A

apopwtacom©

K

1

pipfpcOsanme

1d

Environmental Toxicology and Pharmacology 23 (2007) 279–285

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

E. Mulkiewicz a, B. Jastorff b, A.C. Składanowski c, K. Kleszczynski c, P. Stepnowski a,∗a Faculty of Chemistry, University of Gdansk, Sobieskiego 18, PL-80-952 Gdansk, Poland

b Centre for Environmental Research and Technology (UFT), University of Bremen, D-28359 Bremen, Leobener Str., Germanyc Intercollegiate Faculty of Biotechnology, Medical University of Gdansk & University of Gdansk, PL 80-211 Gdansk, ul. Debinki 1, Poland

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

bstract

The acute biological activity of a homologous series of perfluorinated carboxylic acids – perfluorohexanoic acid (PFHxA), perfluoroheptanoiccid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) – was studied. To analyze theotential risk of the perfluorinated acids to humans and the environment, different in vitro toxicity test systems were employed. The cytotoxicityf the chemicals towards two different types of mammalian cell lines and one marine bacteria was investigated. The viability of cells from theromyelocytic leukemia rat cell line (IPC-81) and the rat glioma cell line (C6) was assayed calorimetrically with WST-1 reagent. The evaluationas combined with the Vibrio fischeri acute bioluminescence inhibition assay. The biological activity of the compounds was also determined at

he molecular level with acetylcholinesterase and glutathione reductase inhibition assays. This is the first report of the effects of perfluorinated

cids on the activity of purified enzymes. The results show these compounds have a very low acute biological activity. The observed effectiveoncentrations lie in the millimole range, which is well above probable intracellular concentrations. A relationship was found between the toxicityf the perfluorinated carboxylic acids and the perfluorocarbon chain length: in every test system applied, the longer the perfluorocarbon chain, theore toxic was the acid. The lowest effective concentrations were thus recorded for perfluorononanoic and perfluorodecanoic acids.2006 Elsevier B.V. All rights reserved.

R

pic(

icttrofi

eywords: Toxicity; Perfluorinated acids; IPC-81; C6; Vibrio fischeri; AChE; G

. Introduction

In recent years, growing concern has been expressed abouterfluorinated compounds, the global production of which hasncreased since the 1970s. With their unique physicochemicalroperties, they have a broad spectrum of applications as sur-actants, refrigerants and polymers, and also as components ofharmaceuticals, fire retardants, lubricants, adhesives, paints,osmetics, agrochemicals and food packaging (Key et al., 1997).wing to the presence of high-energy carbon-fluorine bonds (the

trongest of all covalent bonds), perfluorochemicals are stablend persistent in the environment (Banks et al., 1994). They do

ot undergo photolysis, hydrolysis, defluorination or phase IIetabolism (Kudo and Kawashima, 2003). It is common knowl-

dge that biodegradation is restricted to the non-perfluorinated

∗ Corresponding author. Tel.: +48 58 523 5448; fax: +48 58 523 5472.E-mail address: sox@chem.univ.gda.pl (P. Stepnowski).

a2biaHr

382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.etap.2006.11.002

art of the molecules (Hagen et al., 1981). Furthermore, dur-ng microbial degradation, perfluorochemicals tend to be slowlyonverted to more bioaccumulative and more toxic productsDimitrov et al., 2004).

Perfluorochemicals have been detected not only in the phys-cal environment, but also in humans and wildlife. Theseontaminants have been found in oceanic waters (from severalhousands pg/L in coastal waters to few tens of pg/L in the cen-ral Pacific Ocean) (Yamashita et al., 2005). Several studies haveeported the presence of perfluorinated chemicals in a varietyf wildlife species, including freshwater and marine mammals,sh, birds and shellfish (Giesy and Kannan, 2001; Kannan etl., 2001, 2002a,b). These investigators and others (Bossia et al.,005; Martin et al., 2003) suggested that the chemicals undergoiomagnification at the top levels of the food chain. Increas-

ng concentrations of perfluorochemicals have been observed innimal tissues (Giesy and Kannan, 2001; Kannan et al., 2002a).uman contamination by perfluorinated compounds has been

eported, mostly in blood samples collected in the United States,

2 colog

SLdi(tt(eiettpho

oMaopSiaca(oPoeioi(2fmasleaf(dfl

cpctitat

2

2

bp(b

rRiT(Dtat(bo

2

aotswowNcuw

2

Arc((

eg(

fr

sba

2

I

80 E. Mulkiewicz et al. / Environmental Toxi

outh America, Europe and Asia (Kudo and Kawashima, 2003;au et al., 2004). More recently, these chemicals have beenetected in human seminal plasma (Guruge et al., 2005). Exper-ments with animals have shown that perfluorooctanoic acidPFOA) is well absorbed following oral exposure and inhala-ion, and less so following dermal exposure. Once absorbed inhe body, it is distributed predominantly to the plasma and liverKudo and Kawashima, 2003). Organic fluorine is very slowlyliminated from the human body; the mean half-life of PFOAn human serum ranges from 1.5 to more than 13 years (Burrist al., 2005). This slow elimination of perfluorinated acids fromhe body is believed to be a consequence of them being boundo proteins in the liver and serum (Jones et al., 2003). In Japan,erfluorooctane sulfonate (PFOS) and PFOA concentrations inuman serum have increased by factors of 3 and 14, respectively,ver the past 25 years (Harada et al., 2004).

Furthermore, relatively little is known about the acute toxicityf perfluorinated carboxylic acids towards animals and humans.ost of the toxicity data concern perfluorooctane sulfonate

nd perfluorooctanoic acid, the most intensively researchedf these compounds. Animal studies have suggested theirotential developmental, reproductive and systemic toxicity.ubchronic exposure leads to significant body weight loss, and

ncreased liver weight accompanied by hepatotoxicity (Kudond Kawashima, 2003; Lau et al., 2004). Hepatic PFOS con-entration in fish was positively correlated with serum alanineminotransferase activity, the indicator of liver damage, in fishHoff et al., 2005). PFOA treatment induced xenobiotic metab-lizing enzyme activities in the rat testis (Mehrotra et al., 1999).FOA and perfluorodecanoic acid (PFDA) are known as per-xisome proliferators, and exert morphological and biologicalffects characteristic of this group of compounds. These effectsnclude the beta-oxidation of fatty acids, increased frequencyf several cytochrome P-450 mediated reactions, and inhib-ted secretion of triglycerides and cholesterol from the liverKawashima et al., 1994; Kudo et al., 2000; Kennedy et al.,004). Experiments with rats exposed to perfluorooctane sul-onate have shown that it can impair sperm production andaturation in male rats (Fan et al., 2005). The reproductive

nd developmental toxicity of perfluorinated chemicals has beentudied in fish. Exposure to PFOS caused histopathologicalesions, most prominently in the ovaries of adult females (Ankleyt al., 2005). Altered plasma concentrations of both steroidalndrogens and estrogens after exposure to perfluorooctane sul-onate and perfluorooctanoic acid have been reported in fishOakes et al., 2004, 2005). More recently, in vitro studies haveemonstrated the endocrine disrupting capacity of selected per-uorinated compounds (Maras et al., 2006).

The aim of the present study was to assess the acute biologi-al activity of perfluorohexanoic acid, perfluoroheptanoic acid,erfluorooctanoic acid, perfluorononanoic acid and perfluorode-anoic acid using different in vitro test systems: cytotoxicityowards two mammalian cell lines, bioluminescence inhibition

n the marine bacterium Vibrio fischeri, and enzyme inhibi-ion. The perfluorochemicals to be tested were selected tollow study of the influence of perfluorocarbon chain length onoxicity.

RmNCF

y and Pharmacology 23 (2007) 279–285

. Materials and methods

.1. Enzymes and chemicals

Perfluorinated carboxylic acids: perfluorohexanoic acid (PFHxA, CAS num-er: 307-24-4), perfluoroheptanoic acid (PFHpA, CAS number: 375-85-9),erfluorooctanoic acid (PFOA, CAS number: 335-67-1), perfluorononanoic acidPFNA, CAS number: 375-95-1) and perfluorodecanoic acid (PFDA, CAS num-er: 335-76-2) were purchased from ABCR GmbH (Karlsruhe, Germany).

Liquid-dried luminescent V. fischeri bacteria (NRRLB-11177) and all theeagents used in the test were obtained from Dr. Lange GmbH (Germany).PMI medium, streptomycin and penicillin, glutamine, horse serum (HS), heat-

nactivated fetal bovine serum (FBS Hi) were purchased from Gibco BRL Lifeechnologies (Germany). WST-1 test (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-2,4-disulfophenyl)-2H-tetrazolium monosodium salt) was obtained from Rocheiagnostics (Germany). Acetylcholinesterase (AChE, type VI-S, from Elec-

rophorus electricus, EC 3.1.1.7), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB),cetylcholine iodide, NADPH, oxidized glutathione (GSSG), glutathione reduc-ase (GR, from Saccharomyces cerevisiae, EC 1.6.4.2), bovine serum albuminBSA), dimethylsulfoxide (DMSO), sodium phosphate, phosphoric acid, sodiumicarbonate, methanol HPLC grade were purchased from Sigma (USA). Allther chemicals were of the highest purity commercially available.

.2. Luminescent bacteria acute toxicity test

The standard bioluminescence inhibition assay was performed according tomodified DIN/EN/ISO 11348-2 protocol (Ranke et al., 2004). Stock solutionsf the tested compounds (from 10 to 0.1 mM) were prepared in 2% NaCl. Bac-eria were rehydrated in reactivation solution. Culture suspensions and dilutedamples were preincubated for 15 min at 15 ◦C. After the initial luminescenceas measured, 0.5 ml of the culture suspension was mixed with the same volumef diluted sample. After 30 min incubation at 15 ◦C the final bioluminescenceas measured. Tests were carried out in triplicate using 8 dilutions and 7.5%aCl solution as a control. Perfluorodecanoic acid was excluded from lumines-

ent bacteria assay because of solubility problems. DMSO or methanol weresed to improve the solubility of the compounds in other tests; the co-solventsere shown to be toxic towards V. fischeri.

.3. Acetylcholinesterase inhibition assay

A colorimetric assay based on the reduction of DTNB was used to measureChE inhibition (Ellman et al., 1961; Fisher et al., 2000). This was done in a

eaction mixture consisting of solutions of each of the perfluorinated acids at con-entrations ranging from 4 to 4000 �M, acetylcholinesterase (0.05 U/ml + BSA62.5 �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

ach of the perfluorinated acids at concentrations ranging from 4 to 4000 �M,lutathione reductase (0.04 U/ml), NADPH (0.4 mM), GSSG (0.8 mM), DTNB0.4 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 readeror 5 min at 405 nm. Enzyme activity was expressed as OD/min from the linearegression.

The concentration inhibition curves were fitted with the nonlinear leastquares method using a logistic model representing enzyme inhibition to thease 10 logarithm of the perfluorinated acids concentration. IC50 values werelso derived.

.4. Cell lines

Cytotoxicity was determined using the promyelocytic leukemia rat cell linePC-81 (Lacaze et al., 1983) and the rat glioma cell line C6 (Benda, 1968;

uchaud et al., 1995). IPC-81 cells were cultured in RPMI medium, supple-ented with 1% antibiotic solution (penicillin/streptomycin), 1% glutamine,aHCO3 (3.7 g/l) and 10% HS at 37 ◦C in a humidified atmosphere of 5%O2. The medium was changed every 2 days and the cells were subcultured.or the cytotoxicity assays cells were added to the plates at a concentration

E. Mulkiewicz et al. / Environmental Toxicology and Pharmacology 23 (2007) 279–285 281

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

Perfluorochemical IPC-81 C6 Vibrio fischeri

EC50 (�M) log EC50 (�M) EC50 (�M) log EC50 (�M) EC50 (�M) log EC50 (�M)

Perfluorohexanoic acid 3715.4 ± 85.6 3.57 ± 0.01 7943.3 ± 365.9 3.90 ± 0.02 4265.8 ± 393.5 3.63 ± 0.04Perfluoroheptanoic acid 1778.3 ± 41.9 3.25 ± 0.01 3981.1 ± 275.2 3.60 ± 0.03 3020.0 ± 69.5 3.48 ± 0.01Perfluorooctanoic acid 457.1 ± 10.5 2.66 ± 0.01 676.1 ± 46.7 2.83 ± 0.03 1380.4 ± 138.8 3.14 ± 0.04Perfluorononanoic acid 457.1 ± 21.1 2.66 ± 0.02 741.3 ± 68.4 2.87 ± 0.04 1148.2 ± 130.7 3.06 ± 0.05P 363.1

n

omsFwm

2

S0c

tiisvcfs

Fl

erfluorodecanoic acid 173.8 ± 16.0 2.24 ± 0.04

.a., data not available.

f 15 × 104 cells/ml (in RPMI with 8% FBS Hi). C6 cells were grown as aonolayer in DMEM (high glucose) medium supplemented with 1% antibiotic

olution (penicillin/streptomycin), 1% glutamine, NaHCO3 (3.7 g/l) and 10%BS Hi at 37 ◦C under the same conditions. For the cytotoxicity assays, cellsere seeded in 96-well plates at an initial density of 5 × 104 cells/ml of cultureedium and incubated for 24 h.

.5. Cell viability assay

A colorimetric assay with WST-1 reagent was used for the cell viability tests.tock solutions of the perfluorinated acids were prepared in growth media with.5% DMSO added to improve solubility. Cells were exposed to nine differentoncentrations (from 2 �M to 20 mM) of the perfluorinated acids. Each incuba-

wlell

ig. 1. Exemplary concentration–response relationships for perfluorooctanoic acid foine exposed (logistic model, n = 9) and (c) V. fischeri (linear logistic model, n = 3).

± 8.4 2.56 ± 0.01 n.a. n.a.

ion was performed in triplicate, including the controls and blanks. The cells werencubated for 44 h. After these times, 10 �l of WST-1 reagent, diluted four-foldn phosphate buffer, was added to each well and incubated for 4 h at 37 ◦C. Sub-equently, the optical density at 450 nm was measured in the plate reader. Celliability was calculated as the percentage of the viability of exposed cells versusontrols. These data are the means of three independent experiments conductedor each acid. Concentration response curves were fitted with the nonlinear leastquares method using a linear logistic model for the IPC-81 and V. fischeri cells,

here a hormetic effect was observed, and a logistic model for C6 glioma cell

ine, where hormesis was not observed (Van Ewijk and Hoekstra, 1993; Ranket al., 2004). The log EC50 values were given since it is a model parameter in theogistic as well as in linear logistic model. Calculations were carried out with Ranguage and environment for statistical computing (http://www.r-project.org).

r (a) IPC-81 leukemia cell line (linear logistic model, n = 9), (b) C6 glioma cell

2 colog

3

wvaIowtPpio1

tfnE1

tcbrrrd(

o(it

4

Fanp

82 E. Mulkiewicz et al. / Environmental Toxi

. Results

The biological activity of the perfluorinated carboxylic acidsas studied at the cellular and molecular levels with different initro test systems. At the molecular level it was determined withcetylcholinesterase and glutathione reductase inhibition assays.n the applied concentration range only the AChE inhibition levelf perfluorononanoic acid was high enough to determine IC50,hich was 3526 �M. For the other acids, the enzyme inhibi-

ion was observed only at the highest concentration (4000 �M).erfluorohexanoic, perfluoroheptanoic, perfluorooctanoic anderfluorodecanoic acids, respectively, inhibited enzyme activ-ty by 87.9, 86.6, 76.2 and 84.5%. With glutathione reductase,nly PFNA caused enzyme inhibition; the IC50 for this acid was788 �M.

The IPC-81 leukemia cell line was the most sensitive tohe perfluorinated acids. The EC50 values obtained ranged

rom 173.8 ± 16.0 to 3715.4 ± 85.6 �M (Table 1). Biolumi-escence in V. fischeri bacteria was inhibited with respectiveC50 values of 4265.8 ± 393.5, 3020.0 ± 69.5, 1380.4 ± 138.8,148.2 ± 130.7 �M for PFHxA, PFHpA, PFOA and PFNA. For

sb(b

ig. 2. Influence of perfluorocarbon chain length on toxicity of perfluorinated acids (a)nd substance), (b) C6 glioma cell line (logistic model, n = 9 for each concentration= 3 for each concentration and substance) perfluorohexanoic acid (. . .), perfluoroheerfluorodecanoic acid (—).

y and Pharmacology 23 (2007) 279–285

he IPC-81 viability test and the V. fischeri acute biolumines-ence inhibition assay, the hormetic effect at concentrationselow inhibitory concentrations can be seen on the concentrationesponse curves (Figs. 1 and 2). Only the concentration responseelationship curve for C6 glioma cells showed no stimulatoryesponse. This latter cell line was also less sensitive to the acids,isplaying EC50 values from 363.1 ± 8.4 to 7943.3 ± 365.9 �MTable 1).

These results yielded a relationship between the cytotoxicityf the perfluorinated acids and the perfluorocarbon chain lengthFig. 2). In each test system used, the toxicity increased withncreasing chain length; the lowest effective concentrations werehus recorded for perfluorodecanoic acid.

. Discussion

Since perfluorinated acids are chemically stabilized by the

trong covalent bond between carbon and fluorine, they haveeen historically regarded as metabolically inert and nontoxicSargent and Seffl, 1970). Chemicals that are difficult to degradeiologically may nonetheless bioaccumulate and may affect the

in IPC-81 leukemia cell line (linear logistic model, n = 9 for each concentrationand substance); on V. fischeri luminescence inhibition (linear logistic model,ptanoic acid (—), perfluorooctanoic acid (– –), perfluorononanoic acid (- . -),

colog

hted

tlsAoa

nItcmHADbfeutTft1

patwluwaa

firl2b2

ilcwafebtp

icecacpcdt2ia

hseaf(iiptts

cadsa4istDiotao2herhac(w2h

E. Mulkiewicz et al. / Environmental Toxi

ealth of humans and biota. Because of the global distribu-ion and persistent nature of these chemicals, monitoring theirnvironmental fate and their ecotoxicological profile is highlyesirable (Van de Vijver et al., 2003, 2004).

Usually, the first detectable responses to environmental per-urbation are changes at the molecular, biochemical or cellularevel. During the last decade, the importance of in vitro systemsuch as cell lines has increased in toxicology and ecotoxicology.lthough such systems cannot replicate the complex interactionsf animals in vivo, they provide important predictive informationbout the biological activities of chemical substances.

There are not many studies where the toxicity of perfluori-ated carboxylic acids has been evaluated at the cellular level.n experiments with human hepatoblastoma HepG2 cells, PFOAreatment resulted in apoptosis as well as perturbation of theell cycle. Apoptosis became manifest with 200 �M and maxi-al upon exposure to 450 �M PFOA for 24 h. The cell cycle ofepG2 cells was perturbed by exposure to 50–150 �M PFOA.dditionally, exposure to 500 �M PFOA for 48 h resulted inNA degradation (Shabalina et al., 1999). Apart from the liver,lood is the tissue where perfluorinated chemicals are mostlyound in the human body (Kudo and Kawashima, 2003; Laut al., 2004). To assess the toxicity of perfluorinated acids, wesed the mammalian hematopoietic IPC-81 cell line. This sys-em demonstrated the highest sensitivity to the tested chemicals.he effective concentrations of PFOA recorded in our study

or the IPC-81 cell line were at the same level as those regis-ered for adverse effects in the HepG2 cell line (Shabalina et al.,999).

The effects observed in the two rat cell lines used in theresent study were different. The leukemia cell line displayedhigher sensitivity than the glioma cell line; to observe similar

oxic effects, 50% lower concentrations of the tested compoundsere necessary. Furthermore, in the presence of subinhibitory

evels of all compounds tested C6 glioma cells showed no stim-latory response, whereas for IPC-81 leukemia cells hormesisas observed. These differences between the two cell lines were

lso observed during tests of other types of chemicals (Ranke etl., 2004).

In our study, the hormetic response was also noticed in the V.scheri test. A fundamental component of many dose–responseelationships, hormesis is a reproducible and generalized bio-ogical phenomenon (Calabrese et al., 1999; Stepnowski et al.,004). Though typical of V. fischeri tests, it is very rarely reportedecause of difficulties in estimating EC50 values (Christofi et al.,002).

Different mechanisms of cytotoxicity for this group of chem-cals have been proposed. Perfluorinated acids with carbon chainengths of 7–10 were found to inhibit gap junctional intercellularommunication (GJIC) (Upham et al., 1998; Hu et al., 2002),hich is the major pathway of intracellular signal transduction

nd is thus important for normal cell growth and functioning, andor maintaining tissue homeostasis. A structural relationship was

stablished, indicating that the inhibitory effect was determinedy the length of the fluorinated tail, not by the nature of the func-ional group. In another study (Hu et al., 2003), the effects oferfluorinated compounds on membrane properties such as flu-

pgc1

y and Pharmacology 23 (2007) 279–285 283

dity and permeability was studied. The selectively permeableell membrane forms the first barrier protecting the cell fromxogenous exposure. Effects on the permeability status of theell membrane could play an important role in mediating thedverse effects of number environmental contaminants, espe-ially surface-active compounds. PFOS increased membraneermeability towards hydrophobic ligands, and perfluorinatedompounds increased membrane fluidity in fish leucocytes in aose-dependent manner. The lowest effective concentration forhe membrane fluidity effects of PFOS was 5–15 mg/l (Hu et al.,003). It is hard to speculate about the mechanism of cytotox-city, but we observed in our study that similar concentrationsffected cell viability.

To obtain more comprehensive information on the possiblearmful effects of exposure to these chemicals on the nervousystem, the acetylcholinesterase inhibition assay was used. Thisnzyme is an essential part of the nervous system. Widely useds a biomarker detecting pesticides, it is also inhibited by dif-erent classes of chemicals, including metals and surfactantsHerbert et al., 1995; Guilhermino et al., 1998). More recently,t was used to assess the environmental risk of newly designedndustrial chemicals (Stock et al., 2004). Our results indicate thaterfluorinated acids inhibit AChE only at very high concentra-ions. This low inhibition of AChE activity, in conjunction withhe results obtained for the C6 cell line, suggest that the nervousystem may not be very sensitive to perfluorinated acids.

Many pollutants can exhibit oxidative stress-related toxi-ity. It has been reported that treatment with perfluorinatedcids causes oxidative stress (Van der Oost et al., 2003). Aramatic increase in the cellular content of reactive oxygenpecies (superoxide anions and hydrogen peroxide) was foundfter treatment of human hepatoma HepG2 cells with 200 and00 �M PFOA after 3 h (Panaretakis et al., 2001). An increasen the activities of antioxidant enzymes such as catalase anduperoxide dismutase was observed in rat liver after adminis-ration of PFDA (Glauert et al., 1992). Lipid peroxidation andNA damage, well-known biochemical effects associated with

ncreased fluxes of oxyradicals (very important consequencesf oxidative stress), were observed in animals after exposureo perfluorinated acids (Takagi et al., 1991). Lipid peroxidesre known to be reduced by the action of glutathione per-xidase to alcohols using glutathione (Nordberg and Arner,001). Both increases and decreases in the glutathione levelave been observed after exposure to different chemicals. Annzyme known to have a physiological significance, glutathioneeductase plays an important role in maintaining GSH/GSSGomeostasis under oxidative stress conditions (Van der Oost etl., 2003). It has been shown that treatment with perfluorode-anoic acid significantly increased hepatic reduced glutathioneGSH) content and affected the activities of enzymes associatedith GSH synthesis, utilization and regeneration (Chen et al.,001). In that study, a decrease in glutathione reductase by theighest dose of PFDA (35 mg/kg) was observed. In our study,

erfluorinated acids did not significantly affect the activity oflutathione reductase. Inhibition was observed only at very highoncentrations of perfluoronanoic acid showing IC50 value of788 �M. Similarly, no changes in glutathione reductase activ-

2 colog

ie

l(tocarmtlwa

A

E1d

R

A

B

B

B

B

C

C

C

C

D

E

F

F

G

G

G

G

H

H

H

H

H

H

J

K

K

K

K

K

K

84 E. Mulkiewicz et al. / Environmental Toxi

ty were observed in rat liver after treatment with PFOA (Glauertt al., 1992; Kawashima et al., 1994; Cai et al., 1995).

Although a relationship between chain lengths and the bio-ogical effects of perfluorinated acids has already been suggestedKudo et al., 2000, 2001), it is still unclear since little informa-ion is available except for PFDA and PFOA. The results ofur study show a relation between toxicity and perfluorocarbonhain length, with toxicity being lowest for perfluorohexanoiccid and highest for perfluorodecanoic acid. Kudo et al. (2001)eported that perfluorinated acids with shorter carbon chains areore quickly excreted in the urine, resulting in a lower concen-

ration in the serum and liver. Thus, perfluorinated acids withonger carbon chains will be eliminated in urine less rapidly,hich together with their higher toxicity should be recognized

s a great health and environmental concern.

cknowledgements

Financial support was provided by the Polish Ministry ofducation and Research under grants: 2P04G 083 29, 2P04G18 29 and DS 8390-4-0141-6. Help of Dr. Tomasz Puzyn withata analysis is greatly acknowledged.

eferences

nkley, 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 bio-concentration of perfluorooctanesulfonate in a partial life-cycle test with thefathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 24, 2316.

anks, R.E., Smart, B.E., Tatlow, J.C., 1994. Organofluorine Chemistry Princi-ples and Comercial Applications. Plenum Press, New York.

enda, P., 1968. Differentiated rat glial cell strain in tissue culture. Science 161,370.

ossia, R., Riget, F.F., Dietz, R., Sonne, C., Fauser, P., Dam, M., Vorkamp, K.,2005. Preliminary screening of perfluorooctane sulfonate (PFOS) and otherfluorochemicals in fish, birds and marine mammals from Greenland and theFaroe Islands. Environ. Pollut. 136, 323.

urris, J.M., Lundberg, J.K., Olsen, G., Simpson, C., Mandel, J., 2005. Deter-mination of serum half-lives of several fluorochemicals: Interim Report #2.Study Sponsor: 3M Company, Corporate Occupational Medicine Depart-ment, US EPA AR226-1086.

ai, Y., Appelkvist, E.L., DePierre, J.W., 1995. Hepatic oxidative stress andrelated defenses during treatment of mice with acetylsalicylic acid and otherperoxisome proliferators. J. Biochem. Toxicol. 10, 87.

alabrese, E.J., Baldwin, L.A., Holland, C.D., 1999. Hormesis: a highly gener-alizable and reproducible phenomenon with important implications for riskassessment. Risk Anal. 19, 261.

hen, L.C., Tatum, V., Glauert, H.P., Chow, C.K., 2001. Peroxisome proliferatorperfluorodecanoic acid alters glutathione and related enzymes. J. Biochem.Mol. Toxicol. 15, 107.

hristofi, N., Hoffmann, C., Tosh, L., 2002. Hormesis responses of free andimmobilized light-emitting bacteria. Ecotoxicol. Environ. Saf. 52, 227.

imitrov, S., Kamenska, V., Walker, J.D., Windle, W., Purdy, R., Lewis, M., etal., 2004. Predicting the biodegradation products of perfluorinated chemicalsusing CATABOL. SAR QSAR Environ. Res. 15, 69.

llman, G.L., Courtney, K.O., Andrres, V., Featherstone, R.M., 1961. A newrapid colorimetric determination of acetylcholinesterase activity. Biochem.Pharmacol. 7, 88.

an, Y.O., Jin, Y.H., Ma, Y.X., Zhang, Y.H., 2005. Effects of perfluorooctanesulfonate on spermiogenesis function of male rats. Wei Sheng Yan Jiu. 34,37.

isher, T.C., Crane, M., Callaghan, A., 2000. A quality-controlled and optimisedmicrotitreplate assay to detect acetylcholinesterase activity in individual Chi-

K

K

y and Pharmacology 23 (2007) 279–285

ronomus riparius meigen, for use as a biomarker of pesticide exposure.Environ. Toxicol. Chem. 19, 1749.

iesy, J.P., Kannan, K., 2001. Global distribution of perfluorooctane sulfonatein wildlife. Environ. Sci. Technol. 35, 1339.

lauert, 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 theperoxisome proliferators ciprofibrate and perfluorodecanoic acid on hepaticcellular antioxidants and lipid peroxidation in rats. Biochem. Pharmacol. 43,1353.

uilhermino, L., Barros, P., Silva, M.C., Soares, A.M.V.M., 1998. Should the useof inhibition of cholinesterases as a specific biomarker for organophosphateand carbamate pesticides be questioned? Biomarkers 3, 157.

uruge, K.S., Taniyasu, S., Yamashita, N., Wijeratna, S., Mohotti, K.M., Senevi-ratne, H.R., Kannan, K., Yamanaka, N., Miyazaki, S., 2005. Perfluorinatedorganic compounds in human blood serum and seminal plasma: a study ofurban and rural tea worker populations in Sri Lanka. J. Environ. Monit. 7,371.

agen, D.F., Belisle, J., Johnson, J.D., Venkateswarlu, P., 1981. Charac-terization of fluorinated metabolites by a gas chromatographic-heliummicrowave plasma detector—the biotransformation of 1H, 1H, 2H, 2H-perfluorodecanol to perfluorooctanoate. Anal. Biochem. 118, 336.

arada, K., Saito, N., Inoue, K., Yoshinaga, T., Watanabe, T., Sasaki, S.,Kamiyama, S., Koizumi, A., 2004. The influence of time, sex and geo-graphic factors on levels of perfluorooctane sulfonate and perfluorooctanoatein human serum over the last 25 years. J. Occup. Health 46, 141.

erbert, A., Guilhermino, L., Da Silva de Assis, H.C., Hansen, P.D., 1995.Acetylcholinesterase activity in aquatic organismsas pollution biomarker.Angew. Zool. 3, 1.

off, 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 inliver of three freshwater fish species in Flanders (Belgium): relationshipswith biochemical and organismal effects. Environ. Pollut. 137, 324.

u, W., Jones, P.D., Upham, B.L., Trosko, J.E., Lau, C., Giesy, J.P., 2002.Inhibition of gap junctional intercellular communication by perfluorinatedcompounds in rat liver and dolphin kidney epithelial cell lines in vitro andSprague–Dawley rats in vivo. Toxicol. Sci. 68, 265.

u, W., Jones, P.D., De Coen, W., King, L., Fraker, P., Newsted, J., Giesy,J.P., 2003. Alterations in cell membrane properties caused by perfluorinatedcompounds. Comp. Biochem. Physiol. C 135, 77.

ones, P.D., Hu, W., De Coen, W., Newsted, J.L., Giesy, J.P., 2003. Binding ofperfluorinated. Fatty acids to serum proteins. Environ. Toxicol. Chem. 22,2639.

annan, K., Koistinen, J., Beckman, K., Evans, T., Gorzelany, J.F., Hansen,K.J., Jones, E., Helle, P.D., Nyman, M., Geisey, J.P., 2001. Accumulationof perflurooctane sulfonate in marine mammals. Environ. Sci. Technol. 35,1593.

annan, K., Corsolini, S., Falandysz, J., Oehme, G., Focardi, S., Giesy, J.P.,2002a. Perfluorooctanesulfonate and related fluorinated hydrocarbons inmarine mammals, fishes, and birds from coasts of the Baltic and the Mediter-ranean Seas. Environ. Sci. Technol. 36, 3210.

annan, K., Hansen, K.J., Wade, T.L., Giesy, J.P., 2002b. Perfluorooctane sul-fonate in oyster, Crassostrea virginica, from the Gulf of Mexico and theChesapeake Bay, USA. Arch. Environ. Contam. Toxicol. 42, 313.

awashima, Y., Suzuki, S., Kozuka, H., Sato, M., Suzuki, Y., 1994. Effects ofprolonged administration of perfluorooctanoic acid on hepatic activities ofenzymes, which detoxify peroxide and xenobiotic in the rat. Toxicology 93,85.

ennedy 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 toxicologyof perfluorooctanoate. Crit. Rev. Toxicol. 34, 351.

ey, B.D., Howell, R.D., Criddle, C.S., 1997. Fluorinated organics in the bio-sphere. Environ. Sci. Technol. 31, 2445.

udo, N., Kawashima, Y., 2003. Toxicity and toxicokinetics of perfluorooctanoicacid in humans and animals. J. Toxicol. Sci. 28, 49.

udo, N., Bandai, N., Suzuki, E., Katakura, M., Kawashima, Y., 2000. Inductionby perfluorinated fatty acids with different carbon chain length of peroxiso-mal beta-oxidation in the liver of rats. Chem. Biol. Interact. 124, 119.

colog

K

L

L

M

M

M

N

O

O

P

R

R

S

S

S

S

T

U

V

V

V

E. Mulkiewicz et al. / Environmental Toxi

udo, N., Suzuki, E., Katakura, M., Ohmori, K., Noshiro, R., Kawashima, Y.,2001. Comparison of the elimination between perfluorinated fatty acids withdifferent carbon chain length in rats. Chem. Biol. Interact. 134, 203.

acaze, N., Gombaud-Saintonge, G., Lanotte, M., 1983. Conditions controllinglong-term proliferation of brown Norway rat promyelocytic leukemia invitro: primary growth stimulation by microenvironment and established ofan autonomous brown Norway “leukemic stem cell line”. Leuk. Res. 7,145.

au, C., Butenhoff, J.L., Rogers, J.M., 2004. The developmental toxicity ofperfluoroalkyl acids and their derivatives. Toxicol. Appl. Pharmacol. 198,231.

aras, M., Vanparys, C., Muylle, F., Robbens, J., Berger, U., Barber, J.L., Blust,R., De Coen, W., 2006. Estrogen-like properties of fluorotelomer alcohols asrevealed by mcf-7 breast cancer cell proliferation. Environ. Health Perspect.114, 100–105.

artin, J.W., Mabury, S.A., Solomon, K.R., Muir, D.C., 2003. Bioconcen-tration and tissue distribution of perfluorinated acids in rainbow trout(Oncorhynchus mykiss). Environ. Toxicol. Chem. 22, 196.

ehrotra, K., Morgenstern, R., Ahlberg, M.B., Georgellis, A., 1999. Hypophy-sectomy and/or peroxisome proliferators strongly influence the levels ofphase II xenobiotic metabolizing enzymes in rat testis. Chem. Biol. Interact.122, 73.

ordberg, J., Arner, E.S., 2001. Reactive oxygen species, antioxidants, and themammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287.

akes, K.D., Sibley, P.K., Solomon, K.R., Mabury, S.A., Van der Kraak, G.J.,2004. Impact of perfluorooctanoic acid on fathead minnow (Pimephalespromelas) fatty acyl-CoA oxidase activity, circulating steroids, and repro-duction in outdoor microcosms. Environ. Toxicol. Chem. 23, 1912.

akes, 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 per-fluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity,oxidative stress, and circulating sex steroids. Environ. Toxicol. Chem. 24,1172.

anaretakis, 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 peroxisomeproliferator and hepatocarcinogen, perfluorooctanoic acid. Toxicol. Appl.Pharmacol. 173, 56.

anke, J., Moelter, K., Stock, F., Bottin-Weber, U., Poczobutt, J., Hoffmann,J., Ondruschka, B., Filser, J., Jastorff, B., 2004. Biological effects of imida-

V

Y

y and Pharmacology 23 (2007) 279–285 285

zolium ionic liquids with varying chain lengths in acute Vibrio fischeri andWST-1 cell viability assays. Ecotoxicol. Environ. Saf. 58, 396.

uchaud, S., Zorn, M., Davilar-Villar, E., Genieser, H.-G., Hoffmann, C., Gjer-sten, B.T., Døskeland, S.O., Jastorff, B., Lanotte, M., 1995. Evidence forseveral pathways of biological response to hydrolysable cAMP analogs usinga model system of apoptosis in IPC-81 leukemia cells. J. Cell. Pharmacol.2, 127.

argent, J.W., Seffl, R.J., 1970. Properties of perfluorinated liquids. Fed. Proc.29, 1699.

habalina, I.G., Panaretakis, T., Bergstrand, A., DePierre, J.W., 1999. Effects ofthe rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoicacid, on apoptosis in human hepatoma HepG2 cells. Carcinogenesis 20,2237.

tepnowski, P., Skladanowski, A.C., Ludwiczak, A., Łaczynska, E., 2004. Eval-uating the cytotoxicity of ionic liquids using human cell line HeLa. Hum.Exp. Toxicol. 23, 513.

tock, F., Hoffmann, J., Ranke, J., Stormann, R., Jastorff, B., 2004. Effects ofionic liquids on the acetylcholinesterase—a structure-activity relationshipconsideration. Green Chem. 6, 286.

akagi, A., Sai, K., Umemura, T., Hasegawa, R., Kurokawa, Y., 1991. Short-termexposure to the peroxisome proliferators, perfluorooctanoic acid and perflu-orodecanoic acid, causes significant increase of 8-hydroxydeoxyguanosinein liver DNA of rats. Cancer Lett. 57, 55.

pham, B.L., Deocampo, N.D., Wurl, B., Trosko, J.E., 1998. Inhibition ofgap junctional intercellular communication by perfluorinated fatty acids isdependent on the chain length of the fluorinated tail. Int. J. Cancer. 78, 491.

an 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 lev-els and stable isotope ratios in marine mammals. Environ. Sci. Toxicol.Chem. 22, 2037.

an 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.

an der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation andbiomarkers in environmental risk assessment: a review. Environ. Toxicol.

Pharmacol. 13, 57.

an Ewijk, P.H., Hoekstra, J.A., 1993. Calculation of the EC50 and its confidenceinterval when subtoxic stimulus is present. Ecotoxicol. Environ. Saf. 25, 25.

amashita, 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.