as to understand the underlying molecular mechanisms, wild-type (WT) and PPARα-null mice were treated with perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), or perfluorohexane sulfonate (PFHxS), by oral gavage for 7days, and their effects were compared to that of PPARα agonist WY-14643 (WY), which does not cause steatosis. Increases in liver weight and cell size, and decreases in DNA content per mg of liver, were observed for all compounds in WT mice, and were also seen in PPARα-null mice for PFOA, PFNA, and PFHxS, but not for WY. In Oil Red O stained sections, WT liver showed increased lipid accumulation in all treatment groups, whereas in PPARα-null livers, accumulation was observed after PFNA and PFHxS treatment, adding to the burden of steatosis observed in control (untreated) PPARα-null mice. Liver triglyceride (TG) levels were elevated in WT mice by all PFAAs and in PPARα-null mice only by PFNA. In vitro ß-oxidation of palmitoyl carnitine by isolated rat liver mitochondria was not inhibited by any of the 7 PFAAs tested. Likewise, neither PFOA nor PFOS inhibited palmitate oxidation by HepG2/C3A human liver cell cultures. Microarray analysis of livers from PFAAs-treated mice indicated that the PFAAs induce the expression of the lipid catabolism genes, as well as those involved in fatty acid and triglyceride synthesis, in WT mice and, to a lesser extent, in PPARα-null mice. These results indicate that most of the PFAAs increase liver TG load and promote steatosis in mice We hypothesize that PFAAs increase steatosis because the balance of fatty acid accumulation/synthesis and oxidation is disrupted to favor accumulation.

A systematic evaluation of the potential effects of trichloroethylene exposure on cardiac development.

The 2011 EPA trichloroethylene (TCE) IRIS assessment, used developmental cardiac defects from a controversial drinking water study in rats (Johnson et al. [51]), along with several other studies/endpoints to derive reference values. An updated literature search of TCE-related developmental cardiac defects was conducted. Study quality, strengths, and limitations were assessed. A putative adverse outcome pathway (AOP) construct was developed to explore key events for the most commonly observed cardiac dysmorphologies, particularly those involved with epithelial-mesenchymal transition (EMT) of endothelial origin (EndMT); several candidate pathways were identified. A hypothesis-driven weight-of-evidence analysis of epidemiological, toxicological, in vitro, in ovo, and mechanistic/AOP data concluded that TCE has the potential to cause cardiac defects in humans when exposure occurs at sufficient doses during a sensitive window of fetal development. The study by Johnson et al. [51] was reaffirmed as suitable for hazard characterization and reference value derivation, though acknowledging study limitations and uncertainties.

Identification of modulators of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) in a mouse liver gene expression compendium.

The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents increases liver cancer incidence, whereas suppression of PPARα activity leads to hepatocellular steatosis. Analytical approaches were developed to identify biosets (i.e., gene expression differences between two conditions) in a genomic database in which PPARα activity was altered. A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice after exposure to three structurally diverse PPARα activators (WY-14,643, fenofibrate and perfluorohexane sulfonate). A fold-change rank-based test (Running Fisher's test (p-value ≤ 10(-4))) was used to evaluate the similarity between the PPARα signature and a test set of 48 and 31 biosets positive or negative, respectively for PPARα activation; the test resulted in a balanced accuracy of 98%. The signature was then used to identify factors that activate or suppress PPARα in an annotated mouse liver/primary hepatocyte gene expression compendium of ~1850 biosets. In addition to the expected activation of PPARα by fibrate drugs, di(2-ethylhexyl) phthalate, and perfluorinated compounds, PPARα was activated by benzofuran, galactosamine, and TCDD and suppressed by hepatotoxins acetaminophen, lipopolysaccharide, silicon dioxide nanoparticles, and trovafloxacin. Additional factors that activate (fasting, caloric restriction) or suppress (infections) PPARα were also identified. This study 1) developed methods useful for future screening of environmental chemicals, 2) identified chemicals that activate or suppress PPARα, and 3) identified factors including diets and infections that modulate PPARα activity and would be hypothesized to affect chemical-induced PPARα activity.

The effects of perfluorinated chemicals on adipocyte differentiation in vitro.

The 3T3-L1 preadipocyte culture system has been used to examine numerous compounds that influence adipocyte differentiation or function. The perfluoroalkyl acids (PFAAs), used as surfactants in a variety of industrial applications, are of concern as environmental contaminants that are detected worldwide in human serum and animal tissues. This study was designed to evaluate the potential for PFAAs to affect adipocyte differentiation and lipid accumulation using mouse 3T3-L1 cells. Cells were treated with perfluorooctanoic acid (PFOA) (5-100 µM), perfluorononanoic acid (PFNA) (5-100 µM), perfluorooctane sulfonate (PFOS) (50-300 µM), perfluorohexane sulfonate (PFHxS) (40-250 µM), the peroxisome proliferator activated receptor (PPAR) PPARα agonist Wyeth-14,643 (WY-14,643), and the PPARγ agonist rosiglitazone. The PPARγ agonist was included as a positive control as this pathway is critical to adipocyte differentiation. The PPARα agonist was included as the PFAA compounds are known activators of this pathway. Cells were assessed morphometrically and biochemically for number, size, and lipid content. RNA was extracted for qPCR analysis of 13 genes selected for their importance in adipocyte differentiation and lipid metabolism. There was a significant concentration-related increase in cell number and decreased cell size after exposure to PFOA, PFHxS, PFOS, and PFNA. All four PFAA treatments produced a concentration-related decrease in the calculated average area occupied by lipid per cell. However, total triglyceride levels per well increased with a concentration-related trend for all compounds, likely due to the increased cell number. Expression of mRNA for the selected genes was affected by all exposures and the specific impacts depended on the particular compound and concentration. Acox1 and Gapdh were upregulated by all six compounds. The strongest overall effect was a nearly 10-fold induction of Scd1 by PFHxS. The sulfonated PFAAs produced numerous, strong changes in gene expression similar to the effects after treatment with the PPARγ agonist rosiglitazone. By comparison, the effects on gene expression were muted for the carboxylated PFAAs and for the PPARα agonist WY-14,643. In summary, all perfluorinated compounds increased cell number, decreased cell size, increased total triglyceride, and altered expression of genes associated with adipocyte differentiation and lipid metabolism.

Evaluating the additivity of perfluoroalkyl acids in binary combinations on peroxisome proliferator-activated receptor-α activation.

Perfluoroalkyl acids (PFAAs) are found globally in the environment, detected in humans and wildlife, and are typically present as mixtures of PFAA congeners. Mechanistic studies have found that responses to PFAAs are mediated in part by PPARα. Our previous studies showed that individual PFAAs activate PPARα transfected into COS-1 cells. The goal of the current study was to determine if binary combinations of perfluorooctanoic acid (PFOA) and another PFAA act in an additive fashion to activate PPARα in the mouse one-hybrid in vitro model. COS-1 cells were transiently transfected with mouse PPARα luciferase reporter construct and exposed to either vehicle control (0.1% DMSO or water), PPARα agonist (WY14643, 10 µM), PFOA at 1-128µM, perfluorononanoic acid (PFNA) at 1-128 µM, perfluorohexanoic acid (PFHxA) at 8-1024 µM, perfluorooctane sulfonate (PFOS) at 4-384 µM or perfluorohexane sulfonate (PFHxS) at 8-2048 µM to generate sigmoidal concentration-response curves. In addition, cells were exposed to binary combinations of PFOA+either PFNA, PFHxA, PFOS or PFHxS in an 8×8 factorial design. The concentration-response data for individual chemicals were fit to sigmoidal curves and analyzed with nonlinear regression to generate EC50s and Hillslopes, which were used in response-addition and concentration-addition models to calculate predicted responses for mixtures in the same plate. All PFOA+PFAA combinations produced concentration-response curves that were closely aligned with the predicted curves for both response addition and concentration addition at low concentrations. However, at higher concentrations of all chemicals, the observed response curves deviated from the predicted models of additivity. We conclude that binary combinations of PFAAs behave additively at the lower concentration ranges in activating PPARα in this in vitro system.

Evaluation of perfluoroalkyl acid activity using primary mouse and human hepatocytes.

While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is known about the biological activity of other perfluoroalkyl acids (PFAAs) detected in the environment. Using a transient transfection assay developed in COS-1 cells, our group has previously evaluated a variety of PFAAs for activity associated with activation of peroxisome proliferator-activated receptor alpha (PPARα). Here we use primary heptatocytes to further assess the biological activity of a similar group of PFAAs using custom designed Taqman Low Density Arrays. Primary mouse and human hepatoyctes were cultured for 48h in the presence of varying concentrations of 12 different PFAAs or Wy14,643, a known activator of PPARα. Total RNA was collected and the expression of 48 mouse or human genes evaluated. Gene selection was based on either in-house liver microarray data (mouse) or published data using primary hepatocytes (human). Gene expression in primary mouse hepatocytes was more restricted than expected. Genes typically regulated in whole tissue by PPARα agonists were not altered in mouse cells including Acox1, Me1, Acaa1a, Hmgcs1, and Slc27a1. Cyp2b10, a gene regulated by the constitutive androstane receptor and a transcript normally up-regulated by in vivo exposure to PFAAs, was also unchanged in cultured mouse hepatocytes. Cyp4a14, Ehhadh, Pdk4, Cpt1b, and Fabp1 were regulated as expected in mouse cells. A larger group of genes were differentially expressed in human primary hepatocytes, however, little consistency was observed across compounds with respect to which genes produced a significant dose response making the determination of relative biological activity difficult. This likely reflects weaker activation of PPARα in human versus rodent cells as well as variation among individual cell donors. Unlike mouse cells, CYP2B6 was up-regulated in human hepatocytes by a number of PFAAs as was PPARδ. Rankings were conducted on the limited dataset. In mouse hepatocytes, the pattern was similar to that previously observed in the COS-1 reporter cell assay. With the exception of PFHxA, longer chain PFAA carboxylates were the most active. The pattern was similar in human hepatocytes, although PFDA and PFOS showed higher activity than previously observed while PFOA showed somewhat less activity. These data reflect inherent challenges in using primary hepatocytes to predict toxicological response.

Testing for departures from additivity in mixtures of perfluoroalkyl acids (PFAAs).

This study is a follow-up to a paper by Carr et al. that determined a design structure to optimally test for departures from additivity in a fixed ratio mixture of four perfluoroalkyl acids (PFAAs) using an in vitro transiently-transfected COS-1 PPARα reporter model with a mixing ratio that is based on average serum levels in NHANES subjects. Availability of information regarding potential for additivity of PFAAs in mixtures is critically important for risk assessors who are concerned with the ability of the compounds to affect human health and impact ecological systems. It is clear that exposures are not to single compounds, but to mixtures of the PFAAs. This paper presents the results from the data collected using the design from Carr et al. along with subsequent analyses that were performed to classify the relationships among mixtures of PFAAs. A non-linear logistic additivity model was employed to predict relative luciferase units (RLU), an indicator of PPARα activation. The results indicated a less than additive relationship among the four PFAAs. To determine if the possible "antagonism" is from the competition among or between carboxylates and sulfonates, four different binary mixtures were also studied. There was a less than additive relationship in all four binary mixtures. These findings are generally similar to two other reports of interfering interactions between PFAAs in mixtures. The most conservative interpretation for our data would be an assumption of additivity (and lack of a greater than additive interaction), with a potential for antagonistic interactions.

Transcriptional ontogeny of the developing liver.

BACKGROUND: During embryogenesis the liver is derived from endodermal cells lining the digestive tract. These endodermal progenitor cells contribute to forming the parenchyma of a number of organs including the liver and pancreas. Early in organogenesis the fetal liver is populated by hematopoietic stem cells, the source for a number of blood cells including nucleated erythrocytes. A comprehensive analysis of the transcriptional changes that occur during the early stages of development to adulthood in the liver was carried out. RESULTS: We characterized gene expression changes in the developing mouse liver at gestational days (GD) 11.5, 12.5, 13.5, 14.5, 16.5, and 19 and in the neonate (postnatal day (PND) 7 and 32) compared to that in the adult liver (PND67) using full-genome microarrays. The fetal liver, and to a lesser extent the neonatal liver, exhibited dramatic differences in gene expression compared to adults. Canonical pathway analysis of the fetal liver signature demonstrated increases in functions important in cell replication and DNA fidelity whereas most metabolic pathways of intermediary metabolism were under expressed. Comparison of the dataset to a number of previously published microarray datasets revealed 1) a striking similarity between the fetal liver and that of the pancreas in both mice and humans, 2) a nucleated erythrocyte signature in the fetus and 3) under expression of most xenobiotic metabolism genes throughout development, with the exception of a number of transporters associated with either hematopoietic cells or cell proliferation in hepatocytes. CONCLUSIONS: Overall, these findings reveal the complexity of gene expression changes during liver development and maturation, and provide a foundation to predict responses to chemical and drug exposure as a function of early life-stages.

Activation of mouse and human peroxisome proliferator-activated receptor-alpha (PPARα) by perfluoroalkyl acids (PFAAs): further investigation of C4-C12 compounds.

Perfluorinated alkyl acids (PFAAs) are manufactured surfactants found globally in the environment and in tissues of humans and wildlife. Several PFAAs adversely affect rodents and activation of PPARα is thought to be their mode of action. Our previous study demonstrated that some PFAAs activate mouse and human PPARα in transiently transfected COS-1 cells. Here, we test more PFAAs for PPARα activation in the same system. Cells were transfected with either mouse or human PPARα-luciferase reporter plasmid, exposed the next day to either vehicle, PPARα agonist (WY14643), perfluoropentanoic acid (C5), perfluoroheptanoic acid (C7), perfluorooctanoic acid (C8), perfluoroundecanoic acid (C11), or perfluorododecanoic acid (C12) at concentrations from 0.5µM to 100µM, and luminescence was measured after 24h. C8 induced the highest activity for human PPARα, followed by C7, C5, and C11. C12 had little activity. C8 induced the highest activity for mouse PPARα, followed by C11, C7, C12 and C5. The two studies together found increasing activity of PPARα with increasing chain length of the PFAA up to perfluorononanoic acid (C9) and lower activity with longer chain PFAAs with both mouse and human PPARα.