Evaluation of the acute toxicity of perfluorinated carboxylic acids using eukaryotic cell lines, bacteria and enzymatic assays

Environmental Toxicology and Pharmacology 23 (2007) 279–285

Evaluation of the acute toxicity of perfluorinated carboxylic acids using eukaryotic cell lines, bacteria and enzymatic assays E. Mulkiewicz a , B. Jastorff b , A.C. Składanowski c , K. Kleszczy´nski c , P. Stepnowski a,∗ a

Faculty of Chemistry, University of Gda´nsk, Sobieskiego 18, PL-80-952 Gda´nsk, Poland Centre for Environmental Research and Technology (UFT), University of Bremen, D-28359 Bremen, Leobener Str., Germany c Intercollegiate Faculty of Biotechnology, Medical University of Gda´ nsk & University of Gda´nsk, PL 80-211 Gda´nsk, ul. D˛ebinki 1, Poland b

Received 9 August 2006; received in revised form 30 October 2006; accepted 7 November 2006 Available online 12 November 2006

Abstract The acute biological activity of a homologous series of perfluorinated carboxylic acids – perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) – was studied. To analyze the potential risk of the perfluorinated acids to humans and the environment, different in vitro toxicity test systems were employed. The cytotoxicity of the chemicals towards two different types of mammalian cell lines and one marine bacteria was investigated. The viability of cells from the promyelocytic leukemia rat cell line (IPC-81) and the rat glioma cell line (C6) was assayed calorimetrically with WST-1 reagent. The evaluation was combined with the Vibrio fischeri acute bioluminescence inhibition assay. The biological activity of the compounds was also determined at the molecular level with acetylcholinesterase and glutathione reductase inhibition assays. This is the first report of the effects of perfluorinated acids on the activity of purified enzymes. The results show these compounds have a very low acute biological activity. The observed effective concentrations lie in the millimole range, which is well above probable intracellular concentrations. A relationship was found between the toxicity of the perfluorinated carboxylic acids and the perfluorocarbon chain length: in every test system applied, the longer the perfluorocarbon chain, the more toxic was the acid. The lowest effective concentrations were thus recorded for perfluorononanoic and perfluorodecanoic acids. © 2006 Elsevier B.V. All rights reserved. Keywords: Toxicity; Perfluorinated acids; IPC-81; C6; Vibrio fischeri; AChE; GR

1. Introduction In recent years, growing concern has been expressed about perfluorinated compounds, the global production of which has increased since the 1970s. With their unique physicochemical properties, they have a broad spectrum of applications as surfactants, refrigerants and polymers, and also as components of pharmaceuticals, fire retardants, lubricants, adhesives, paints, cosmetics, agrochemicals and food packaging (Key et al., 1997). Owing to the presence of high-energy carbon-fluorine bonds (the strongest of all covalent bonds), perfluorochemicals are stable and persistent in the environment (Banks et al., 1994). They do not undergo photolysis, hydrolysis, defluorination or phase II metabolism (Kudo and Kawashima, 2003). It is common knowledge that biodegradation is restricted to the non-perfluorinated

∗

Corresponding author. Tel.: +48 58 523 5448; fax: +48 58 523 5472. E-mail address: [email protected] (P. Stepnowski).

1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.11.002

part of the molecules (Hagen et al., 1981). Furthermore, during microbial degradation, perfluorochemicals tend to be slowly converted to more bioaccumulative and more toxic products (Dimitrov et al., 2004). Perfluorochemicals have been detected not only in the physical environment, but also in humans and wildlife. These contaminants have been found in oceanic waters (from several thousands pg/L in coastal waters to few tens of pg/L in the central Pacific Ocean) (Yamashita et al., 2005). Several studies have reported the presence of perfluorinated chemicals in a variety of wildlife species, including freshwater and marine mammals, fish, birds and shellfish (Giesy and Kannan, 2001; Kannan et al., 2001, 2002a,b). These investigators and others (Bossia et al., 2005; Martin et al., 2003) suggested that the chemicals undergo biomagnification at the top levels of the food chain. Increasing concentrations of perfluorochemicals have been observed in animal tissues (Giesy and Kannan, 2001; Kannan et al., 2002a). Human contamination by perfluorinated compounds has been reported, mostly in blood samples collected in the United States,

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South America, Europe and Asia (Kudo and Kawashima, 2003; Lau et al., 2004). More recently, these chemicals have been detected in human seminal plasma (Guruge et al., 2005). Experiments with animals have shown that perfluorooctanoic acid (PFOA) is well absorbed following oral exposure and inhalation, and less so following dermal exposure. Once absorbed in the body, it is distributed predominantly to the plasma and liver (Kudo and Kawashima, 2003). Organic fluorine is very slowly eliminated from the human body; the mean half-life of PFOA in human serum ranges from 1.5 to more than 13 years (Burris et al., 2005). This slow elimination of perfluorinated acids from the body is believed to be a consequence of them being bound to proteins in the liver and serum (Jones et al., 2003). In Japan, perfluorooctane sulfonate (PFOS) and PFOA concentrations in human serum have increased by factors of 3 and 14, respectively, over the past 25 years (Harada et al., 2004). Furthermore, relatively little is known about the acute toxicity of perfluorinated carboxylic acids towards animals and humans. Most of the toxicity data concern perfluorooctane sulfonate and perfluorooctanoic acid, the most intensively researched of these compounds. Animal studies have suggested their potential developmental, reproductive and systemic toxicity. Subchronic exposure leads to significant body weight loss, and increased liver weight accompanied by hepatotoxicity (Kudo and Kawashima, 2003; Lau et al., 2004). Hepatic PFOS concentration in fish was positively correlated with serum alanine aminotransferase activity, the indicator of liver damage, in fish (Hoff et al., 2005). PFOA treatment induced xenobiotic metabolizing enzyme activities in the rat testis (Mehrotra et al., 1999). PFOA and perfluorodecanoic acid (PFDA) are known as peroxisome proliferators, and exert morphological and biological effects characteristic of this group of compounds. These effects include the beta-oxidation of fatty acids, increased frequency of several cytochrome P-450 mediated reactions, and inhibited secretion of triglycerides and cholesterol from the liver (Kawashima et al., 1994; Kudo et al., 2000; Kennedy et al., 2004). Experiments with rats exposed to perfluorooctane sulfonate have shown that it can impair sperm production and maturation in male rats (Fan et al., 2005). The reproductive and developmental toxicity of perfluorinated chemicals has been studied in fish. Exposure to PFOS caused histopathological lesions, most prominently in the ovaries of adult females (Ankley et al., 2005). Altered plasma concentrations of both steroidal androgens and estrogens after exposure to perfluorooctane sulfonate and perfluorooctanoic acid have been reported in fish (Oakes et al., 2004, 2005). More recently, in vitro studies have demonstrated the endocrine disrupting capacity of selected perfluorinated compounds (Maras et al., 2006). The aim of the present study was to assess the acute biological activity of perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid and perfluorodecanoic acid using different in vitro test systems: cytotoxicity towards two mammalian cell lines, bioluminescence inhibition in the marine bacterium Vibrio fischeri, and enzyme inhibition. The perfluorochemicals to be tested were selected to allow study of the influence of perfluorocarbon chain length on toxicity.

2. Materials and methods 2.1. Enzymes and chemicals Perfluorinated carboxylic acids: perfluorohexanoic acid (PFHxA, CAS number: 307-24-4), perfluoroheptanoic acid (PFHpA, CAS number: 375-85-9), perfluorooctanoic acid (PFOA, CAS number: 335-67-1), perfluorononanoic acid (PFNA, CAS number: 375-95-1) and perfluorodecanoic acid (PFDA, CAS number: 335-76-2) were purchased from ABCR GmbH (Karlsruhe, Germany). Liquid-dried luminescent V. fischeri bacteria (NRRLB-11177) and all the reagents used in the test were obtained from Dr. Lange GmbH (Germany). RPMI medium, streptomycin and penicillin, glutamine, horse serum (HS), heatinactivated fetal bovine serum (FBS Hi) were purchased from Gibco BRL Life Technologies (Germany). WST-1 test (2-(4-iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) was obtained from Roche Diagnostics (Germany). Acetylcholinesterase (AChE, type VI-S, from Electrophorus electricus, EC 3.1.1.7), 5,5 -dithiobis (2-nitrobenzoic acid) (DTNB), acetylcholine iodide, NADPH, oxidized glutathione (GSSG), glutathione reductase (GR, from Saccharomyces cerevisiae, EC 1.6.4.2), bovine serum albumin (BSA), dimethylsulfoxide (DMSO), sodium phosphate, phosphoric acid, sodium bicarbonate, methanol HPLC grade were purchased from Sigma (USA). All other chemicals were of the highest purity commercially available.

2.2. Luminescent bacteria acute toxicity test The standard bioluminescence inhibition assay was performed according to a modified DIN/EN/ISO 11348-2 protocol (Ranke et al., 2004). Stock solutions of the tested compounds (from 10 to 0.1 mM) were prepared in 2% NaCl. Bacteria were rehydrated in reactivation solution. Culture suspensions and diluted samples were preincubated for 15 min at 15 ◦ C. After the initial luminescence was measured, 0.5 ml of the culture suspension was mixed with the same volume of diluted sample. After 30 min incubation at 15 ◦ C the final bioluminescence was measured. Tests were carried out in triplicate using 8 dilutions and 7.5% NaCl solution as a control. Perfluorodecanoic acid was excluded from luminescent bacteria assay because of solubility problems. DMSO or methanol were used to improve the solubility of the compounds in other tests; the co-solvents were shown to be toxic towards V. fischeri.

2.3. Acetylcholinesterase inhibition assay A colorimetric assay based on the reduction of DTNB was used to measure AChE inhibition (Ellman et al., 1961; Fisher et al., 2000). This was done in a reaction mixture consisting of solutions of each of the perfluorinated acids at concentrations ranging from 4 to 4000 ␮M, acetylcholinesterase (0.05 U/ml + BSA (62.5 ␮g/ml)), acetylcholine iodide (0.5 mM), DTNB (0.5 mM) and NaHCO3 (47 ␮g/ml) in 0.02 M phosphate buffer, pH 8.0. GR inhibition was determined in a reaction mixture containing solutions of each of the perfluorinated acids at concentrations ranging from 4 to 4000 ␮M, glutathione reductase (0.04 U/ml), NADPH (0.4 mM), GSSG (0.8 mM), DTNB (0.4 mM) and NaHCO3 (38 ␮g/ml) in 0.1 M phosphate buffer, pH 7.6. The enzyme kinetics was measured at 30-s intervals in a microplate reader for 5 min at 405 nm. Enzyme activity was expressed as OD/min from the linear regression. The concentration inhibition curves were fitted with the nonlinear least squares method using a logistic model representing enzyme inhibition to the base 10 logarithm of the perfluorinated acids concentration. IC50 values were also derived.

2.4. Cell lines Cytotoxicity was determined using the promyelocytic leukemia rat cell line IPC-81 (Lacaze et al., 1983) and the rat glioma cell line C6 (Benda, 1968; Ruchaud et al., 1995). IPC-81 cells were cultured in RPMI medium, supplemented with 1% antibiotic solution (penicillin/streptomycin), 1% glutamine, NaHCO3 (3.7 g/l) and 10% HS at 37 ◦ C in a humidified atmosphere of 5% CO2 . The medium was changed every 2 days and the cells were subcultured. For the cytotoxicity assays cells were added to the plates at a concentration

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Table 1 EC50 obtained for IPC-81 and C6 cell lines and Vibrio fischeri Perfluorochemical

IPC-81

C6

EC50 (␮M) Perfluorohexanoic acid Perfluoroheptanoic acid Perfluorooctanoic acid Perfluorononanoic acid Perfluorodecanoic acid

3715.4 1778.3 457.1 457.1 173.8

± ± ± ± ±

85.6 41.9 10.5 21.1 16.0

log EC50 (␮M) 3.57 3.25 2.66 2.66 2.24

± ± ± ± ±

0.01 0.01 0.01 0.02 0.04

Vibrio fischeri

EC50 (␮M) 7943.3 3981.1 676.1 741.3 363.1

± ± ± ± ±

365.9 275.2 46.7 68.4 8.4

log EC50 (␮M) 3.90 3.60 2.83 2.87 2.56

± ± ± ± ±

0.02 0.03 0.03 0.04 0.01

EC50 (␮M)

log EC50 (␮M)

4265.8 ± 393.5 3020.0 ± 69.5 1380.4 ± 138.8 1148.2 ± 130.7 n.a.

3.63 ± 0.04 3.48 ± 0.01 3.14 ± 0.04 3.06 ± 0.05 n.a.

n.a., data not available.

of 15 × 104 cells/ml (in RPMI with 8% FBS Hi). C6 cells were grown as a monolayer in DMEM (high glucose) medium supplemented with 1% antibiotic solution (penicillin/streptomycin), 1% glutamine, NaHCO3 (3.7 g/l) and 10% FBS Hi at 37 ◦ C under the same conditions. For the cytotoxicity assays, cells were seeded in 96-well plates at an initial density of 5 × 104 cells/ml of culture medium and incubated for 24 h.

2.5. Cell viability assay A colorimetric assay with WST-1 reagent was used for the cell viability tests. Stock solutions of the perfluorinated acids were prepared in growth media with 0.5% DMSO added to improve solubility. Cells were exposed to nine different concentrations (from 2 ␮M to 20 mM) of the perfluorinated acids. Each incuba-

tion was performed in triplicate, including the controls and blanks. The cells were incubated for 44 h. After these times, 10 ␮l of WST-1 reagent, diluted four-fold in phosphate buffer, was added to each well and incubated for 4 h at 37 ◦ C. Subsequently, the optical density at 450 nm was measured in the plate reader. Cell viability was calculated as the percentage of the viability of exposed cells versus controls. These data are the means of three independent experiments conducted for each acid. Concentration response curves were fitted with the nonlinear least squares method using a linear logistic model for the IPC-81 and V. fischeri cells, where a hormetic effect was observed, and a logistic model for C6 glioma cell line, where hormesis was not observed (Van Ewijk and Hoekstra, 1993; Ranke et al., 2004). The log EC50 values were given since it is a model parameter in the logistic as well as in linear logistic model. Calculations were carried out with R language and environment for statistical computing (http://www.r-project.org).

Fig. 1. Exemplary concentration–response relationships for perfluorooctanoic acid for (a) IPC-81 leukemia cell line (linear logistic model, n = 9), (b) C6 glioma cell line exposed (logistic model, n = 9) and (c) V. fischeri (linear logistic model, n = 3).

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3. Results The biological activity of the perfluorinated carboxylic acids was studied at the cellular and molecular levels with different in vitro test systems. At the molecular level it was determined with acetylcholinesterase and glutathione reductase inhibition assays. In the applied concentration range only the AChE inhibition level of perfluorononanoic acid was high enough to determine IC50 , which was 3526 ␮M. For the other acids, the enzyme inhibition was observed only at the highest concentration (4000 ␮M). Perfluorohexanoic, perfluoroheptanoic, perfluorooctanoic and perfluorodecanoic acids, respectively, inhibited enzyme activity by 87.9, 86.6, 76.2 and 84.5%. With glutathione reductase, only PFNA caused enzyme inhibition; the IC50 for this acid was 1788 ␮M. The IPC-81 leukemia cell line was the most sensitive to the perfluorinated acids. The EC50 values obtained ranged from 173.8 ± 16.0 to 3715.4 ± 85.6 ␮M (Table 1). Bioluminescence in V. fischeri bacteria was inhibited with respective EC50 values of 4265.8 ± 393.5, 3020.0 ± 69.5, 1380.4 ± 138.8, 1148.2 ± 130.7 ␮M for PFHxA, PFHpA, PFOA and PFNA. For

the IPC-81 viability test and the V. fischeri acute bioluminescence inhibition assay, the hormetic effect at concentrations below inhibitory concentrations can be seen on the concentration response curves (Figs. 1 and 2). Only the concentration response relationship curve for C6 glioma cells showed no stimulatory response. This latter cell line was also less sensitive to the acids, displaying EC50 values from 363.1 ± 8.4 to 7943.3 ± 365.9 ␮M (Table 1). These results yielded a relationship between the cytotoxicity of the perfluorinated acids and the perfluorocarbon chain length (Fig. 2). In each test system used, the toxicity increased with increasing chain length; the lowest effective concentrations were thus recorded for perfluorodecanoic acid. 4. Discussion Since perfluorinated acids are chemically stabilized by the strong covalent bond between carbon and fluorine, they have been historically regarded as metabolically inert and nontoxic (Sargent and Seffl, 1970). Chemicals that are difficult to degrade biologically may nonetheless bioaccumulate and may affect the

Fig. 2. Influence of perfluorocarbon chain length on toxicity of perfluorinated acids (a) in IPC-81 leukemia cell line (linear logistic model, n = 9 for each concentration and substance), (b) C6 glioma cell line (logistic model, n = 9 for each concentration and substance); on V. fischeri luminescence inhibition (linear logistic model, n = 3 for each concentration and substance) perfluorohexanoic acid (. . .), perfluoroheptanoic acid (—), perfluorooctanoic acid (– –), perfluorononanoic acid (- . -), perfluorodecanoic acid (—).

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health of humans and biota. Because of the global distribution and persistent nature of these chemicals, monitoring their environmental fate and their ecotoxicological profile is highly desirable (Van de Vijver et al., 2003, 2004). Usually, the first detectable responses to environmental perturbation are changes at the molecular, biochemical or cellular level. During the last decade, the importance of in vitro systems such as cell lines has increased in toxicology and ecotoxicology. Although such systems cannot replicate the complex interactions of animals in vivo, they provide important predictive information about the biological activities of chemical substances. There are not many studies where the toxicity of perfluorinated carboxylic acids has been evaluated at the cellular level. In experiments with human hepatoblastoma HepG2 cells, PFOA treatment resulted in apoptosis as well as perturbation of the cell cycle. Apoptosis became manifest with 200 ␮M and maximal upon exposure to 450 ␮M PFOA for 24 h. The cell cycle of HepG2 cells was perturbed by exposure to 50–150 ␮M PFOA. Additionally, exposure to 500 ␮M PFOA for 48 h resulted in DNA degradation (Shabalina et al., 1999). Apart from the liver, blood is the tissue where perfluorinated chemicals are mostly found in the human body (Kudo and Kawashima, 2003; Lau et al., 2004). To assess the toxicity of perfluorinated acids, we used the mammalian hematopoietic IPC-81 cell line. This system demonstrated the highest sensitivity to the tested chemicals. The effective concentrations of PFOA recorded in our study for the IPC-81 cell line were at the same level as those registered for adverse effects in the HepG2 cell line (Shabalina et al., 1999). The effects observed in the two rat cell lines used in the present study were different. The leukemia cell line displayed a higher sensitivity than the glioma cell line; to observe similar toxic effects, 50% lower concentrations of the tested compounds were necessary. Furthermore, in the presence of subinhibitory levels of all compounds tested C6 glioma cells showed no stimulatory response, whereas for IPC-81 leukemia cells hormesis was observed. These differences between the two cell lines were also observed during tests of other types of chemicals (Ranke et al., 2004). In our study, the hormetic response was also noticed in the V. fischeri test. A fundamental component of many dose–response relationships, hormesis is a reproducible and generalized biological phenomenon (Calabrese et al., 1999; Stepnowski et al., 2004). Though typical of V. fischeri tests, it is very rarely reported because of difficulties in estimating EC50 values (Christofi et al., 2002). Different mechanisms of cytotoxicity for this group of chemicals have been proposed. Perfluorinated acids with carbon chain lengths of 7–10 were found to inhibit gap junctional intercellular communication (GJIC) (Upham et al., 1998; Hu et al., 2002), which is the major pathway of intracellular signal transduction and is thus important for normal cell growth and functioning, and for maintaining tissue homeostasis. A structural relationship was established, indicating that the inhibitory effect was determined by the length of the fluorinated tail, not by the nature of the functional group. In another study (Hu et al., 2003), the effects of perfluorinated compounds on membrane properties such as flu-

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idity and permeability was studied. The selectively permeable cell membrane forms the first barrier protecting the cell from exogenous exposure. Effects on the permeability status of the cell membrane could play an important role in mediating the adverse effects of number environmental contaminants, especially surface-active compounds. PFOS increased membrane permeability towards hydrophobic ligands, and perfluorinated compounds increased membrane fluidity in fish leucocytes in a dose-dependent manner. The lowest effective concentration for the membrane fluidity effects of PFOS was 5–15 mg/l (Hu et al., 2003). It is hard to speculate about the mechanism of cytotoxicity, but we observed in our study that similar concentrations affected cell viability. To obtain more comprehensive information on the possible harmful effects of exposure to these chemicals on the nervous system, the acetylcholinesterase inhibition assay was used. This enzyme is an essential part of the nervous system. Widely used as a biomarker detecting pesticides, it is also inhibited by different classes of chemicals, including metals and surfactants (Herbert et al., 1995; Guilhermino et al., 1998). More recently, it was used to assess the environmental risk of newly designed industrial chemicals (Stock et al., 2004). Our results indicate that perfluorinated acids inhibit AChE only at very high concentrations. This low inhibition of AChE activity, in conjunction with the results obtained for the C6 cell line, suggest that the nervous system may not be very sensitive to perfluorinated acids. Many pollutants can exhibit oxidative stress-related toxicity. It has been reported that treatment with perfluorinated acids causes oxidative stress (Van der Oost et al., 2003). A dramatic increase in the cellular content of reactive oxygen species (superoxide anions and hydrogen peroxide) was found after treatment of human hepatoma HepG2 cells with 200 and 400 ␮M PFOA after 3 h (Panaretakis et al., 2001). An increase in the activities of antioxidant enzymes such as catalase and superoxide dismutase was observed in rat liver after administration of PFDA (Glauert et al., 1992). Lipid peroxidation and DNA damage, well-known biochemical effects associated with increased fluxes of oxyradicals (very important consequences of oxidative stress), were observed in animals after exposure to perfluorinated acids (Takagi et al., 1991). Lipid peroxides are known to be reduced by the action of glutathione peroxidase to alcohols using glutathione (Nordberg and Arner, 2001). Both increases and decreases in the glutathione level have been observed after exposure to different chemicals. An enzyme known to have a physiological significance, glutathione reductase plays an important role in maintaining GSH/GSSG homeostasis under oxidative stress conditions (Van der Oost et al., 2003). It has been shown that treatment with perfluorodecanoic acid significantly increased hepatic reduced glutathione (GSH) content and affected the activities of enzymes associated with GSH synthesis, utilization and regeneration (Chen et al., 2001). In that study, a decrease in glutathione reductase by the highest dose of PFDA (35 mg/kg) was observed. In our study, perfluorinated acids did not significantly affect the activity of glutathione reductase. Inhibition was observed only at very high concentrations of perfluoronanoic acid showing IC50 value of 1788 ␮M. Similarly, no changes in glutathione reductase activ-

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ity were observed in rat liver after treatment with PFOA (Glauert et al., 1992; Kawashima et al., 1994; Cai et al., 1995). Although a relationship between chain lengths and the biological effects of perfluorinated acids has already been suggested (Kudo et al., 2000, 2001), it is still unclear since little information is available except for PFDA and PFOA. The results of our study show a relation between toxicity and perfluorocarbon chain length, with toxicity being lowest for perfluorohexanoic acid and highest for perfluorodecanoic acid. Kudo et al. (2001) reported that perfluorinated acids with shorter carbon chains are more quickly excreted in the urine, resulting in a lower concentration in the serum and liver. Thus, perfluorinated acids with longer carbon chains will be eliminated in urine less rapidly, which together with their higher toxicity should be recognized as a great health and environmental concern. Acknowledgements Financial support was provided by the Polish Ministry of Education and Research under grants: 2P04G 083 29, 2P04G 118 29 and DS 8390-4-0141-6. Help of Dr. Tomasz Puzyn with data analysis is greatly acknowledged. References Ankley, G.T., Kuehl, D.W., Kahl, M.D., Jensen, K.M., Linnum, A., Leino, R.L., Villeneuvet, D.A., 2005. Reproductive and developmental toxicity and bioconcentration of perfluorooctanesulfonate in a partial life-cycle test with the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 24, 2316. Banks, R.E., Smart, B.E., Tatlow, J.C., 1994. Organofluorine Chemistry Principles and Comercial Applications. Plenum Press, New York. Benda, P., 1968. Differentiated rat glial cell strain in tissue culture. Science 161, 370. Bossia, R., Riget, F.F., Dietz, R., Sonne, C., Fauser, P., Dam, M., Vorkamp, K., 2005. Preliminary screening of perfluorooctane sulfonate (PFOS) and other fluorochemicals in fish, birds and marine mammals from Greenland and the Faroe Islands. Environ. Pollut. 136, 323. Burris, J.M., Lundberg, J.K., Olsen, G., Simpson, C., Mandel, J., 2005. Determination of serum half-lives of several fluorochemicals: Interim Report #2. Study Sponsor: 3M Company, Corporate Occupational Medicine Department, US EPA AR226-1086. Cai, Y., Appelkvist, E.L., DePierre, J.W., 1995. Hepatic oxidative stress and related defenses during treatment of mice with acetylsalicylic acid and other peroxisome proliferators. J. Biochem. Toxicol. 10, 87. Calabrese, E.J., Baldwin, L.A., Holland, C.D., 1999. Hormesis: a highly generalizable and reproducible phenomenon with important implications for risk assessment. Risk Anal. 19, 261. Chen, L.C., Tatum, V., Glauert, H.P., Chow, C.K., 2001. Peroxisome proliferator perfluorodecanoic acid alters glutathione and related enzymes. J. Biochem. Mol. Toxicol. 15, 107. Christofi, N., Hoffmann, C., Tosh, L., 2002. Hormesis responses of free and immobilized light-emitting bacteria. Ecotoxicol. Environ. Saf. 52, 227. Dimitrov, S., Kamenska, V., Walker, J.D., Windle, W., Purdy, R., Lewis, M., et al., 2004. Predicting the biodegradation products of perfluorinated chemicals using CATABOL. SAR QSAR Environ. Res. 15, 69. Ellman, G.L., Courtney, K.O., Andrres, V., Featherstone, R.M., 1961. A new rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88. Fan, Y.O., Jin, Y.H., Ma, Y.X., Zhang, Y.H., 2005. Effects of perfluorooctane sulfonate on spermiogenesis function of male rats. Wei Sheng Yan Jiu. 34, 37. Fisher, T.C., Crane, M., Callaghan, A., 2000. A quality-controlled and optimised microtitreplate assay to detect acetylcholinesterase activity in individual Chi-

ronomus riparius meigen, for use as a biomarker of pesticide exposure. Environ. Toxicol. Chem. 19, 1749. Giesy, J.P., Kannan, K., 2001. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 35, 1339. Glauert, H.P., Srinivasan, S., Tatum, V.L., Chen, L.C., Saxon, D.M., Lay, L.T., Borges, T., Baker, M., Chen, L.H., Robertson, L.W., et al., 1992. Effects of the peroxisome proliferators ciprofibrate and perfluorodecanoic acid on hepatic cellular antioxidants and lipid peroxidation in rats. Biochem. Pharmacol. 43, 1353. Guilhermino, L., Barros, P., Silva, M.C., Soares, A.M.V.M., 1998. Should the use of inhibition of cholinesterases as a specific biomarker for organophosphate and carbamate pesticides be questioned? Biomarkers 3, 157. Guruge, K.S., Taniyasu, S., Yamashita, N., Wijeratna, S., Mohotti, K.M., Seneviratne, H.R., Kannan, K., Yamanaka, N., Miyazaki, S., 2005. Perfluorinated organic compounds in human blood serum and seminal plasma: a study of urban and rural tea worker populations in Sri Lanka. J. Environ. Monit. 7, 371. Hagen, D.F., Belisle, J., Johnson, J.D., Venkateswarlu, P., 1981. Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector—the biotransformation of 1H, 1H, 2H, 2Hperfluorodecanol to perfluorooctanoate. Anal. Biochem. 118, 336. Harada, K., Saito, N., Inoue, K., Yoshinaga, T., Watanabe, T., Sasaki, S., Kamiyama, S., Koizumi, A., 2004. The influence of time, sex and geographic factors on levels of perfluorooctane sulfonate and perfluorooctanoate in human serum over the last 25 years. J. Occup. Health 46, 141. Herbert, A., Guilhermino, L., Da Silva de Assis, H.C., Hansen, P.D., 1995. Acetylcholinesterase activity in aquatic organismsas pollution biomarker. Angew. Zool. 3, 1. Hoff, P.T., Van Campenhout, K., Van de Vijver, K., Covaci, A., Bervoets, L., Moens, L., Huyskens, G., Goemans, G., Belpaire, C., Blust, R., De Coen, W., 2005. Perfluorooctane sulfonic acid and organohalogen pollutants in liver of three freshwater fish species in Flanders (Belgium): relationships with biochemical and organismal effects. Environ. Pollut. 137, 324. Hu, W., Jones, P.D., Upham, B.L., Trosko, J.E., Lau, C., Giesy, J.P., 2002. Inhibition of gap junctional intercellular communication by perfluorinated compounds in rat liver and dolphin kidney epithelial cell lines in vitro and Sprague–Dawley rats in vivo. Toxicol. Sci. 68, 265. Hu, W., Jones, P.D., De Coen, W., King, L., Fraker, P., Newsted, J., Giesy, J.P., 2003. Alterations in cell membrane properties caused by perfluorinated compounds. Comp. Biochem. Physiol. C 135, 77. Jones, P.D., Hu, W., De Coen, W., Newsted, J.L., Giesy, J.P., 2003. Binding of perfluorinated. Fatty acids to serum proteins. Environ. Toxicol. Chem. 22, 2639. Kannan, K., Koistinen, J., Beckman, K., Evans, T., Gorzelany, J.F., Hansen, K.J., Jones, E., Helle, P.D., Nyman, M., Geisey, J.P., 2001. Accumulation of perflurooctane sulfonate in marine mammals. Environ. Sci. Technol. 35, 1593. Kannan, K., Corsolini, S., Falandysz, J., Oehme, G., Focardi, S., Giesy, J.P., 2002a. Perfluorooctanesulfonate and related fluorinated hydrocarbons in marine mammals, fishes, and birds from coasts of the Baltic and the Mediterranean Seas. Environ. Sci. Technol. 36, 3210. Kannan, K., Hansen, K.J., Wade, T.L., Giesy, J.P., 2002b. Perfluorooctane sulfonate in oyster, Crassostrea virginica, from the Gulf of Mexico and the Chesapeake Bay, USA. Arch. Environ. Contam. Toxicol. 42, 313. Kawashima, Y., Suzuki, S., Kozuka, H., Sato, M., Suzuki, Y., 1994. Effects of prolonged administration of perfluorooctanoic acid on hepatic activities of enzymes, which detoxify peroxide and xenobiotic in the rat. Toxicology 93, 85. Kennedy Jr., G.L., Butenhoff, J.L., Olsen, G.W., O’Connor, J.C., Seacat, A.M., Perkins, R.G., Biegel, L.B., Murphy, S.R., Farrar, D.G., 2004. The toxicology of perfluorooctanoate. Crit. Rev. Toxicol. 34, 351. Key, B.D., Howell, R.D., Criddle, C.S., 1997. Fluorinated organics in the biosphere. Environ. Sci. Technol. 31, 2445. Kudo, N., Kawashima, Y., 2003. Toxicity and toxicokinetics of perfluorooctanoic acid in humans and animals. J. Toxicol. Sci. 28, 49. Kudo, N., Bandai, N., Suzuki, E., Katakura, M., Kawashima, Y., 2000. Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats. Chem. Biol. Interact. 124, 119.

E. Mulkiewicz et al. / Environmental Toxicology and Pharmacology 23 (2007) 279–285 Kudo, N., Suzuki, E., Katakura, M., Ohmori, K., Noshiro, R., Kawashima, Y., 2001. Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats. Chem. Biol. Interact. 134, 203. Lacaze, N., Gombaud-Saintonge, G., Lanotte, M., 1983. Conditions controlling long-term proliferation of brown Norway rat promyelocytic leukemia in vitro: primary growth stimulation by microenvironment and established of an autonomous brown Norway “leukemic stem cell line”. Leuk. Res. 7, 145. Lau, C., Butenhoff, J.L., Rogers, J.M., 2004. The developmental toxicity of perfluoroalkyl acids and their derivatives. Toxicol. Appl. Pharmacol. 198, 231. Maras, M., Vanparys, C., Muylle, F., Robbens, J., Berger, U., Barber, J.L., Blust, R., De Coen, W., 2006. Estrogen-like properties of fluorotelomer alcohols as revealed by mcf-7 breast cancer cell proliferation. Environ. Health Perspect. 114, 100–105. Martin, J.W., Mabury, S.A., Solomon, K.R., Muir, D.C., 2003. Bioconcentration and tissue distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem. 22, 196. Mehrotra, K., Morgenstern, R., Ahlberg, M.B., Georgellis, A., 1999. Hypophysectomy and/or peroxisome proliferators strongly influence the levels of phase II xenobiotic metabolizing enzymes in rat testis. Chem. Biol. Interact. 122, 73. Nordberg, J., Arner, E.S., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287. Oakes, K.D., Sibley, P.K., Solomon, K.R., Mabury, S.A., Van der Kraak, G.J., 2004. Impact of perfluorooctanoic acid on fathead minnow (Pimephales promelas) fatty acyl-CoA oxidase activity, circulating steroids, and reproduction in outdoor microcosms. Environ. Toxicol. Chem. 23, 1912. Oakes, K.D., Sibley, P.K., Martin, J.W., MacLean, D.D., Solomon, K.R., Mabury, S.A., Van Der Kraak, G.J., 2005. Short-term exposures of fish to perfluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids. Environ. Toxicol. Chem. 24, 1172. Panaretakis, T., Shabalina, I.G., Grander, D., Shoshan, M.C., DePierre, J.W., 2001. Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid. Toxicol. Appl. Pharmacol. 173, 56. Ranke, J., Moelter, K., Stock, F., Bottin-Weber, U., Poczobutt, J., Hoffmann, J., Ondruschka, B., Filser, J., Jastorff, B., 2004. Biological effects of imida-

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zolium ionic liquids with varying chain lengths in acute Vibrio fischeri and WST-1 cell viability assays. Ecotoxicol. Environ. Saf. 58, 396. Ruchaud, S., Zorn, M., Davilar-Villar, E., Genieser, H.-G., Hoffmann, C., Gjersten, B.T., Døskeland, S.O., Jastorff, B., Lanotte, M., 1995. Evidence for several pathways of biological response to hydrolysable cAMP analogs using a model system of apoptosis in IPC-81 leukemia cells. J. Cell. Pharmacol. 2, 127. Sargent, J.W., Seffl, R.J., 1970. Properties of perfluorinated liquids. Fed. Proc. 29, 1699. Shabalina, I.G., Panaretakis, T., Bergstrand, A., DePierre, J.W., 1999. Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells. Carcinogenesis 20, 2237. Stepnowski, P., Skladanowski, A.C., Ludwiczak, A., Ł˛aczy´nska, E., 2004. Evaluating the cytotoxicity of ionic liquids using human cell line HeLa. Hum. Exp. Toxicol. 23, 513. Stock, F., Hoffmann, J., Ranke, J., St¨ormann, R., Jastorff, B., 2004. Effects of ionic liquids on the acetylcholinesterase—a structure-activity relationship consideration. Green Chem. 6, 286. Takagi, A., Sai, K., Umemura, T., Hasegawa, R., Kurokawa, Y., 1991. Short-term exposure to the peroxisome proliferators, perfluorooctanoic acid and perfluorodecanoic acid, causes significant increase of 8-hydroxydeoxyguanosine in liver DNA of rats. Cancer Lett. 57, 55. Upham, B.L., Deocampo, N.D., Wurl, B., Trosko, J.E., 1998. Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail. Int. J. Cancer. 78, 491. Van de Vijver, K.I., Hoff, P.T., Van Dongen, W., Blust, R., De Coen, W.M., 2003. Perfluorinated chemicals infiltrate ocean waters: link between exposure levels and stable isotope ratios in marine mammals. Environ. Sci. Toxicol. Chem. 22, 2037. Van de Vijver, K.I., Hoff, P.T., Das, K., Van Dongen, W., Esmans, E.L., Siebert, U., et al., 2004. Baseline study of perfluorochemicals in harbour porpoises (Phocoena phocoena) from Northern Europe. Mar. Pollut. Bull. 48, 992. Van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13, 57. Van Ewijk, P.H., Hoekstra, J.A., 1993. Calculation of the EC50 and its confidence interval when subtoxic stimulus is present. Ecotoxicol. Environ. Saf. 25, 25. Yamashita, N., Kannan, K., Taniyasu, S., Horii, Y., Petrick, G., Gamo, T., 2005. A global survey of perfluorinated acids in oceans. Mar. Pollut. Bull. 51, 658.