John L. Newsted

Binding of perfluorinated fatty acids to serum proteins

Perfluorooctane sulfonic acid (PFOS) accumulates in the liver and blood of exposed organisms. The potential for these surfactant molecules to interfere with hormone/protein interactions in blood is of concern given the importance of these interactions. The PFOS binding to serum proteins was investigated by assessing its ability to displace a variety of steroid hormones from specific binding proteins in the serum of birds and fishes. Perfluorooctane sulfonic acid had only a weak ability to displace estrogen or testosterone from carp serum steroid binding proteins. Displacement of cortisone in avian sera occurred at relatively low PFOS concentrations. Corticosterone displacement potency increased with chain length, and sulfonic acids were more potent than carboxylic acids. The PFOS concentrations estimated to cause these effects were 320 microM or greater, equivalent to serum concentrations greater than 160 mg/L. Using mass spectrometry and direct in vitro binding assays, PFOS was demonstrated to bind strongly to bovine serum albumin (BSA) in a 1:1 stoichiometric ratio. It appears that PFOS in serum is in general bound to albumins. Concentrations of PFOS required to saturate albumin would be in excess of 50 to 100 mg/L. Based on current environmental concentrations, it is unlikely that PFOS would cause displacement of hormones from serum proteins in wildlife.

Alterations in cell membrane properties caused by perfluorinated compounds.

The recent detection of perfluorinated compounds (PFCs) in wildlife from even remote locations has spurred interest in the environmental occurrence and effects of these chemicals. While the global distribution of PFCs is increasingly understood, there is still little information available on their effects on wildlife. The amphiphillic nature of PFCs suggests that their effects could be primarily on cell membranes. In this study we measured the effects of PFCs on membrane fluidity and mitochondrial membrane potential using flow cytometry and effects on membrane permeability using cell bioassay procedures (H4IIE, MCF-7, PLHC-1). Of the PFCs tested, only perfluorooctane sulfonic acid (PFOS) increased the permeability of cell membranes to the hydrophobic ligands used. Three PFCs were tested in the membrane fluidity assay: PFOS, perfluorohexane sulfonic acid (PFHS), and perfluorobutane sulfonic acid (PFBS). PFOS increased membrane fluidity in fish leukocytes in a dose-dependent fashion, while PFHS and PFBS had no effect in the concentration range tested. The lowest effective concentrations for the membrane fluidity effects of PFOS were 5-15 mg/l. Effects on mitochondrial membrane potential occurred in the same concentration range as effects on membrane fluidity. This suggests that PFOS effects membrane properties at concentrations below those associated with other adverse effects.

Aquatic Toxicology of Perfluorinated Chemicals

Perfluorinated compounds (PFCs) are fluorinated at all of the valence electrons of the carbon atoms in organic molecules, or at least a portion of the molecule is perfluorinated (Fig. 1). All PFCs are synthetic and many have been used in commercially available products or released as byproducts. A partial list of the compounds that are known to have been manufactured and/or released into the environment is given in Table 1. These compounds vary in structure, and thus exhibit different environmental fates and toxicities. Unfortunately, there is presently little information on the chemical–physical properties of most PFCs, and even less toxicity information is available on these compounds.

Ecotoxicological Evaluation of Perfluorooctanesulfonate (PFOS)

Based on available toxicity data, protective screening-level concentrations of PFOS were calculated for aquatic and terrestrial organisms. Using the Great Lakes Initiative, water concentrations of PFOS were calculated to protect aquatic plants and animals. The screening plant value (SPV) protective of aquatic algae and macrophytes was calculated as 2.3 mg PFOS/L. The secondary chronic value protective of aquatic organisms was 1.2 microg PFOS/L. The screening-value water concentrations less than or equal to 1.2 microg PFOS/L would not pose a potential risk to aquatic organisms. Because the aquatic benchmark is based on the most sensitive species, this benchmark should also be protective of other aquatic organisms, including amphibians. The tissue-based TRV for fish was determined to be 87 mg PFOS/kg ww. For terrestrial plants, a screening benchmark was determined to be 1.3 mg PFOS/kg soil ww or 1.5 mg PFOS/kg soil dw, whereas for soil invertebrates such as earthworms the benchmark value was 39 mg PFOS/kg dw soil or 33 mg PFOS/kgww soil. For avian species, dietary, ADI, and egg yolk-based benchmarks were determined as 0.28mg PFOS/kg diet, 0.021mg PFOS/kg bw/d, and 1.7 microg PFOS/mL yolk, respectively. Benchmarks for serum and liver for the protection of avian species were 1.0 microg PFOS/mL and 0.6 microg PFOS/gww, respectively. However, no-effect levels in laboratory studies suggest actual population-level effects would not be expected to occur until a concentration of 6.0mg PFOS/kg in the diet, 5.0 microg PFOS/gww in the liver, or 9.0 microg PFOS/mL in the serum was exceeded, thus indicating the conservative nature of the benchmarks.

Perfluorinated compounds in streams of the Shihwa industrial zone and Lake Shihwa, South Korea

Concentrations of perfluorinated alkyl compounds (PFAs), including perfluorooctane sulfonate (PFOS), perfluorohexanesulfonate, perfluorobutanesulfonate, perfluorooctanesulfonamide, perfluorodecanoate, perfluorononanoic acid, perfluorooctanoate (PFOA), perfluoroheptanoate, and perfluorohexanoate, were measured in the streams of the Shihwa and Banweol industrial areas on the west coast of South Korea as well as the adjacent Lake Shihwa (an artificial lake) and Gyeonggi Bay. Perfluorinated alkyl compounds were concentrated from water using solid-phase extraction and were identified and quantified by liquid chromatography/ triple-quadrapole tandem mass spectrometry. Of the PFAs measured, PFOS and PFOA occurred at the greatest concentrations. Concentrations of PFOS ranged from 2.24 to 651 ng/L, and concentrations of PFOA ranged from 0.9 to 62 ng/L. The concentrations of PFOS observed in Lake Shihwa were among the greatest ever measured in the environment. These results suggest local industrial sources of PFOS and PFOA as well as other PFAs. Because of dilution, the greatest concentrations occur in a rather restricted area, near the points of discharge of the streams that empty into the lake. The greatest measured concentration of PFOS exceeded the threshold for effects predicted for predatory birds consuming aquatic organisms continuously exposed to this level.

Real-time PCR array to study effects of chemicals on the Hypothalamic-Pituitary-Gonadal axis of the Japanese medaka

This paper describes the development and validation of a PCR array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic-pituitary-gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka (Oryzias latipes). The Japanese medaka HPG-PCR array combines the quantitative performance of SYBR Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG-PCR array was evaluated by examining effects of two model compounds, the synthetic estrogen, 17alpha-ethinylestradiol (EE2) and the anabolic androgen, 17beta-trenbolone (TRB) on the HPG axis of the Japanese medaka. Four-month-old medaka was exposed to three concentrations of EE2 (5, 50, 500 ng/L) or TRB (50, 500, 5000 ng/L) for 7d in a static renewal exposure system. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH RI and testicular CYP17. The down-regulation of AR-alpha expression in brain of EE2-exposed males was associated with suppression of male sexual behavior. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH RII and ovary steroidogenic CYP19A. Overall, the results suggested that the Japanese medaka HPG-PCR array has potential not only as a screening tool of potential endocrine-disrupting chemicals but also in elucidating mechanisms of action.

Contaminants in fishes from Great Lakes-influenced sections and above dams of three Michigan rivers. II: Implications for health of mink

Populations of mink (Mustela vison) have declined in many areas of the world. Such declines have been linked to exposures to synthetic, halogenated hydrocarbons. In the Great Lakes region, mink are fewer in areas along the shore of the Great Lakes and their tributaries where mink have access to fish from the Great Lakes. Recently, there has been discussion of the relative merits of passage of fishes around hydroelectric dams on rivers in Michigan. A hazard assessment was conducted to determine the potential for adverse effects on mink, which could consume such fishes from above or below dams on the rivers. Concentrations of organochlorine insecticides, polychlorinated biphenyls (PCBs), 2,3,7,8-tetrachloridibenzo-p-dioxin equivalents (TCDD-EQ), and total mercury were measured in composite samples of fishes from above or below hydroelectric dams on the Manistee and Muskegon Rivers, which flow into Lake Michigan, and the Au Sable River, which flows into Lake Huron. Concentrations of organochlorine insecticides, PCBs, and TCDD-EQ were all greater in fishes from below the dams than those from above. Concentrations of neither organochlorine insecticides nor mercury in fishes are currently a risk to mink above or below the dams. All of the species of fishes collected from downstream of the dams contained concentrations of PCBs and TCDD-EQ, which represent a hazard to mink. The hazard index for PCBs was less than one for the average of all species from the upstream reaches of the Manistee and Au Sable Rivers, but not the Muskegon. The hazard index (concentration in fish/NOAEC) was greater than 1 for all of the species collected from below the dams, in all three rivers. The greatest hazard index was observed for carp (Cyprinus carpio) downstream on the Muskegon River. Because the concentrations of PCBs used in the hazard assessment were corrected for relative toxic potencies, the hazard ratios based on PCBs should be similar to those based on TCDD-EQ. This was found to be true. Thus, either total PCBs or TCDD-EQ could be used as the critical toxicant in the hazard assessment. However, if uncorrected concentrations of PCBs, expressed as Aroclors®, were used in the hazard assessment, the toxicity of the weathered mixture would have been underestimated by approximately five-fold, and, in that instance, TCDD-EQ would be the critical contaminant for the hazard assessment. The average maximum allowable percentage of fish from above the dams, which would result in no observable adverse effects of TCDD-EQ, was 70%. Based on the average TCDD-EQ concentrations in the fishes, an average of 8.6% of the diet could be made up of fishes from below dams on the rivers. The most restrictive daily allowable intakes were for carp on the Muskegon and steelhead trout (Onchorhyncus mykiss) on the Manistee Rivers. Only 2.7% of the diet could be made up of these two species from influenced portion of the Au Sable River, they would be exposed to 390 μg PCBs and 8.55 ng of TCDD-EQ per day, respectively (Giesy et al. 1994b). Thus, it would take 15.1 or 77 days for mink to receive their total annual dose of PCBs or TCDD-EQ, respectively. At least for chinook salmon, the critical contaminant for the purposes of hazard assessment would be total concentrations of PCBs. Consuming chinook salmon for as little as 2 weeks would deliver the annual allowable dose of PCBs to mink.

Effects of perfluorooctane sulfonate on mallard and northern bobwhite quail exposed chronically via the diet

Adult mallard ducks and northern bobwhite quail were exposed to 0, 10, 50, or 150mg perfluorooctane sulfonate (PFOS)/kg in the diet for up to 21 weeks. Adult health, body and liver weight, feed consumption, gross morphology and histology of body organs, and reproduction were examined. Due to mortality, birds exposed to 50 or 150mg PFOS/kg feed were terminated by Week 7. In quail, the lowest observable adverse effect level (LOAEL) was 10mg PFOS/kg feed based on decreased survivorship of 14-day-old quail offspring. For adult female quail fed 10mg/kg feed, there was a slight but statistically significantly PFOS-related increase in liver weight when compared to controls. When liver weight was normalized to body weight, the statistically significant differences were still observed indicating that PFOS affected liver size. However, no other pathological effects were observed livers of quail from this treatment group which suggests that this enlargement may have been an adaptive response. For adult mallards, no treatment-related effects on feed consumption, body or liver weight, growth, or reproductive performance were observed. There was a slightly greater incidence of small testes (length) in adult male mallards and quail exposed to 10mg PFOS/kg, feed when compared to controls. However, spermatogenesis was not affected and there was no effect on the rates of egg fertilization. Due to transfer to eggs, concentrations of PFOS measured in the liver and blood at study termination were greater in male birds than female birds.

2,3,7,8-Tetrachlorodibenzo-p-dioxin equivalents in tissues of birds at Green Bay, Wisconsin, USA

The environment has become contaminated with complex mixtures of planar, chlorinated hydrocarbons (PCHs) such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and structurally similar compounds. Because the potencies of individual congeners to cause the same adverse effects vary greatly and the relative as well as absolute concentrations of individual PCH vary among samples from different locations, it is difficult to assess the toxic effects of these mixtures on wildlife. These compounds can cause a number of adverse effects, however, because the toxic effects which occur at ecologically-relevant concentrations such as embryo-lethality and birth defects appear to be mediated through the same mechanism, the potency of individual congeners can be reported relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) which is the most toxic congener in the PCH class. The concentations of 2,3,7,8-TCDD Equivalents (TCDD-EQ) were determined in the tissues of aquatic and terrestrial birds of Green Bay, Wisconsin by the H4IIE bioassay system and compared toxic equivalency factors (TEFs) with the concentration predicted by the use of toxic equivalency factors applied to concentrations of PCH, which were determined by instrumental analyses. Concentrations of TCDD-EQ ranged from 0.52 to 440 ng/kg, wet weight. The greatest concentrations occurred in the fish-eating birds. Concentrations of TCDD-EQ, which were determined by the two methods were significantly correlated, but the additive model which used the TEFs with concentrations of measured PCB, PCDD and PCDF congeners underestimated the concentrations of TCDD-EQ measured by the H4IIE bioassay by an average of 57%. This is thought to be due to contributions from un-quantified PCH, which are known to occur in the environment. Of the quantified PCH congeners, PCDD and PCDF contributed a small portion of the TCDD-EQ in the aquatic birds, while most of the TCDD-EQ were due to non-ortho-substituted PCBs. In the terrestrial birds, the proportion of the TCDD-EQ contributed by the PCDD and PCDF was greater.

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Background, goals, and scopeIn response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.MethodsA previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and “negative” chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.ResultsWith a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.Discussion and conclusionsWith one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).PerspectivesBased on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.