Dermal penetration and metabolism of five glycidyl ethers in human, rat and mouse skin

1. Glycidyl ethers (GE), an important class of industrial chemicals, are considered to be potentially mutagenic in vivo because some GE have been shown to be direct mutagens in short-term in vitro tests. 2. The percutaneous penetration and metabolism of representatives of different classes of GE was studied in the fresh, full-thickness C3H mouse, and dermatomed human and Fisher 344 rat skin to determine the apparent permeability constants, lag times and metabolic profiles. 3. Five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C12GE) and o-cresol (o-CGE), were synthesized by reaction of their alcohols with epichlorohydrin. Their radiolabelled analogues were synthesized with a 14C-label using [U-14C]-epichlorohydrin. 4. There was a large variation (four orders of magnitude) in percutaneous penetration between the five GE. In general, penetration through full-thickness mouse skin was higher than through dermatomed rat skin, whereas dermatomed human skin was the least permeable. The permeability increased in the order YX4000 < BADGE < C12GE < o-CGE < HDDGE. 5. The relative skin permeability of the five GE could be explained for a significant part by the lipophilicity, expressed as log P(o/w), in combination with the molecular weight of the compounds. 6. During skin penetration, all GE were extensively metabolized to their corresponding (bis-)diols. Virtually no YX4000, and only very small amounts of C12GE and BADGE, penetrated the skin unchanged, but significant amounts of HDDGE and o-CGE penetrated the skin unchanged. For o-CGE, but none of the other GE, the percentage of the applied dose that penetrated the skin unchanged increased over time. 7. The large variation in response observed with the five selected GE indicates that GE should not be considered as a single class of compounds but rather on the basis of their individual properties.     

Dermal penetration and metabolism of five glycidyl ethers in human,rat and mouse skin

1. Glycidyl ethers (GE), an important class of industrial chemicals, are considered to be potentially mutagenic in vivo because some GE have been shown to be direct mutagens in short-term in vitro tests. 2. The percutaneous penetration and metabolism of representatives of different classes of GE was studied in the fresh, full-thickness C3H mouse, and dermatomed human and Fisher 344 rat skin to determine the apparent permeability constants, lag times and metabolic profiles. 3. Five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C₁₂GE) and o-cresol (o-CGE), were synthesized by reaction of their alcohols with epichlorohydrin. Their radiolabelled analogues were synthesized with a ¹⁴Clabel using [U-¹⁴C]-epichlorohydrin. 4. There was a large variation (four orders of magnitude) in percutaneous penetration between the five GE. In general, penetration through full-thickness mouse skin was higher than through dermatomed rat skin, whereas dermatomed human skin was the least permeable. The permeability increased in the order YX4000<BADGE<C₁₂GE<o CGE<HDDGE. 5. The relative skin permeability of the five GE could be explained for a significant part by the lipophilicity, expressed as log Po/w, in combination with the molecular weight of the compounds. 6. During skin penetration, all GE were extensively metabolized to their corresponding (bis-)diols. Virtually no YX4000, and only very small amounts of C₁₂GE and BADGE, penetrated the skin unchanged, but significant amounts of HDDGE and o-CGE penetrated the skin unchanged. For o-CGE, but none of the other GE, the percentage of the applied dose that penetrated the skin unchanged increased over time. 7. The large variation in response observed with the five selected GE indicates that GE should not be considered as a single class of compounds but rather on the basis of their individual properties.     

Mode of Action analysis of perfluorooctanoic acid (PFOA) tumorigenicity and Human Relevance

Perfluorooctanoic acid (PFOA) is an environmentally persistent chemical used in the manufacturing of a wide array of industrial and commercial products. PFOA has been shown to induce tumors of the liver, testis and pancreas (tumor triad) in rats following chronic dietary administration. PFOA belongs to a group of compounds that are known to activate the PPARα receptor. The PPARα activation Mode of Action was initially addressed in 2003 [9] and further refined in subsequent reviews [92-94]. In the intervening time, additional information on PFOA effects as well as a further refinement of the Mode of Action framework warrants a re-examination of this compound for its cancer induction Mode of Action. This review will address the rodent (rat) cancer data and cancer Mode of Action of PFOA for tumors of the liver, testes and pancreas.     

Dietary exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS): a review of recent literature

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) are part of a family of perfluorinated chemicals that are used in protective coatings of all sorts. Due to their unique physicochemical characteristics, such as chemical and thermal inertness, low surface energy, and high surface-active properties, they have been used in a very wide variety of consumer and industrial applications. However, several perfluorinated compounds have been shown to be persistent, bioaccumulative, and toxic. Therefore, the determination of amounts of PFOA and PFOS in the food stuffs is very important. The information reviewed in this paper suggests that continued human exposure to even relatively low concentrations of PFOA and PFOS in foodstuffs results in elevated body burdens that may increase the risk of health effects.     

Gene expression profiles in rat liver treated with perfluorooctanoic acid (PFOA).

Perfluorooctanoic acid (PFOA; Pentadecafluorooctanoic acid) is widely used in various industrial applications. It is persistent in the environment and does not appear to undergo further degradation or transformation. PFOA is found in tissues including blood of wildlife and humans; however, the environmental fate and biological effects of PFOA remain unclear. Microarray techniques of gene expression have become a powerful approach for exploring the biological effects of chemicals. Here, the Affymetrix, Inc. rat genome 230 2.0 GeneChip was used to identify alterations in gene regulation in Sprague-Dawley rats treated with five different concentrations of PFOA. Male rats were exposed by daily gavage to 1, 3, 5, 10, or 15 mg PFOA/kg, body weight (bw)/day for 21 days and at the end of the exposure, liver was isolated and total liver RNA were used for the gene chip analysis. Over 500 genes, whose expression was significantly (p < 0.0025) altered by PFOA at two-fold changes compared to control, were examined. The effects were dose-dependent with exposure to 10 mg PFOA/kg, bw/day, causing alteration in expression of the greatest number of genes (over 800). Approximately 106 genes and 38 genes were consistently up- or down-regulated, respectively, in all treatment groups. The largest categories of induced genes were those involved in transport and metabolism of lipids, particularly fatty acids. Other induced genes were involved in cell communication, adhesion, growth, apoptosis, hormone regulatory pathways, proteolysis and peptidolysis and signal transduction. The genes expression of which was suppressed were related to transport of lipids, inflammation and immunity, and especially cell adhesion. Several other genes involved in apoptosis; regulation of hormones; metabolism; and G-protein coupled receptor protein signaling pathways were significantly suppressed.     

Effects of perfluorooctanoic acid (PFOA) on expression of peroxisome proliferator-activated receptors (PPAR) and nuclear receptor-regulated genes in fetal and postnatal CD-1 mouse tissues

PPARs regulate metabolism and can be activated by environmental contaminants such as perfluorooctanoic acid (PFOA). PFOA induces neonatal mortality, developmental delay, and growth deficits in mice. Studies in genetically altered mice showed that PPARα is required for PFOA-induced developmental toxicity. In this study, pregnant CD-1 mice were dosed orally from GD1 to 17 with water or 5mg PFOA/kg to examine PPARα, PPARβ, and PPARγ expression and profile the effects of PFOA on PPAR-regulated genes. Prenatal and postnatal liver, heart, adrenal, kidney, intestine, stomach, lung, spleen, and thymus were collected at various developmental ages. RNA and protein were examined using qPCR and Western blot analysis. PPAR expression varied with age in all tissues, and in liver PPARα and PPARγ expression correlated with nutritional changes as the pups matured. As early as GD14, PFOA affected expression of genes involved in lipid and glucose homeostatic control. The metabolic disruption produced by PFOA may contribute to poor postnatal survival and persistent weight deficits of CD-1 mouse neonates.     

In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL‐2 production in human T‐cells

Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)‐2 production in the human Jurkat T‐cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD‐3/anti CD‐28, or anti CD‐3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml^?1 PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml^?1 PFOA. Jurkat cells stimulated with PHA/PMA or anti CD‐3 exhibited decreased IL‐2 production beginning at 50 µg PFOS ml^?1 and 5 µg PFOS ml^?1 respectively, but stimulation with anti‐CD3/anti‐CD28 resulted in no changes compared with the control. Addition of the PPAR‐alpha antagonist GW6471 to PFOS‐dosed cells stimulated with PHA/PMA resulted in decreases in IL‐2 production starting at 50 µg PFOS ml^?1, which suggests PFOS affected T‐cell IL‐2 production via PPAR‐alpha‐independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL‐2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL‐2 production, but PFOS suppressed IL‐2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL‐2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

The relationship between birth weight, gestational age and perfluorooctanoic acid (PFOA)-contaminated public drinking water

BackgroundRecent studies have examined the associations between perfluorooctanoic acid (PFOA) levels in cord blood and maternal plasma with lowered birth weight and gestational age in humans; however, no study has examined these effects in a population of known high PFOA exposure. Residents drinking PFOA-contaminated water from the Little Hocking Water Association (LHWA) in Washington County, Ohio have serum PFOA levels approximately 80 times those in the general U.S. population.ObjectivesTo compare birth weights and gestational ages of neonates born to mothers residing in zip codes with water service provided completely, partially or not at all by the LHWA.MethodsMultiple logistic and linear regression analyses were performed on singleton neonatal birth weight data supplied by the Ohio Department of Health to examine the associations between LHWA water service category (used as a surrogate for PFOA exposure) with mean birth weight, mean gestational age, the likelihood of low birth weight (<2500 g), and the likelihood of preterm birth (<37 completed weeks of gestation). All models were adjusted for maternal age, gestational age, sex, race and population-level socioeconomic status.ResultsThe incidence of low birth weight, preterm birth, mean birth weight and mean gestational age of neonates did not significantly differ among water service categories.ConclusionMarkedly elevated PFOA exposure, as categorized by water service category, is not associated with increased risk of lowered birth weight or gestational age. This study does not confirm earlier findings of an association between PFOA and lowered birth weight observed at normal population levels.     

The role of transcription factor Nrf2 in the toxicity of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in C57BL/6 mouse astrocytes

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are members of perfluoroalkyl substances (PFAS). This study aimed to determine the protective role of Nrf2 against the toxicity of these agents. Nrf2-/- and wild-type astrocytes were exposed to PFOS (75−600 μM) and PFOA (400−1000 μM) for 24 h. Lactate dehydrogenase (LDH) release was significantly higher in nrf2-/- than in the wild-type astrocytes. Exposure to 600 μM PFOS and 800 μM PFOA showed significant increases in reactive oxygen species, lipid peroxidation, and apoptosis in nrf2-/- astrocytes as compared to wild-type astrocytes. The GSH/GSSG ratio was significantly decreased in nrf2-/- astrocytes when compared to wild-type astrocytes. Additionally, PFOS and PFOS caused dramatic ultrastructural alterations to the mitochondria. BHT pretreatment in wild-type cells decreased ROS production with exposure to both agents. Results of the present study conclude that PFOS and PFOA are cytotoxic to astrocytes and that nrf2 -/- cells are more sensitive to toxicity by these agents.     

A methodology for estimating human exposure to perfluorooctanoic acid (PFOA): a retrospective exposure assessment of a community (1951-2003)

Perfluorooctanoic acid (PFOA) is a persistent chemical that was recently shown to be widely distributed in the ambient environment. Because of concerns about the possible adverse health effects on persons exposed to PFOA, a retrospective exposure assessment was conducted for a population of about 50,000 persons who reside near one of the facilities where this chemical was used. No similar study of any chemical with the properties of PFOA had ever been performed; thus, several novel methods were developed and applied in this analysis. Historical records of the emissions from the facility were the basis for the estimates of the potential intake of (PFOA) by residents over the past 53 yr. Various well-accepted environmental models were dynamically combined in order to estimate the concentrations in all relevant environmental media including ambient air, surface soil, drinking water, and homegrown vegetables. Following considerable analyses, particulate deposition from facility air emissions to soil and the subsequent transfer of the chemical through the soil was determined to be the most likely source of PFOA that was detected in groundwater. The highest off-site environmental concentrations were predicted to occur about 1 mile away. For this approximately square mile area, during the time period 1951-2003, the model-estimated average air concentration was 0.2 microg/m3, the estimated surface soil concentration was 11 microg/kg, and the estimated drinking water concentration was 4 microg/L. Similar data were generated for 20 additional geographical areas around the facility. Comparison of measured PFOA concentrations in groundwater in the various water districts indicated that the models appeared to overpredict recent groundwater concentrations by a factor of 3 to 5. The predicted historical lifetime and average daily estimates of PFOA intake by persons who lived within 5 miles of the plant over the past 50 yr were about 10,000-fold less than the intake of the chemical not considered as a health risk by an independent panel of scientists who recently studied PFOA.     

Dermal penetration of bisphenol A in human skin contributes marginally to total exposure

Bisphenol A (BPA) is ubiquitous and many exposure scenarios have been described during the last decades. While oral uptake is considered as the major route of exposure, the contribution of skin penetration has been recently discussed. In the present study, the dermal penetration rate of BPA has been determined in human skin in an in vitro test method according to the OECD Test Guideline 428. This analysis resulted in penetration of 8.6% and a total amount of bio-available BPA of 9.3% of the dose applied after 24h incubation under realistic exposure conditions. This confirms that the systemic exposure to BPA via the skin contributes in a negligible way to total systemic BPA exposure.     

Congenital anomalies,labor/delivery complications,maternal risk factors and their relationship with perfluorooctanoic acid (PFOA)-contaminated public drinking water

BackgroundWe have previously examined the associations between perfluorooctanoic acid (PFOA) exposure, birth weight and gestational age in individuals exposed to PFOA-contaminated residential drinking water from the Little Hocking Water Association (LHWA). In this investigation, we expand the scope of our analysis to examine the associations between PFOA, congenital anomalies, labor and delivery complications and maternal risk factors.ObjectivesTo compare the likelihood of congenital anomalies, labor and delivery complications and maternal risk factors in neonates and their mothers residing in zip codes with public water service provided completely, partially or not at all by the LHWA.MethodsLogistic regression analyses were performed on singleton neonatal birth outcome data supplied by the Ohio Department of Health to examine the associations between LHWA water service category and the outcomes of interest. When possible, models were adjusted for maternal age, preterm birth, neonatal sex, race, maternal education, alcohol use, tobacco use and diabetic status.ResultsIncreased PFOA exposure, as assessed by water service category, was not associated with an overall increase in the likelihood of congenital anomalies or any specific diagnosis (adjusted OR: 1.4, 95% CI: 0.34–3.3). The overall likelihood of labor and delivery complications was significantly lower among mothers with water service provided by the LHWA, as compared to mothers not serviced by the LHWA (adjusted OR: 0.65, 95% CI: 0.46–0.92). A significant increase in the likelihood of anemia (crude OR: 11, 95% CI: 1.8–64) and dysfunctional labor (crude OR: 5.3, 95% CI: 1.2–24) was noted for mothers residing within zip codes serviced by the LHWA, but the number of reported cases was very small.ConclusionAt the levels measured in the LHWA, we conclude that PFOA is not associated with increased risk of congenital anomalies, most labor and delivery complications and maternal risk factors. Additional research is required to assess the observed associations between PFOA, anemia and dysfunctional labor.     

Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo

This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.     

Effects of perfluorooctanoic acid exposure during pregnancy in the mouse.

Perfluorooctanoic acid (PFOA), a member of the perfluoroalkyl acids that have wide commercial applications, has recently been detected in humans and wildlife. The current study characterizes the developmental toxicity of PFOA in the mouse. Timed-pregnant CD-1 mice were given 1, 3, 5, 10, 20, or 40 mg/kg PFOA by oral gavage daily from gestational day (GD) 1 to 17; controls received an equivalent volume (10 ml/kg) of water. PFOA treatment produced dose-dependent full-litter resorptions; all dams in the 40-mg/kg group resorbed their litters. Weight gain in dams that carried pregnancy to term was significantly lower in the 20-mg/kg group. At GD 18, some dams were sacrificed for maternal and fetal examinations (group A), and the rest were treated once more with PFOA and allowed to give birth (group B). Postnatal survival, growth, and development of the offspring were monitored. PFOA induced enlarged liver in group A dams at all dosages, but did not alter the number of implantations. The percent of live fetuses was lower only in the 20-mg/kg group (74 vs. 94% in controls), and fetal weight was also significantly lower in this group. However, no significant increase in malformations was noted in any treatment group. The incidence of live birth in group B mice was significantly lowered by PFOA: ca. 70% for the 10- and 20-mg/kg groups compared to 96% for controls. Postnatal survival was severely compromised at 10 or 20 mg/kg, and moderately so at 5 mg/kg. Dose-dependent growth deficits were detected in all PFOA-treated litters except the 1-mg/kg group. Significant delays in eye-opening (up to 2-3 days) were noted at 5 mg/kg and higher dosages. Accelerated sexual maturation was observed in male offspring, but not in females. These data indicate maternal and developmental toxicity of PFOA in the mouse, leading to early pregnancy loss, compromised postnatal survival, delays in general growth and development, and sex-specific alterations in pubertal maturation.     

Activation of mouse and human peroxisome proliferator-activated receptors (alpha, beta/delta, gamma) by perfluorooctanoic acid and perfluorooctane sulfonate.

This study evaluates the potential for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) to activate peroxisome proliferator-activated receptors (PPARs), using a transient transfection cell assay. Cos-1 cells were cultured in Dulbecco's Minimal Essential Medium (DMEM) with fetal bovine serum in 96-well plates and transfected with mouse or human PPARalpha, beta/delta, or gamma reporter plasmids. Transfected cells were exposed to PFOA (0.5-100 microM), PFOS (1-250 microM), positive controls (i.e., known agonists and antagonists), and negative controls (i.e., DMEM, 0.1% water, and 0.1% dimethyl sulfoxide). Following treatment for 24 h, activity was measured using the Luciferase reporter assay. In this assay, PFOA had more transactivity than PFOS with both the mouse and human PPAR isoforms. PFOA significantly increased mouse and human PPARalpha and mouse PPARbeta/delta activity relative to vehicle. PFOS significantly increased activation of mouse PPARalpha and PPARbeta/delta isoforms. No significant activation of mouse or human PPARgamma was observed with PFOA or PFOS. The PPARalpha antagonist, MK-886, significantly suppressed PFOA and PFOS activity of mouse and human PPARalpha. The PPARgamma antagonist, GW9662, significantly suppressed PFOA activity on the human isoform. In conclusion, this study characterized the dose response and differential activation of mouse and human PPARalpha, beta/delta, gamma by PFOA and PFOS. While this model allows opportunities to compare potential activation by perfluoroalkyl acids, it only evaluates the interaction and activation of the PPAR reporter constructs and is not necessarily predictive of a toxicological response in vivo.     

The mammary gland is a sensitive pubertal target in CD-1 and C57Bl/6 mice following perinatal perfluorooctanoic acid (PFOA) exposure

Perfluorooctanoic acid (PFOA) is a developmental toxicant in mice, with varied strain outcomes depending on dose and period of exposure. The impact of PFOA on female mouse pubertal development at low doses (≤1 mg/kg) has yet to be determined. Therefore, female offspring from CD-1 and C57Bl/6 dams exposed to PFOA, creating serum concentrations similar to humans, were examined for pubertal onset, including mammary gland development. Pups demonstrated a shorter PFOA elimination half-life than that reported for adult mice. Prenatal exposure to PFOA caused significant mammary developmental delays in female offspring in both strains. Delays started during puberty and persisted into young adulthood; severity was dose-dependent. Also an evaluation of female serum hormone levels and pubertal timing onset revealed no effects of PFOA compared to controls in either strain. These data suggest that the mammary gland is more sensitive to early low level PFOA exposures compared to other pubertal endpoints, regardless of strain.     

Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay

The estrogenic and antiestrogenic potential of perfluorooctanoic acid (PFOA) was assessed using an immature mouse uterotrophic assay and by histologic evaluation of the uterus, cervix and vagina following treatment. Female offspring of CD-1 dams were weaned at 18days old and assigned to groups of equal weight, and received 0, 0.01, 0.1, or 1mg PFOA/kg BW/d by gavage with or without 17-β estradiol (E(2), 500μg/kg/d) from PND 18-20 (n=8/treatment/block). At 24h after the third dose (PND 21), uteri were removed and weighed. Absolute and relative uterine weights were significantly increased in the 0.01mg/kg PFOA only group. Characteristic estrogenic changes were present in all E(2)-treated mice; however, they were minimally visible in the 0.01 PFOA only mice. These data suggest that at a low dose PFOA produces minimal histopathologic changes in the reproductive tract of immature female mice, and does not antagonize the histopathologic effects of E(2).