Use of hepatocytes from Hoplias malabaricus to characterize the toxicity of a complex mixture of lipophilic halogenated compounds

Toxicology in Vitro 21 (2007) 706–715 www.elsevier.com/locate/toxinvit

Use of hepatocytes from Hoplias malabaricus to characterize the toxicity of a complex mixture of lipophilic halogenated compounds F. Filipak Neto a, S.M. Zanata b, H.C. Silva de Assis c, D. Bussolaro a, M.V.M. Ferraro e, M.A.F. Randi a, J.R.M. Alves Costa a, M.M. Cestari d, H. Roche f, C.A. Oliveira Ribeiro a,¤ a b

Departamento de Biologia Celular, Universidade Federal do Paraná, Cx. Postal 19031, CEP: 81.531-990, Curitiba, PR, Brazil Departamento de Patologia Básica, Universidade Federal do Paraná, Cx. Postal 19031, CEP: 81.531-990, Curitiba, PR, Brazil c Departamento de Farmacologia, Universidade Federal do Paraná, Cx. Postal 19031, CEP: 81.531-990, Curitiba, PR, Brazil d Departamento de Genética, Universidade Federal do Paraná, Cx. Postal 19031, CEP: 81.531-990, Curitiba, PR, Brazil e Colégio de Aplicação, Universidade Federal de Santa Catarina, Cx. Postal 5188, CEP: 88040-570, Florianópolis, SC, Brazil f Université Paris-Sud XI, ESE UMR 8079 – Ecologie Systématique et Evolution, Bât 362 F91405 Orsay Cedex, France Received 4 September 2006; accepted 12 December 2006 Available online 10 January 2007

Abstract Organisms are continuously exposed to a plethora of anthropogenic toxicants daily released to the environment. In the present study, the eVects of a mixture of halogenated organic compounds (HOCs) extracted from hepatic lipids were evaluated on the primary hepatocyte culture from Wsh Hoplias malabaricus. Cells were isolated through non-enzymatic perfusion protocol and cultured during 3 days to allow attachment. Two concentrations of the mixture of HOCs (10 ng ml¡1 [Mix10] and 50 ng ml¡1 [Mix50]) were tested in cells for 2 days by medium replacement. The control groups, with and without solvent (DMSO) were run in the same conditions. Both tested concentrations of HOCs increased the catalase and GST activities, but only the Mix50 increase the DNA damage and decreased the GSH concentration and cell viability. Lipid peroxidation increased in the Mix10 group, but it seems to be more a consequence of DMSO presence than the HOCs themselves. The DMSO at 0.1% increased the lipid peroxidation, GSH concentration, apoptosis and DNA damage. The present data suggest that DMSO interferes with the hepatocytes of H. malabaricus in culture and that the mixture of HOCs tested alters the redox state of the hepatocytes. © 2007 Elsevier Ltd. All rights reserved. Keywords: Halogenated organic compounds; Organochlorine pesticides; DMSO; Primary hepatocytes culture; Hoplias malabaricus

1. Introduction Organochlorine pesticides (OCPs) are a broad class of organochlorine compounds (OCs) that were widely used as insecticides between the 1950s and 1970s. Due to their gloAbbreviations: DMSO, dimethylsulfoxide; EDTA, ethylenedinitrilotetraacetic acid; GSH, reduced glutathione; GST, glutathione S-transferase; HCH, hexachlorocyclohexane; HOCs, halogenated organic compounds; LPO, lipid peroxidation; OCs, organochlorine compounds; OCPs, organochlorine pesticides; PAHs, polycyclic aromatic hydrocarbons; PBS, phosphate buVered saline; PCBs, polychlorinated biphenyls; ROS, reactive oxygen species. * Corresponding author. Tel.: +55 41 3361 1680; fax: +55 41 3266 2042. E-mail address: [email protected] (C.A. Oliveira Ribeiro). 0887-2333/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2006.12.011

bal distribution (Warren et al., 2003) and persistent contamination of the environment, the restriction to their use increased in several countries. OCPs are implicated with several biological abnormalities. For example, dieldrin and lindane (-HCH, -hexachlorocyclohexane) have been implicated with Parkinsonism in humans (Uversky et al., 2002) and DDT (Scippo et al., 2004), dieldrin and endosulfan (Andersen et al., 2002) have been classiWed as potentially estrogenic compounds. In addition, the exposure to some OCs that exhibit endocrine-disrupting properties can cause eVects from decrease in sperm counts in humans to feminization of Wsh (Royal Society, 2000). The presence of OCs and some other halogenated organic compounds (HOCs) in remote and non-urbanized

F. Filipak Neto et al. / Toxicology in Vitro 21 (2007) 706–715

regions as the Arctic (Li and Macdonald, 2005) and Everest (Li et al., 2006) emphasizes their global dispersion. Concentrations as high as 32–59 and 17–102 ng g¡1 wet weight liver of total OCPs have been detected in striped weakWsh (Cynoscion guatucupa) from Bahia Blanca Estuary, Argentinean coast (Lanfranchi et al., 2006) and in european eel (Anguilla anguilla) from Camargue Nature Reserve, France (Oliveira Ribeiro et al., 2005; Buet et al., 2006), respectively. Despite The Stockholm Convention on POPs (persistent organic pollutants) established the immediate banishment of some of the identiWed OCPs in these studies (aldrin, endrin, dieldrin, chlordane, heptachlor and hexachlorobenzene), residues of some of them may still persist in the environment during the following years. In Wsh, as in other vertebrates, the liver is an important site of biotransformation and bioaccumulation of several HOCs, as well as the target to toxic eVects of these chemicals and byproducts. Important eVects of HOCs on the liver are the induction of hepatic drug-metabolizing enzymes, i.e., isoforms of cytochrome P450 (Li et al., 1995; Coumoul et al., 2002), interaction with cellular macromolecules such as lipids and thus, disruption of the function of cellular and subcellular membranes, and generation of prooxidant species. Oxidative stress (imbalance between the production of oxidants and the respective defense systems of an organism) and suppression of hepatic mitochondrial energy metabolism has been suggested as one of the mechanisms of several HOCs-induced hepatotoxicity, like HCHs, chlordane, aldrin, dieldrin and endosulfan (see ATSDR, 2006 for review). Hoplias malabaricus is a voracious freshwater teleost predator, widely distributed in South America (Fowler, 1950). This species occupies a high trophic level in aquatic food chain and it is a valuable biological model of Wsh for tropical Brazilian’s ecosystems. Due to human activities, atmospheric transport and environmental persistence of several HOCs such as some OCs, it was detected high levels of these halogenated compounds in the liver and muscle of H. malabaricus from lakes in the South of Brazil (Miranda, 2006, personal communication). These anthropogenic halogenated compounds, which include several OCPs, are important toxicants to worldwide natural biota, including the Brazilian’s. They bioaccumulate at menacing levels in fat tissues of aquatic organisms such as A. anguilla and H. malabaricus, as previously reported to other teleosts from diVerent continents. Thus, it is indispensable to generate data about HOCs toxicity in native Wsh species from Brazil, to compare and complement studies being conducted with exotic Wsh species (Oncorhynchus mykiss, Cyprinus carpio, A. anguilla etc.) as counterparts. According to Yang (1994) and Groten et al. (1999), the majority of the studies involving the pesticide toxicity, which can be extrapolated to HOCs in general, has been conducted to evaluate the eVects of an individual chemical or a speciWc residue. More recently, emphasis has been

707

given on chemical mixtures studies (Olgun et al., 2004) thanks to the possibility of simulating a more realistic situation. Extracted lipophilic xenobiotics from animal organs constitute the “end-result” of the biotransformation and bioaccumulation processes of contaminants in biological systems, oVering a reasonable proWle of biota contamination. Then, the aim of the present study is to investigate the cellular eVects of a pool of HOCs obtained from hepatic lipids of A. anguilla on primary hepatocyte culture from H. malabaricus, through some biomarkers of oxidative stress (lipid peroxidation, catalase and glutathione S-transferase activities, reduced glutathione concentration), genotoxicity (DNA integrity) and cell status (apoptosis and cell viability). The use of extracted lipophilic pollutants on cultured hepatocytes represents a valuable new approach in an attempt to reproduce the chemical form and concentration of these xenobiotics, and allow important interactions of many naturally occurring chemicals in a mixture, as close as currently possible to the hepatic situation of chronically and environmentally exposed Wshes of some impacted areas. The liver is of key importance at this point, not only because it is an essential site to the action of HOCs, but also because of the fact that during periods of food privation or increase in energy demand (e.g., during reproduction), hepatic fat storage are “used”. As a consequence, bioaccumulated HOCs are remobilized from lipids, which facilitates their interaction with subcellular compartments of hepatic cells and perhaps, with several sites of the Wshes’ body as a result of their transport in blood stream. 2. Materials and methods 2.1. Fishes Specimens of H. malabaricus (350–800 g of body mass) were obtained from a private farm at Joinville city (Santa Catarina State, South of Brazil) and kept in 30 l individual aquaria under controlled temperature (20–22 °C) and photoperiod (12:12 h). Every 3 days, they were fed with one young live carp C. carpio (8–15 g of body mass) until they were sacriWced. 2.2. Hepatocytes preparation A non-enzymatic protocol (Filipak Neto et al., 2006) was performed to isolate hepatocytes from H. malabaricus. BrieXy, Wshes were anesthetized (0.02% MS222 in water) and sacriWced. The liver was removed and perfused through portal vein and arterial system (ice cold 2 mM EDTA in PBS – phosphate-buVered saline, pH 7.8), aseptically minced with stainless-steel blades and gently pressed through a stainless-steel mesh for mechanical disruption. Cells were collected in PBS, centrifuged at low speed (100–120g) and washed twice in PBS and once in culture medium to remove debris.

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2.3. Culture conditions Cellular suspensions were adjusted to a density of 0.5 £ 106 cells ml¡1 in RPMI 1640 medium (pH 7.8) supplemented with HEPES (15 mM), NaHCO3 (6 mM), bovine– porcine insulin (0.2 U ml¡1), gentamycin sulfate (40 mg l¡1) and fetal bovine serum (10%). Then, they were seeded onto 75 cm2 culture Xasks (PY430720, Corning-Costar) at a density of 1.2–1.6 £ 105 cells cm¡2 and kept at 24 °C in a common incubator (at atmospheric pCO2). 2.4. Preparation of a mixture of the halogenated organic compounds A large pool of HOCs (mainly OCPs) extracted from hepatic lipids of 28 Wshes A. anguilla captured at the Camargue Biosphere Reserve, France (Oliveira Ribeiro et al., 2005) was used in the present study (Table 1). These compounds were puriWed by solid phase extraction on Xorisil, an extremely polar, magnesium-loaded silica gel, following the EPA method 3620, Wrst with hexane, to eliminate other lipophilic compounds, then with hexane/ diethyl ether (90/10) for OCs clean-up. OCs were analyzed by gas chromatography with AutoSystem XL (Perkin– Elmer), using electron capture detection (63Ni Source) and nitrogen as the carrier gas following an adapted procedure of the EPA Method 8081a (Oliveira Ribeiro et al., 2005). Reference organochlorines were purchased from CIL Cluzeau (Ste-Foy-la-Grande, France) and used to identify and to quantify each component of the mixture of HOCs. The concentration of compounds was evaluated on the basis of

the GC analysis as described by Oliveira Ribeiro et al. (2005). The procedure used assures that if there are other (not identiWed) lipophilic substances in the extracts, these compounds are halogenated and have polarities equivalent to that of the analyzed chlorinated ones. Some examples of the eliminated compounds in the extracts are the PCBs – polychlorinated biphenyls – and the least polar substances (eliminated with hexane or retained on the column of Xorisil), the PAHs – polycyclic aromatic hydrocarbons – (normally extracted with a more polar solvent (dichloromethane) or retained on the column) and the pyrethroids (more polar, used at very low dose at Camargue and quickly degraded). The substituted ureas, triazines and organophosphorous are also more polar, but one cannot be sure that a part of them did not pass (very low) together with the OCs. A certain number of peaks were detected but not identiWed on the chromatograph. They could correspond to metabolites or unknown or unidentiWed OCs. In addition, one can also think that the most volatile and least stable molecules disappeared during the preparation and the processing. However, the OCs (excluded the least polar like the DDT) are the most logical molecules found in the analyzed phase. In the pool it was identiWed Wfteen HOCs and metabolites (Table 1). They were dissolved in dimethylsulfoxide (DMSO) before the exposure of cultured hepatocytes from H. malabaricus at a Wnal medium concentration of 0.1% of DMSO. DMSO was used because HOCs are highly lipophilic compounds and the checked literature did not indicate cytotoxic eVects of this solvent to hepatocytes at low concentrations (60.5%).

Table 1 Concentrations of DMSO and identiWed halogenated organic compounds (HOCs) in the culture medium for the Control, DMSO, Mix10 and Mix50 groups Tested groups Mix50 ¡1

Mix10 ¡1

DMSO

Control

IdentiWed HOC (class of pollutant) -HCH (OCP) -HCH + -HCH (OCP) -HCH (OCP) Heptachlor (OCP) Heptachlor epoxide (OCP metabolite) -Endosulfan (OCP) -Endosulfan (OCP) Endosulfan sulfate (OCP metabolite) Aldrin (OCP) Dieldrin (OCP/OCP metabolite) Endrin (OCP) Endrin aldehyde (OCP metabolite) -Chlordane (OCP) -Chlordane (OCP) Fipronil (phenyl pyrazole pesticide)

ng ml 0.57 4.69 2.13 1.47 4.92 3.33 6.14 1.93 1.91 1.99 1.76 3.14 1.51 9.08 5.44

Final concentration of HOCs

50 ng ml¡1

10 ng ml¡1

0 ng ml¡1

0 ng ml¡1

Final concentration of DMSO

0.1%

0.1%

0.1%

0%

nM 1.97 16.11 7.33 3.94 12.64 8.17 15.09 4.56 5.23 5.22 4.62 8.25 3.68 22.16 12.45

ng ml 0.11 0.94 0.43 0.29 0.98 0.67 1.23 0.39 0.38 0.40 0.35 0.63 0.30 1.82 1.09

nM 0.39 3.22 1.47 0.79 2.60 1.64 3.02 0.91 1.05 1.04 0.92 1.65 0.74 4.43 2.49

0

0

Most of the identiWed HOCs are OCPs (organochlorine pesticides). Some unidentiWed halogenated compounds possibly present in the pool of HOCs may include other OCs and OCs metabolites, and at a very low concentration (if any), ureas, triazines and organophosphorous compounds. The extraction protocol allows eliminating the PCBs, PAHs, pyrethroids and least stable or most volatile lipophilic compounds from extracts.

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3. Experimental design First, the cells were cultured for 3 days to allow cell’s attachment and recovery from isolation procedure. The majority of unattached or dead cells were removed from Xasks by a mild washing with PBS. The attached cells were exposed to diVerent concentrations of an mixture of HOCs (plus 0.1% of DMSO) dissolved in the culture medium (10 and 50 ng HOCs ml¡1) during 48 h. Two control groups (cells cultured in HOCs-free and in DMSO/HOCs-free media) were also established (Table 1). For each experiment, one Wsh was killed and the same hepatocyte’s population was seeded onto several culture Xasks, which were employed for the tested groups. On this way, it was possible to compare the control, DMSO and HOCs-exposed groups without interferences of Wsh-to-Wsh variability inside the same experiment. After 48 h, the cells were harvested by scrapping oV for biochemical assays and by trypsinization (0.02% EDTA, 0.05% trypsin in PBS) at room temperature for cell viability, TUNEL and Comet assays. 3.1. Cell viability The viability of cells was determined by Trypan blue dye (0.4%) exclusion method after 48 h of exposure, using a Neubauer chamber. It was visually counted 200 cells per sample, which were classiWed as viable (without intake of dye) or non-viable (with intake of dye). 3.2. Biochemical procedures Hepatocytes were washed twice in PBS and centrifuged at low speed. Pelleted cells were suspended in 0.5 ml of icecold Tris–sucrose buVer (10 mM Tris–HCl, 250 mM sucrose; pH 7.8), shock frozen in liquid nitrogen and stored at ¡76 °C. Prior to the essays, samples were thawed and soniWed on ice, during 2 min (2 cycles of 1 min each) for lipid peroxidation and 6 cycles of 4 s each for the other biochemical assays. 3.2.1. Catalase activity Cell homogenates were centrifuged at 9000g for 20 min at 4 °C and catalase activity was assayed spectrophotometrically in supernatant (S9) fraction by measuring the decrease in absorbance of H2O2 (Aebi, 1984). Shortly, 20 l of supernatant was added to 980 l of reaction medium (20 mM H2O2, 50 mM Tris–base, 0.25 mM EDTA, pH 8.0, 25 °C) in a quartz cuvette and the decrease in absorbance was immediately measured at 240 nm wave length during 1 min 20 s. For the determination of catalase activity, it was selected the Wrst interval of 1 min (r2 > 0.98) and used the molar extinction coeYcient for H2O2 of 40 M¡1 cm¡1.

709

(GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrata (Keen et al., 1976). First, 50 l of supernatant was added to 96-well microplate and then, 100 l of reaction medium (1.5 mM GSH, 2.0 mM CDNB, 0.1 M potassium phosphate buVer, pH 6.5) was rapidly added using a multichannel micropipette. Absorbance increase was immediately measured at 340 nm wave length during 3 min. For the determination of GST activity, it was selected the Wrst interval of 2 min (r2 > 0.99) and used the molar extinction coeYcient for CDNB of 9.6 mM¡1 cm¡1. 3.2.3. Lipid peroxidation measure Lipid peroxidation (LPO) was determined by FOX (ferrous oxidation-xylenol) assay (Jiang et al., 1991, 1992) with minor modiWcations. Cell homogenates were centrifuged at 1000g for 10 min at 4 °C. For the assay, 30 l of supernatant and 270 l of a solution containing 100 M xylenol orange, 25 mM H2SO4, 4 mM BHT (butylated hydroxytoluene) and 250 M FeSO4 · NH4 (ammonium ferrous sulfate) in 90% grade methanol were, respectively, added to a 96-well microplate. Blanks were prepared by replacing the supernatant for Tris–sucrose buVer. Samples were incubated at room temperature for 30 min until the reaction is complete and absorbances were measured at 570 nm wave length. To determine the hydroperoxides concentration, it was used the apparent molar extinction coeYcient for H2O2 and cumene hydroperoxide of 4.3 £ 104 M¡1 cm¡1. 3.2.4. Reduced GSH measurement For GSH content determination, proteins from samples were precipitated (10% trichloroacetic acid), and supernatant were centrifuged at 1000g for 15 min at 4 °C. Then, 50 l of supernatant was added to a 96-well microplate, followed by addition of 230 l of Tris (0.4 M, pH 8.9). Right before absorbance measurement, 20 l of 2.5 mM DTNB [5,5⬘-dithiobis(2-nitrobenzoic acid) in 25% methanol] was added using a multichannel micropipette. Absorbance was determined at 415 nm wave length and GSH concentration was calculated through comparison with GSH standard curve (Sedlak and Lindsay, 1968). Protein content was determined in aliquots of sample separated before protein precipitation and blanks consisted of 0.4 M Tris instead of samples. 3.2.5. Protein content Total proteins were quantiWed in the samples according to Bradford (1976). Shortly, 10 l of supernatant samples plus 250 l of Bradford reagent (Sigma®) were added to a 96-well microplate and absorbance was measured at 595 nm wave length. Protein content was calculated thought comparison with a standard curve of bovine serum albumin. 3.3. Comet assay

3.2.2. GST activity S9 fraction was obtained and the global glutathione S-transferase (GST) activity was determined by measuring the increase in absorbance, using reduced glutathione

DNA damage (unspeciWc DNA cleavage) was analyzed by means of the Comet assay (Singh et al., 1988). Trypsinized cells were suspended in 0.5% low melting point agarose

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(LMPA), layered onto precoated 1.5% normal melting point agarose microscope slides and spread with a coverslip. After LMPA solidiWcation, the coverslips were removed and the slides were kept in a cold lysing solution (220 mM NaCl, 9 mM EDTA, 0.9 mM Tris, 1% Triton X-100 and 10% DMSO, 0.9% sodium sarcosinate, pH 10.0) for 1 h at 4 °C. Then, the slides were placed in electrophoresis buVer (300 mM NaOH, 1 mM EDTA, 2% DMSO, pH > 13) for 25 min to allow DNA unwinding. Electrophoresis was performed at 25 V and 300 mA for 25 min. After electrophoresis, the slides were neutralized in 0.4 M Tris (pH 7.5), Wxed in ethanol during 10 min and stained with ethidium bromide (2 g ml¡1). All procedures were performed under red light. For analyzes, one hundred nuclei per slide were blindly classiWed by their tail length (0, 1, 2 or 3) under a ZEISS Axiophot HBO 50 microscope and scored as follow: score D (damage class £ percentage of incidence). The classiWcation of the severity of DNA damage was the following: not fragmented DNA, which migrates toward the positive electrical pole as a single body was classiWed into class 0; slightly fragmented DNA in which a very small tail is visible because some fragments migrate faster than the main DNA structure was included into class 1; the classes 2 and 3 were attributed, respectively, to nuclei that have moderately and severely fragmented DNA as distinguishable by the length of the tails (Fig. 2).

Kruskal–Wallis Test (non-parametric ANOVA) was performed. The HOCs mixtures were tested in two independent experiments for all assays performed except by TUNEL assay. Within a single experiment at least Wfteen replicas per experimental group were used for biochemical assays and at least three replicas for viability and comet assays. TUNEL assay was performed once and contained four replicas (4 £ 104 events each) per experimental group.

3.4. Determination of apoptosis by TUNEL assay

When compared, the control and DMSO groups had similar catalase and GST activities. On the other hand, Mix10 and Mix50 groups showed, respectively, an increase of 25% and 36% of catalase activity and of 22% and 42% of GST activity in comparison with them. For both enzymatic activities, there were no diVerences between Mix10 and Mix50 groups (Fig. 1C and D).

One of the later steps in apoptosis is the total fragmentation of DNA by endonucleases. Thus, TUNEL assay was carried out to detect the presence of broken DNA extremities. Deoxynucleotides conjugated to FITC Xuorochrome (FITC-dUTP) – APO-DIRECT™ KIT (BD Biosciences Pharmingen, USA) was used to label DNA breaks and detect apoptotic cells by Xow cytometry. Dissociated cells were Wxed in 1% paraformaldehyde in PBS (1 h at 4 °C), suspended in ice-cold 70% ethanol and maintained in freezer at ¡20 °C until staining. Then, a number of 1.0– 2 £ 106 cells were washed twice with the kit’s washing solution and incubated in 50 l of staining solution (0.75 l TdT Enzyme, 8 l FITC-dUTP, 10 l reaction buVer and 32 l of deionized water per sample) during 2 h at 37 °C. The cells were washed twice with rinse buVer, suspended in 1 ml of PBS and analyzed under FACSCalibur Xow cytometer. The cells were excited with a blue Argon laser (15 mW) at 488 nm and the FITC-dUTP Xuorescence was recorded in the FL-1 Wlter set (530/30 nm). Data from 4 £ 104 cells were collected and analyzed using the CellQuest® software (Becton–Dickinson). 3.5. Statistical procedures For all the biochemical, cell viability and TUNEL assays, One-way analysis of variance (ANOVA) followed by Tukey–Kramer Multiple Comparisons Test was carried out. To evaluate DNA breaks scored in the Comet assay,

4. Results 4.1. Cell viability and programmed cell death Cell viability after 48 h of exposure was decreased in about 6% in the Mix50 group (Fig. 1A), but no diVerences in viable cells were observed among the control, DMSO and Mix10 groups. The apoptotic cells quantiWed were more frequent in the DMSO group than in the control group (about 19% higher) and there was no diVerence among Mix10, Mix50 and control groups (Fig. 1B). Then, there was a decrease in the incidence of apoptosis in the Mix10 and Mix50 groups in comparison to the DMSO group. 4.2. Catalase and GST activities

4.3. Reduced GSH concentration Glutathione concentration in the DMSO and Mix50 groups were 14% higher and 25% lower than in the control group, respectively. The Mix10 and control groups had similar GSH concentrations. When compared to DMSO group, both Mix10 and Mix50 had statistically lower GSH contents (Fig. 1D). 4.4. Production of lipid hydroperoxides – lipid peroxidation The production of lipid hydroperoxides increased in about 22% in the DMSO group and 15% in the Mix10 group and these two groups had no diVerences between themselves (Fig. 1F). Likewise, Mix50 and control groups had similar lipid peroxidation levels, which were lower than that from DMSO group. 4.5. DNA damage The nuclei from all groups were classiWed according to their tail lengths (Table 2, see also Fig. 2). The fragmenta-

F. Filipak Neto et al. / Toxicology in Vitro 21 (2007) 706–715

Cell Viability

A

% of FITC-dUTP labelled cells

% of viable cells

a

a

a

90

b**

80 70 60

Control

DMSO

Apoptosis - TUNEL

B

100

Mix10

711

5

b* 4

a

a

a

Mix10

Mix50

3

2

1

Mix50

Control

DMSO

D nmol GSH-CDNB.min-1.mg prot -1

μmol H2O 2.min-1.mg prot -1

Catalase Activity 600

b***

500 400

b* a a

300 200

Control

DMSO

Mix10

Concentration of GSH

E

F

nmol GSH.mg prot-1

15

b** 12

a

a

9

c*** 6 3

Control

DMSO

Mix10

Glutathione S-Tranferase Activity 450

b*** b*

350

a

a

Control

DMSO

250

150

50

Mix50

nmol hydroperoxides.mg prot-1

C

Mix10

Mix50

Lipid Peroxidation 4

b*** 3

b***

a

a

2

1 0

Control

Mix50

DMSO

Mix10

Mix50

Fig. 1. Cell viability (A) [percentage of viable cells], apoptosis (B) [percentage of positive FITC–dUTP labeled cells], catalase activity (C) [mol of H2O2 degraded per min per mg of protein], glutathione S-transferase activity (D) [nmol of GSH–CDNB produced per min per mg of protein] activities, reduced GSH concentration (E) [nmol of GSH per mg of protein], lipid peroxidation (F) [nmol of lipid hydroperoxides per mg of protein] for Control, DMSO, Mix10 and Mix50 groups. Total number of replicas: catalase ( D 30), GST ( D 46), GSH ( D 42), lipid peroxidation ( D 60), cell viability ( D 7), apoptosis ( D 4 £ 40.000 events). DiVerent letters (i.e., a, b, c) mean statistical diVerences; *p < 0.05, ¤¤p < 0.01, ¤¤¤p < 0.001 (asterisks in comparison to control group).

Table 2 DNA damage determined through comet assay Groups

DNA damage classes 0

Control DMSO Mix10 Mix50

70.17 § 10.8 16.29 § 4.4 54.89 § 8.7 20.67 § 4.0

Score 1 21.00 § 6.6 48.49 § 5.0 28.21 § 4.9 48.17 § 5.7

2

3

6.33 § 3.1 26.2 § 4.3 10.86 § 3.1 17.33 § 3.6

2.50 § 1.3 9.02 § 2.4 6.04 § 1.3 13.83 § 2.3

41.17 § 16.3a 127.95 § 11.6b** 68.06 § 14.6a 124.33 § 4.6b*

The numbers represent the mean values of incidence of the particular DNA damage class (0, 1, 2 and 3), followed by the standard error of mean. The DNA damage class refers to not fragmented (0), slightly (1), moderately (2) and severely (3) fragmented DNA, respectively. Scores were calculated through the formula: Score D (damage class £ percentage of incidence). Statistical analysis were performed only among the scores (diVerent letters (i.e., a, b) mean statistical diVerences; *p < 0.05, ¤¤p < 0.01, asterisks in comparison to control group). Total number of replicas D six per group.

tion of DNA had higher scores in the DMSO and Mix50 groups, but not in Mix10 group when compared to Control group. When compared to DMSO group, Mix10 had lower and Mix50 had similar scores.

5. Discussion H. malabaricus and A. anguilla are abundant predator Wshes found in several countries (respectively, South America

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Fig. 2. Nuclei from hepatocytes stained with ethidium bromide exemplifying the classes of DNA fragmentation: (A) class 0 (no fragmentation), (B) class 1 (lower), (C) class 2 (moderate) and (D) class 3 (severe fragmentation of DNA). Bar D 8 m.

and North Hemisphere). High concentrations of HOCs, in special those included into the POPs, can be found on naturally occurring specimens from both species once these xenobiotics are highly bioaccumulated and biomagniWed in aquatic organisms. As an innovative experimental design, the HOCs previously extracted from A. anguilla were used to expose primary cultured hepatocytes from H. malabaricus. The focus of the present study was to investigate the eVects of the HOCs mixture on the hepatocyte cell, even though the donor of the cell and donor of the HOCs belongs to diVerent species of Wsh. Also, the contamination of natural biota by several HOCs is a global problem, in Brazil, France or almost any part of the globe. Literature mentions the occurrence of apoptotic cell death following individual chemical exposure to several HOCs. Since neither Mix10 nor Mix50 showed HOCsinduced apoptosis (Fig. 1B), one possible explanation may be the fact that hepatocytes were exposed to a mixture of chemicals in which some may act synergistically or additively, while others may act antagonistically. For instance, studies mentioned that dieldrin (one of the HOCs present in the mixture) aVects mitochondrial function irreversibly (Bergen, 1971) and activates caspases-3 (Kannan et al., 2000), whereas heptachlor (another HOC also present in the mixture) stimulates protein kinase C activity and induces expression of bcl-2 and inhibition of cytochrome c release (Okoumassoun et al., 2003). Also, negative response of heptachlor genotoxicity suggests carcinogenicity through epigenetic pathways (Okoumassoun et al., 2003); while dieldrin produces carcinogenic and reactive intermediates that cause DNA adduct formation and mutations, which develop into hepatic tumors (Ashwood-Smith, 1981; Bachowski et al., 1997). The decrease in viable cells and the absent indication of induction of apoptosis in Mix50 group suggest that cells may be dying by necrosis in this group (Fig. 1A).

Increase in the apoptotic cell death was one astonishing Wnding in the DMSO group, because according to several studies, DMSO at low concentrations (60.5%) seems to not cause cytotoxic eVects to cultured cells. At higher concentrations, DMSO can alter the permeability and stability of lysosomal membrane (Mackie et al., 1989) and lead to activation of caspase-9 and -3 genes (Liu et al., 2001), collapse of mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol (Liu et al., 2001) and increase in cytoplasmic Ca2+ concentration (Nygren et al., 1987). Hypothetically, all of these eVects could be causative of apoptosis. Compared to DMSO group, Mix10 and Mix50 had lower incidence of apoptotic cells. Thus, even though the HOCs can be toxic to cells (e.g., cause decrease in cell viability), the cellular response through increase of catalase and GST activities (maybe also through the increase of other enzymatic activities not measured in the present study) may allow cells to at least avoid the induction of apoptosis during the course of the experiments (i.e., up to 48 h of exposure to HOCs). The induction of Wsh hepatic enzymes such as catalase (Rodriguez-Ariza et al., 1993) and GST (Pesonen et al., 1999; Lindström-Seppä et al., 1996) by OCPs and some other HOCs was already reported for both, Weld and laboratory studies. Here, the activities of these antioxidant and phase II enzymes were evaluated for cultured H. malabaricus hepatocytes. Both enzymatic activities were increased in comparison with the control and DMSO groups (Fig. 1C and D). Physiologically, the antioxidative role of catalases is to decrease the risk of hydroxyl radical («OH) formation from hydrogen peroxide (H2O2), which is vital since hydrogen peroxide can easily cross biological membranes (Nordberg and Arnér, 2001; Cui et al., 2004) and consequently, compromises the integrity of macromolecules within the

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cell (Halliwell and Gutteridge, 1989; Cochrane, 1991; Buet et al., 2006). GSTs are involved with both, catalyzing the conjugation of GSH with a wide variety of organic molecules (e.g., some HOCs) and with the reduction of organic hydroperoxides (ROOH) (Arteel and Sies, 2001). Thus, the increased enzymatic activities detected for both enzymes (in the Mix10 and Mix50 groups) may be a mechanism to compensate the presence of “abnormal” concentrations of hydrogen peroxide (in the case of catalases) and ROOHs and HOCs (in the case of GSTs). Furthermore, a higher production of hydrogen peroxide and the increase in the GST activity after exposure to the mixture of HOCs may be responsible for the decrease in reduced GSH concentration (in the Mix10 group [compared with DMSO group] and in the Mix50 group [in comparison to both control and DMSO groups]), since GSH serves to detoxify compounds via conjugation reactions catalyzed by GSTs (Armstrong, 1997; Van Bladeren, 2000), and in the case of hydrogen peroxide, in the glutathione peroxidases catalyzed reactions. It is well known that by catalyzing thioether products formation with cysteine of GSH, most toxic threats can be reduced or neutralized (Van Bladeren et al., 1979) and that GSH is almost the sole non-enzymatic determinant of cellular redox environment (Schafer and Buettner, 2001). Thus, the reduction of reduced glutathione concentration represents a serious threat to the hepatocytes, because intracellular redox environment may become, sometimes inappropriate for cell survival, as indicated by the decrease in cell viability in the Mix50 group. In the case of DMSO group, because of DMSO is a potent hydroxyl radical scavenging agent (Dorfman and Adams, 1973; Qian et al., 2005), it may decrease the total concentration of hydrogen peroxide and some other prooxidant species by «OH scavenging and then, aids to maintain high concentration of reduced glutathione. Since DMSO group had higher levels of GSH than Mix10 (plus DMSO) and Mix50 (plus DMSO) groups (Fig. 1E), DMSO and HOCs have antagonistic eVects on GSH concentration, which may indicate an even higher decrease in GSH levels in the case of cells cultured in the absence of DMSO. The production of methyl radicals (Byvoet et al., 1995; Halliwell and Gutteridge, 1999) by interaction of DMSO and hydroxyl radical, and subsequent generation of methoxy radical («OCH3) and methylperoxy radical («OOCH3) under aerobic conditions (Britigan et al., 1990; Burkitt and Mason, 1991) or formaldehyde during NADPH-dependent electron transfer (Cohen and Cederbaum, 1980), may explain the increase of LPO in the DMSO group (Fig. 1F). Also, despite there were an increase in the catalase and GST activities in the Mix10 group, these additional enzymatic activities were unsatisfactory to avoid an increase in the peroxidation of lipids (resultant from the presence of the solvent DMSO), which did not occur in the Mix50 group. The Mix50 group had LPO similar to the control group, but lower than the solvent (DMSO) group. Once again, DMSO and HOCs had antagonistic eVects, but in this case in relation to LPO. The decrease in the peroxida-

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tion of hepatic lipids was already reported for the Wsh species Mugil sp. (grey mullet) collected from OCPs contaminated environmental (Rodriguez-Ariza et al., 1993). Despite GSH concentration was slightly higher, lipid peroxidation increased in the DMSO group. Since LPO are generally free radical-driven chain reactions in which one radical can induce the oxidation of a comparatively large number of substrate molecules (Abuja and Albertini, 2001), the structural modiWcation of complex lipid–protein assemblies could be associated with cellular malfunction (Kühn and Borchert, 2002). It indicates the harshness of peroxidative damage to the hepatocytes consequent from the presence of DMSO. As cell viability was not aVected in this solvent group, two hypothesis could be proposed: either cells can bear a slight increase in the peroxidation of lipids (DMSO group) but not decrease in GSH concentration (Mix50 group), or they are highly aVected by LPO and break up completely, thus not being accounted by Trypan blue exclusion method. For DMSO group, the production of methyl radicals and aldehydes by DMSO and/or lipid hydroperoxides may be associated with its genotoxicity. The direct eVect of the hydroxyl radical is conWned (Cui et al., 2004) to regions immediately in the vicinity of its formation (i.e., mainly near mitochondria), but some other radicals may be able to travel longer distances within the cell, also due to their less reactivity in comparison to hydroxyl radicals. Thus, DNA damage observed in the DMSO group may be a consequence of formation of DNA adducts by some radicals, alteration of the permeability and stability of lysosome membranes (Mackie et al., 1989) with leakage of some lysosomal DNAses. Also, it is recognized that reactive oxygen species (ROS) can react with several amino acids such as histidine, proline, arginine and lysine (Çakatay et al., 2001) and directly aVect the conformation and/or activities of all sulfhydryl-containing molecules by oxidation of their thiol moiety (Nordberg and Arnér, 2001). Oxidative modiWcation of proteins could contribute to secondary damage to other biomolecules, for instance, to DNA by inactivation of DNA repair enzymes or loss of Wdelity of DNA polymerases in replicating the DNA (Evans et al., 1999). Repair enzymes, like endonuclease III (Fu et al., 1992) and poly ADP–ribose polymerase-I (Hughes, 2002), have critical sulfhydryl groups that, if aVected, could reduce the recognition of DNA damage and consequently weaken DNA repair. It is likely that the DNA damage scored in the DMSO and Mix50 groups may not be only a consequence of direct damage of some ROS or free radicals, but also malfunction of DNA repair system. It was noted that impacts on LPO and DNA damage were diVerent in the Mix10 and Mix50 groups, i.e., LPO increased but DNA damage did not in the Mix10 group while the opposite happened in the Mix50 group. To explain this discrepancy, it can only be evoked the diVerential susceptibility of DNA and fatty acids to oxidative damage as corroborating factors, since a throughout investigation should be necessary. This way, higher amounts of

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ROS could aVect DNA repair system (Mix50 group) and it would be expected the maintenance of some DNA breaks unrepaired. However, lipid peroxides could be neutralized by antioxidant defense systems in a later step and phospholipids replaced in cell membranes. Conversely, smaller amounts of ROS (Mix10 group) could cause direct damage to DNA and membrane lipids, but not signiWcant damage to the DNA repair proteins. In this case, in a later step (i.e., after 48 h of exposure) of reestablishment of the balance within the cell, DNA breaks could be repaired, but some lipid hydroperoxides could keep unblocked since the increase in the antioxidant defense enzymes was not enough to remove completely this threat. The Wndings of the current study support that changes in the hepatocyte antioxidant system may be happening to rebalance the cellular redox status following exposure to HOCs. Important eVects induced by the mixture of HOCs were in the enzymatic defense systems catalase and GST, GSH concentration and cell viability. Other eVects (LPO, DNA damage and apoptosis) seem to be caused or strongly inXuenced by DMSO. Despite DMSO is frequently used to solubilize several lipophilic pollutants for application in culture, it seems not to be an appropriate solvent for assays employing H. malabaricus hepatocytes. The exposure of cells to a biological extracted mixture of HOCs may allow important interactions and reproduces as close as possible the “factual” situation, even thought it makes diYcult the attribution of a speciWc eVect to one causing agent in particular. Acknowledgements This research was supported in part by CNPq and CAPES (Brazilian Agencies for Science and Technology). The authors acknowledge Dr. Luis Cláudio Fernandes and Dra. Dorly de Freitas Buchi for scientiWc assistance and The Electron Microscopy Center of Federal University of Paraná for the technical support. References Abuja, P.M., Albertini, R., 2001. Methods for monitoring oxidative stress, lipid peroxidation and oxidation resistance of lipoproteins. Clinica Chimica Acta 306, 1–17. Aebi, H., 1984. Catalase in vitro. Methods in Enzymology 105, 121–126. Andersen, H.R., Vinggaard, A.M., Rasmussen, T.H., Gjermandsen, I.M., Bonefeld-Jorgensen, E.C., 2002. EVects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and Applied Pharmacology 179, 1–12. Armstrong, R.N., 1997. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chemical Research in Toxicology 10, 2–18. Arteel, G.E., Sies, H., 2001. The biochemistry of selenium and the glutathione system. Environmental Toxicology and Pharmacology 10, 153– 158. Ashwood-Smith, M.J., 1981. The genetic toxicology of aldrin and dieldrin. Mutation Research 86, 137–154. ATSDR, 2006. Agency for toxic substances and disease registry. http:// www.atsdr.cdc.gov/toxproWles/. Bachowski, S., Kolaja, K.L., Xu, Y., Ketcham, C.A., Stevenson, D.E., Walborg Jr., E.F., 1997. Role of oxidative stress in the mechanism of diel-

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