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Journal of Hazardous Materials 260 (2013) 593– 601

Contents lists available at SciVerse ScienceDirect

Journal of Hazardous Materials

jou rn al hom epage: www.elsev ier .com/ locate / jhazmat

nvironmental and human risk assessment of landfill leachate: Anntegrated approach with the use of cytotoxic and genotoxic stressndices in mussel and human cells

irini Toufexia, Vasiliki Tsarpali a, Ioanna Efthimioub, Maria-Sophia Vidalib,imitris Vlastosb, Stefanos Dailianisa,∗

Section of Animal Biology, Department of Biology, School of Natural Sciences, University of Patras, GR 26500 Patras, GreeceDepartment of Environmental and Natural Resources Management, University of Patras, 2 Seferi Str., GR 30100 Agrinio, Greece

i g h l i g h t s

Landfill leachate poses a threat for aquatic biota and humans.Leachate induces cytotoxic and oxidative effects on mussel hemocytes.Increased levels of DNA damage were observed both in vivo and in vitro in hemocytes.Leachate low doses enhance MN formation in human lymphocyte cultures.Potential leachate aneugenic activity was detected in human lymphocytes.

a r t i c l e i n f o

rticle history:eceived 19 March 2013eceived in revised form 17 May 2013ccepted 28 May 2013vailable online 5 June 2013

eywords:

a b s t r a c t

The present study investigates leachate hazardous effects on marine biota and human cells, with the useof a battery of assays, both under in vivo and in vitro conditions. According to the results, mussels exposedfor 4 days to 0.01 and 0.1% (v/v) of leachate showed increased levels of DNA damage and micronuclei(MN) frequencies in their hemocytes. Similarly, enhanced levels of DNA damage were also observed inhemocytes treated in vitro with relevant concentrations of leachate, followed by a significant enhance-ment of both superoxide anions (•O2

−) and lipid peroxidation products (malondialdehyde/MDA). On the

uman lymphocytesenotoxicityeachateussel hemocytesicronuclei

other hand, human lymphocyte cultures treated with such a low concentrations of leachate (0.1, 0.2 and1%, v/v), showed increased frequencies of MN formation and large MN size ratio, as well as decreased cellproliferation, as indicated by the use of the cytokinesis block micronucleus (CBMN) assay and Cytoki-nesis Block Proliferation Index (CBPI) respectively. These findings showed the clear-cut genotoxic andcytotoxic effects of leachate on both cellular types, as well as its potential aneugenic activity in humanlymphocytes.

. Introduction

During the last decades, ecotoxicological and toxicologicalethodologies have become a priority worldwide, in order to

ssess the human and environmental risk of pollutants andixtures of contaminants [1]. Among them, landfill leachate, com-only generated by the precipitation and penetration of water

nto the mass of residues in disposal landfill areas [2], is consid-

red an environmental matter of concern [3]. This type of liquidffluent consists of a huge number of pollutants, such as dis-olved organic matter, inorganic salts, heavy metals and xenobiotic

∗ Corresponding author. Tel.: +32 610 969213; fax: +32 610 969213.E-mail address: sdailianis@upatras.gr (S. Dailianis).

304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jhazmat.2013.05.054

© 2013 Elsevier B.V. All rights reserved.

organic compounds [4] which could be toxic and carcinogenic [5]and able to induce potential risk for biota and humans.

The entrance of leachate into ground- and surface waters couldrender them unusable for drinking and other domestic purposes [6].On the other hand, bearing in mind that leachate chemical com-pounds can easily assimilate by aquatic organisms, pass throughthe food chain and bioaccumulate over long-term exposure [7], thehuman population is increasingly under threat, after consumptionof contaminated water and/or food [8,9].

Landfill leachate ability to cause deleterious and genotoxiceffects upon organisms of various trophic levels, including crus-

taceans [10–12], fish species [13,14], bacteria [15], plants [16–19],mice [20] and human [6,21–23] is well known, but only recentlyreported its ability to enhance oxidative and genotoxic effectson marine bivalve molluscs, such as Mytilus galloprovincialis [24].

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94 E. Toufexi et al. / Journal of Haza

hose bivalves accumulate large amount of contaminants in theirissues, thus allowing their use as biological models for assessingater and sediment quality [25]. Since mussels are widely culti-

ated and consumed by humans, their use as target organisms foronitoring biological responses, commonly called stress indices,

iscloses the environmental impact of contaminants, such aseachate, not usually and/or hardly analyzed in the real environ-

ent [24,26], as well as their potential human risk [27].Regarding that leachate environmental and human risk depends

n its physico-chemical characteristics, the estimation of stress-ndices in organisms from different trophic levels could assesshe adverse effects of leachate chemical compounds on humannd ecosystem health. According to the latter, the present studynvestigates leachate composition and its toxic effects on musselemocytes and human lymphocytes, both involved in the biolog-

cal/immune response against xenobiotics. In specific, a batteryf cytotoxic, oxidative and genotoxic stress indices were deter-ined for a first time in mussel hemocytes, after in vivo and in vitro

xposure to different concentrations of leachate, since this cellu-ar type is widely used for investigating cellular responses, thusndicating the immunosurveillance and the concomitant health sta-us of the organism [28,29]. On the other hand, leachate-mediatedytotoxic and genotoxic effects were investigated in human lym-hocytes by the use of the cytokinesis block micronucleus (CBMN)ssay, widely performed for assessing the risk of different types ofhemical substances and their ability to cause genetic damage andarcinogenetic processes in humans [30,31].

. Experimental

.1. Chemicals and reagent

All reagents and solvents used were of the highest analyticalrade and purity (see SM 2.1).

.2. Leachate collection and handling

Both in vivo and in vitro studies were conducted with the use ofell-characterized leachate [24] (see also SM Table 1), collected

very 10 days within November 2011 from the landfill (active)ite of Aigeira (Eastern Aigialeia, Peloponissos, Greece). In specific,qual volumes of all samples (at least 5 L) were pooled, filteredhrough sterilized filter membranes (diameter 0.2 �m) and frozent −28 ◦C in the dark until used.

.3. Mussel collection and handling

.3.1. Ethics statementThe Mediterranean mussel M. galloprovincialis (Lmk. 1819) is

ommon and not endangered invertebrate species. Since no per-its were required for their use in both in vitro and in vivo

tudies, the experimental procedure (in terms of acclimationeriod, mussel handling and exposure procedure) was appropri-tely carried out in order to minimize animal suffering. In brief,ussels (4–5 cm long) were collected from a mussel-farm located

o the north side of Korinthiakos Gulf (Gulf of Kontinova, Galaxidi,reece) in March 2012 (no interference of massive spawning isxpected), transferred and acclimated under laboratory conditionsor 7 days in static tanks, containing aerated (dissolved oxygen–8 mg L−1 at 15 ◦C and 35‰ salinity), recirculated UV-sterilized

nd filtered artificial sea water (ASW). Mussels maintained withouteeding during the acclimation period and then fed daily (approxi-

ately 30 mg of dry-microencapsules, Myspat, Inve AquacultureNV,elgium/mussel).

Materials 260 (2013) 593– 601

2.4. In vivo exposure of mussels to sublethal concentrations ofleachate

After the acclimation period, four groups of mussels (30 mus-sels/tank) were placed in static glass-tanks (30 L) and exposed for 4days to sublethal concentrations (0.01 and 0.1%, v/v) of leachate, asrecently mentioned [24]. Seawater was changed every day and newquantities of leachate and food were added in each tank. The afore-mentioned experimental procedure was repeated twice (N = 2).

2.4.1. MN assayAfter the end of the exposure period, live mussels were removed

and hemolymph was withdrawn from the posterior adductor mus-cle of 10 individuals of each group (control and leachate-exposedmussels), using a sterile 1 mL syringe with an 18 G1/2 in. needle,containing equal volume of Alseve buffer (ALS buffer; 20.8 g L−1

glucose, 8 g L−1 sodium citrate, 3.36 g L−1 EDTA and 22.5 g L−1 NaCl,pH 7 and 1000 mOsm L−1). Thereafter, 40 �L of hemolymph sus-pension from each individual were spread on slides, in duplicate,transferred to a lightproof humidity chamber in order to attach,stained with Giemsa and finally scanned under an optical micro-scope (Zeiss microscope, 100× magnification) for the presence ofmicronuclei (MN) formation, according to the procedure and crite-ria proposed by UNEP/RAMOGE [25].

The hemolymph collected from the rest of 20 individuals pergroup of mussels was pooled, further divided in 3 subgroups (whichmean 3 replicates/group of mussels and 6 replicates for the wholeexperimental design) and small portions were finally used for con-ducting Comet assay method (estimation of DNA damage).

2.5. In vitro treatment of mussel hemocytes with leachate

Hemolymph of laboratory acclimated mussels was with-drawn from the posterior adductor muscle of 10 individuals,filtered through sterile gauze and pooled in Falcon tubes at 4 ◦C.Hemolymph serum, used for hemocytes suspension, was obtainedby centrifugation of whole hemolymph at 150 × g for 10 min andsterilization through a 0.22 �m-pore filter. Hemocyte suspen-sions/monolayers (at least 106 cells mL−1) were incubated at 18 ◦Cwith different concentrations of leachate (0, 0.01, 0.1, 1, 10, 20, 40and 80%, v/v, from stock solutions diluted in hemolymph serum) for1 h. All experiments were performed in quadruplicate. Cell viability,tested with Eosin exclusion test (see SM 2.5) after cell dissociationand re-suspension in sterilized and filtered hemolymph, was about95%.

2.5.1. Lysosomal membrane stability (neutral red retention assay)Lysosomal membrane stability was evaluated with the use of

neutral red retention time (NRRT) assay [32], based on the proce-dure and criteria proposed by UNEP/RAMOGE [25]. In brief, 40 �Lof hemocytes suspension were spread on slides, in triplicate, trans-ferred to a lightproof humidity chamber and allowed to attach. Allslides containing cell monolayer were incubated with 40 �L of thecationic dye neutral red probe (40 �g mL−1) for 15 min in dark, andthereafter were examined systematically under a light microscopeevery 15 min (at least 200 granular hemocytes examined in eachslide). The time period between the NR probe application and theappearance of the first evidence of dye loss from the lysosomes tothe cytosol or of other lysosomal abnormalities (Fig. 1) in at least50% of the examined cells represented the NRR time.

2.5.2. Superoxide anion (•O2−) detection and lipid peroxidation

(in terms of MDA equivalents) content in leachate-treatedhemocytes

Superoxide anions (•O2−) were detected intracellularly by a

modification of a previously described method [33], with the use of

E. Toufexi et al. / Journal of Hazardous

Fig. 1. Representative light microscopic view (magnification 1000×) of hemocytesafter the neutral red staining, displaying enlarged lysosomes due to the penetrationolr

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f neutral red, as well as dye loss from the lysosomes to the cytosol. N: nucleus; L:ysosomes. (For interpretation of the references to color in this figure legend, theeader is referred to the web version of the article.)

itroblue tetrazolium (NBT). Briefly, hemocytes (suspended in ster-lized haemolymph) were exposed to sublethal concentrations ofeachate (0.01, 0.1, 1 and 10%, v/v) for 1 h. Thereafter, samples wereentrifuged at 150 × g for 10 min, at 4 ◦C, and the cell pellet wasmmediately suspended in 1 mL ALS buffer, containing 1 mg mL−1

BT and finally incubated for 2 h in the dark. After the end of thencubation period, cell suspensions were centrifuged (150 × g for0 min at 4 ◦C) and washed with 300 �L TBS (0.05 M Tris–chlorideuffer, pH 7.6, containing 2% NaCl) in duplicate, in order to removextracellular NBT. Hemocytes were then fixed with 300 �L of 70%ethanol for 10 min, centrifuged (150 × g, 10 min, at 4 ◦C) and the

ells were air-dried for 5 min in room temperature. Afterwards, mL of extraction fluid (2 M KOH in DMSO) was added and afterolubilization for 30 min, the samples were measured spectropho-ometrically at 620 nm.

The extent of lipid peroxidation (LPO) was measured as malon-ialdehyde (MDA) equivalents, a reliable indicator of oxidativeamage of cellular membranes [34], according to method describedy Chatziargyriou and Dailianis [35]. Briefly, leachate-treatedemocytes were centrifuged at 150 × g (10 min, at 4 ◦C) and theupernatant was removed carefully. Packed cells were mixed with

mL of trichloroacetic acid (TCA)–thiobarbituric acid (TBA)–HCl15%, w/v TCA, 0.375%, w/v TBA in HCl 0.25 N). After vortexingor 5 s, butylated hydroxytoluene (BHT) at final concentration of.02% was finally added, in order to prevent further peroxida-ion of lipids. Finally, samples were incubated at 90–100 ◦C for5 min and cooled at room temperature. When appropriate, theamples were centrifuged at 10,000 × g for 10 min and were mea-ured spectrophotometrically at 535 nm. A molecular co-efficient

5 −1 −1

ε = 1.5 × 10 M cm ) [36] was used for the determination ofDA concentration.In each case, the results are means ± SD from 6 different mea-

urements and expressed as the value of optical density obtained at

Materials 260 (2013) 593– 601 595

620 nm per milligram of protein and nmol MDA/mg protein respec-tively. In any case, protein content was measured with the use ofBradford reagent and bovine serum albumin (BSA) as a standard.

2.6. Estimation of DNA damage in mussel hemocytes (in vitro andin vivo studies) with the use of alkaline single-cell gelelectrophoresis (Comet assay) method

Hemocytes of leachate-exposed mussels (in vivo exposure to0.01 and 0.1%, v/v of leachate), as well as leachate-treated hemo-cytes (in vitro exposure to 0.01, 0.1, 0.2, 0.5 and 1%, v/v) wereresuspended in 180 �L of 1% low-melting-point agarose (LMA) inKenny’s salt solution (0.4 M NaCl, 9 mM KCl, 0.7 mM K2HPO4, 2 mMNaHCO3), layered on glass slides coated with 200 �L of 2% normal-melting-point-agarose (NMA) in TAE solution (40 mM Tris–acetate,1 mM EDTA), and covered with a coverslip. The aforementionedprocess was repeated 4 times and thereafter the alkaline single-cell gel electrophoresis (Comet assay) method was performed [37](see also SM 2.6), in order to investigate DNA damage. In all cases,cell viability measured with the use of Eosin Y exclusion test, wasalmost 80%, which is considered appropriate for conducting Cometanalysis [38].

All slides were coded and randomly scanned for the presence ofcomets, using an Axio Carl Zeiss fluorescent microscope (Carl ZeissMicro-imaging, Germany, 100× magnification), equipped with Isisfluorescence imaging system analysis (Metasystems, Germany).Comet scoring was conducted with Tritek Comet scoreTM soft-ware, according to criteria established by Ritter and Knebel [39],in order to exclude abnormal comets from comet counting andscoring. Comets from each treatment (Fig. 2) were pooled togetherand analyzed for the percentage of DNA in tail (% DNA in tail) andolive tail moment (OTM), which are considered as the most reliableparameter for measuring DNA damage [40–42]. Each measurementindicates a single mean value obtained by the analysis of 4 slides forthe same cell suspension, in each case. Background levels of DNAdamage in control cells showed low variability, thus ranging withinsimilar levels with those measured in both mussel hemocytes andother cellular types previously reported [40–45].

2.7. Evaluation of leachate-induced genotoxic and cytotoxiceffects on human lymphocyte cultures (CBMN assay and CBPIindex)

The CBMN assay was performed according to standard proce-dures [30,46,47], with minor modifications. Briefly, human bloodsamples were obtained from two non-smokers, healthy individ-uals (20 and 25 years old) not undergoing any drug treatment, viralinfection or X-ray exposure for over a year. Whole blood (0.5 mL)was added to 6.5 mL Ham’s F-10 medium, 1.5 mL fetal bovine serumand 0.3 mL phytohaemagglutinin to stimulate cell division. Twoindependent cultures were set up for each donor. The leachate sam-ples were added to final concentrations of 0.02, 0.1, 0.2 and 1% inculture volume (8.8 mL) 24 h after culture initiation. After 44 h ofincubation, cytochalasin-B (Cyt-B, final concentration of 6 �g mL−1)was added to the cultures in order to block cytokinesis of dividingcells.

Cultures were incubated at 37 ◦C in a humidified atmosphereof 5% CO2 for 72 h and then cells were harvested and collected bycentrifugation. A mild hypotonic treatment with 3:1 solution ofHam’s medium and milli-q H2O was left for 3 min at room temper-ature which was followed by 10 min fixation (for at least 3 times)with a fresh 5:1 solution of methanol/acetic acid. Cells were stained

for 10 min with 7% Giemsa, as recently mentioned [48]. At least4000 binucleated (BN) cells with preserved cytoplasm were scoredin each case, in order to calculate the MN frequency, according tostandard criteria [49,50].

596 E. Toufexi et al. / Journal of Hazardous Materials 260 (2013) 593– 601

Fig. 2. Comet assay method. (A) Control hemocytes showing a comet head with no DNA migrating into the tail region (nucleoid core); (B) Comet heads with DNA fragmentso

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Cytokinesis Block Proliferation Index (CBPI) was evaluated byounting at least 2000 cells for each experimental point (500 cellser culture of each donor), as previously mentioned [51] (see alsoM 2.7). The MN size ratio, also calculated for indicating potentialeachate-induced clastogenic or aneugenic activity, was expresseds the ratio of MN diameter to the cell nucleus diameter (small MNize ratio: ≤1/10 and large MN size ratio: ≈1/3 of nuclear diameter)52,53].

.8. Statistical analysis

The statistical analysis of the obtained data was conducted withhe use of the Minitab statistical software (Minitab Inc., PA, USA)nd the SPSS Inc.17. In specific, after checking for homogeneity ofariance (Levene’s test of equality of error variances), the significantifferences among stress indices were assessed by post hoc multipleomparison test (Bonferroni test, ANOVA) in all cases, except fromata for DNA damage (% DNA in tail and OTM), which were testedith Kruskall–Wallis non-parametric test. In case of human lym-hocyte cultures, MN data analysis was conducted using the G-testor independence on 2 × 2 tables, while CBPI data set was analyzedsing the chi-square (�2) test. Significant levels were establisheds p < 0.05 in all cases.

. Results

.1. Neutral red retention (NRR) time values in leachate-treatedussel hemocytes

Since the NRRT assay is applied for monitoring alterations in theysosomal membrane permeability, thus providing a quantitative

stimation of cell viability, the results of the present study showedlmost a dose-dependent decrease of NRRT values in leachate-reated hemocytes (Table 1). Specifically, hemocytes treated with.01% (v/v) of leachate for 1 h showed negligible levels of NRRT

reduction, while treatment with higher concentrations (0.1, 1 and10%, v/v) revealed lower NRRT values than those in control cells.Since NRRT values obtained in hemocytes treated with leachate atconcentrations ranged within 20 and 80% (v/v) showed almost a50% reduction, the rest of the parameters tested were investigatedin hemocytes treated with leachate at concentrations of 0.01, 0.1,1 and 10% (v/v), thus excluding the interference of cell death withthe obtained results.

3.2. Oxidative effects of leachate on mussel hemocytes

Leachate ability to enhance oxidative effects on mussel hemo-cytes was investigated via the determination of superoxide anions(•O2

−) and lipid peroxidation products (in terms of MDA equiv-alents). Specifically, there was a significant increase of •O2

− inhemocytes treated with 0.1, 1 and 10% (v/v) of leachate (Fig. 3A),followed by significantly increased MDA levels, compared to thoseoccurred in control cells in any case (Fig. 3B).

3.3. Genotoxic effects on hemocytes of leachate-exposed mussels(in vivo study) and leachate-treated hemocytes (in vitro study)

According to the results, a significant dose-dependent DNAimpairment was observed in hemocytes of mussels exposed bothin vivo and in vitro to relevant concentrations of leachate. In spe-cific, hemocytes of mussels exposed for 4 days to 0.01 and 0.1%(v/v) of leachate showed increased levels of % DNA in tail andOTM, as well as increased MN frequencies (almost 3.4 and 6.6-fold

increase, respectively), compared to those occurred in leachate-free mussels (Table 2). Similarly, in vitro treated hemocytes, showedalmost a dose-dependent increase DNA damage, compared to thoseoccurred in control cells in all cases (Fig. 4A and B).

E. Toufexi et al. / Journal of Hazardous Materials 260 (2013) 593– 601 597

Table 1Estimation of neutral red retention (NRR) time values in leachate-treated hemocytes. Values (expressed as min) are the mean NRR time value ± SD from 6 independentmeasurements in each case. Values in the parenthesis correspond to the percentage (%) of NRR value of treated cells in respect to control value. Asterisks indicate significantdifference from control. Values that share the same letter are significant different from each other.

Leachate (%, v/v)

0 0.01 0.1 1 10 20 40 80

NRRT (min)130 ± 11.6 120 ± 15.5abcd 100 ± 11.3*efgh 95 ± 15.5*ik 77 ± 8.6*aelm 70 ± 7.3*bfn 55 ± 7.7*cgil 20 ± 7.8*dhkmn

(100) (90.7) (76.1) (71.8) (58.6) (53.4) (41.6) (15.1)

Table 2Genotoxic effects of leachate on mussel hemocytes. In case of DNA damage, the results are mean ± SD from 4 independent measurements. In case of MN, the results are themean frequency value ± SD obtained by the analysis of each slide (N = 2 × 10) for each group (control and leachate-exposed mussels). Asterisks in each raw indicate significantdifference from the respective control. Values in each raw that share the same letter are statistically different from each other.

Leachate (%, v/v)

0 0.01 0.1

DNA damage%DNA in tail 4.74 ± 1.1 11.43 ± 0.7*a 19.95 ± 0.17*a

OTM 0.88 ± 0.16 2.19 ± 0.18*a 5.65 ± 0.33*a

MN frequencyArbitrary units/1000 cells 2.1 ± 0.6 7.2 ± 1.05*a 13.8 ± 3.13*a

FRa 1 3.4* 6.6*

a Frequency ratio = values in hemocytes of leachate-exposed mussels per values in hemocytes of control mussels.

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Fig. 3. Determination of (A) superoxide anions and (B) MDA in leachate-treatedmussel hemocytes. Results (expressed as OD620nm mg−1 protein and nmol MDA mg−1

protein) are mean ± SD from 6 independent measurements. Asterisks indicate signif-icant difference from control in each case. Values that share the same letter indicatesignificant difference from each other.

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Fig. 4. DNA damage, expressed as (A) %DNA in tail and (B) olive tail moment/OTM inleachate-treated mussel hemocytes. The results are mean ± SD from 4 independentmeasurements. Asterisks indicate significant difference from the control. Values thatshare the same letter are statistically different from each other.

598 E. Toufexi et al. / Journal of Hazardous Materials 260 (2013) 593– 601

0

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2

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Fig. 5. Genotoxic effects of leachate on human lymphocytes treated with differentconcentrations of leachate (0.02, 0.1, 0.2 and 1%, v/v). (A) Micronucleous frequencyand (B) frequency ratio (FR = values obtained in treated cells per values obtainedic

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n control cells) in binucleated cells. Asterisks indicate significant difference fromontrol in each case.

.4. Cytotoxic and genotoxic effects of leachate on humanymphocyte cultures

Leachate-treated human lymphocyte cultures showed signif-cantly increased levels of genotoxicity. In specific, lymphocytesreated with 0.1, 0.2 and 1% (v/v) of leachate showed increased MNrequencies and frequency ratios (Fig. 5A and B). Regarding the sizeatio of MN (‰) induced by different doses of leachate (Table 3),here was a significant increase in large size MN frequencies in all

ested doses, except for the treatment of 0.02% (v/v). On the otherand, CBPI values measured in leachate-treated lymphocytes (at

able 3N frequencies (‰) per size in human lymphocytes treated with different con-

entrations of leachate (0.02, 0.1, 0.2 and 1%, v/v). Results (MN per 1000 cells) areean ± SD from 4 different experiments. In each experiment 1000 cells were scored.sterisks indicate significant difference from control. Values in each column thathare the same letter are significant different from each other.

Leachate (%, v/v) BN cells scored MN size ratio (‰)

Small Large

0 4000 1.50 ± 0.71 1.25 ± 0.500.02 4000 1.60 ± 0.89 2.00 ± 1.410.1 4000 2.67 ± 1.15 2.00 ± 0.00a

0.2 4000 2.40 ± 0.89 2.00 ± 1.00b

1 4000 1.50 ± 0.71 4.00 ± 1.00*ab

N: binucleated cells; MN: micronuclei.

Fig. 6. Cytokinesis Block Proliferation Index (CBPI) in human lymphocytes treatedwith different concentrations of leachate (0.02, 0.1, 0.2 and 1%, v/v). Asterisks indi-cate significant difference from control in each case.

least in case of 0.1, 0.2 and 1%, v/v) were significantly lower thanthose measured in control cells (Fig. 6).

4. Discussion

Bearing in mind the huge number of contaminants being presentinto landfill leachates, most of them not yet identified, as well asthe lack of knowledge of their risk to human and environmentalreceptors, a battery of both in vivo and in vitro assays was per-formed in order to estimate the cytotoxic and genotoxic effects ofleachate. In specific, hemocytes of bivalve molluscs, possessing acritical thesis in aquatic environments, not only via their involve-ment within trophic chain but also as a useful sensitive markerfor the monitoring of human-derived effects on the aquatic healthstatus, and human lymphocyte cultures, widely used for assessinghuman risk of genotoxic substances, were shown almost relevantbiological responses against leachate. In addition, the assessmentof leachate potential human risk clearly showed its genotoxic andcytotoxic effects.

4.1. Leachate-induced cytotoxic effects on mussel hemocytes

Hemocytes of mussels have become a useful biological modelin order to investigate cellular responses under controlled condi-tions [54,55], since due to the fact that their viability and functionintegrity could affect immunosurveillance and the concomitanthealth status of the organism [24,28,29,37]. In fact, since their lyso-somes’ membrane impairment constitutes a very useful index ofcellular damage thus linking with many aspects of pathology andtoxicity in bivalve mollusks [56–58], the results of the present studyshowed almost a dose-dependent lysosomal membrane impair-ment in hemocytes exposed to different concentrations (0.01, 0.1,1 and 10%, v/v) of leachate for 1 h. These findings could lead to thesuggestion that leachate could promote cell death even at such lowconcentrations as those currently used, thus confirming recentlypublished studies, concerning leachate ability to impair lysosomalmembranes in leachate-exposed mussels [24].

Leachate ability to promote cell death was seemed to be relatedwith its mediated oxidative stress effects, as clearly indicated bythe increased levels of both •O2

− and lipid peroxides (such asMDA) measured in leachate-treated hemocytes. In fact, high lev-

els of O2

− could lead to the production of oxidative stress-relatedfree radicals, such as H2O2, OH−, OH• and HNO2

− and the con-comitant enhancement of lipid peroxidation products, like MDA,thus revealing the presence of pro-oxidant substances, such as

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henolic compounds, into the leachate complex mixture. Indeed,igh concentrations of phenolic compounds could promoteytotoxic effects, such as lysosomal membrane destabilization,robably via the formation of hydroxyl radicals and highly reactiveetabolites, commonly occurred during their degradation process

59]. On the other hand, the presence of metal ions, ammonia andther organic contaminants, could also mediate the enhancementf oxidative and cytotoxic effects [9,56,60,61], but more studies areeeded in order to clarify their additive, synergistic and antagonis-ic effects.

.2. Leachate-induced genotoxic effects on mussel hemocytes anduman lymphocytes

Although a lot of studies have been focused on leachate geno-oxic potential (for further details see Baderna et al. [9]), onlyecently Tsarpali and Dailianis [24] showed the enhancement ofenotoxic effects on tissues of mussels. In accordance with the lat-er, the present study showed for a first time leachate ability toisturb DNA integrity, as indicated by the increased levels of DNAamage (%DNA in tail and OTM) in hemocytes of mussels, exposedoth in vivo and in vitro to relevant low leachate concentrations.

n addition, the enhanced MN formation observed in hemocytesf leachate-exposed mussels, in combination with the increasedell proliferation inhibition and MN frequencies observed in humanymphocyte cultures, clearly revealed leachate genotoxic and cyto-oxic potency at concentrations even lower than those previously

entioned [6,9,21]. Since the MN test represents a sensitive indica-or of the presence of both organic and inorganic mutagens and/orlastogens into the environmental media [62,63], thus linking withhe induction of carcinogenetic processes in humans [30,31,64], thencreased MN frequencies observed in both cellular types, could be

first evidence of the presence of clastogenic and/or mutagenicubstances into the leachate. Leachate dose-dependent genotoxicariations occurred in the literature could be due to the fact thathe leachate chemical characterization routinely followed, doesot allow us to infer about the presence as well as the amountf genotoxic compounds, commonly formatted through biologicalnd chemical transformations in leachate. Moreover, climatic con-itions, the degradation stage of wastes and the type of wastes and

andfill technology [2,65,66] could alter leachate toxic potency [12],hus leading to variations among studies performed worldwide.

Although metal ions and ammonia, as well as the presencef dioxins, PCBs and PAHs (not measured in the present study)ould impair DNA integrity [9,14], one possible mechanism thatould explain leachate-induced genotoxic effects on both cellu-ar types is related with its ability to enhance oxidative stress

ithin cells. In specific, the impairment of DNA integrity coulde a result of free radical formation occurred by either autooxi-ation or enzyme-catalyzed oxidation of organic compounds, suchs phenols, chlorinated and nonchlorinated hydrocarbons. In fact,henolic compounds, having similar properties to humic sub-tances, can chelate many metal ions [67], which in turn couldediate their autooxidation, the generation of semiquinone and

eactive oxygen species, such as NO, O2− and •OH as recently men-

ioned [68]. These free radicals are able to react with membraneipids, leading to lipid peroxidation [69] and affect the struc-ure proteins and nucleic acids, thus leading to base substitution,NA breakage [24,70] and the induction of mutation [71]. On thether hand, leachate absorption into the cells could lead to pHhanges within and outside cells, which might affect the activitiesf enzymes and change the structures of DNA [72].

Although the latter could explain the genotoxic effects ofeachate observed in both cellular types, the nature of contaminantseing responsible for the induction of aneugenic and clastogenicffects remains still unclear. However, regarding MN size ratio

Materials 260 (2013) 593– 601 599

observed in leachate-treated human lymphocytes, it was revealedfor a first time a significant increase of large MN in lymphocytestreated with 0.1, 0.2 and 1% (v/v) of leachate. These findings areof great interest, since the MN size ratio estimated by the use ofCBMN assay is considered an alerting index (as effective as theFluorescence In Situ Hybridization/FISH analysis) for the discrim-ination of clastogenic and aneugenic effects [52,53]. According tothe latter, the large MN observed in leachate-treated lymphocytes,might contain whole chromosomes, thus revealing a first evidenceof leachate aneugenic potency, while the presence of small MN ismore likely to contain acentric chromosome fragments indicatingits clastogenic effect [48,73,74]. Although Vlastos et al. [48] andSkoutelis et al. [73] reported that the presence of 2-chloropyridineand 2-hydroxypyridine in wastes could exhibit aneugenic effectson cultured human lymphocytes, chemical substances that could beresponsible for leachate-induced clastogenic and aneugenic behav-ior remains still unclear and need further investigation.

5. Conclusion

The present study showed the clear-cut genotoxic and cyto-toxic effect occurred in either hemocytes of mussels or humanlymphocyte cultures, after in vivo and/or in vitro exposure to rel-evant concentrations of leachate. DNA damage and MN frequencyobserved in both cellular types, showed leachate ability to enhancegenotoxic effects, while there was a first evidence of the poten-tial aneugenic effects of leachate on human lymphocytes. Takinginto account that the examined concentrations were very low,leachate should be handled with great care, in order to minimizeits environmental and human risk. Moreover, studies on the addi-tive, synergistic and/or antagonistic effect of leachate contaminantsshould be applied in parallel with various methods performed forthe implementation of landfill site remediation, the installationof practicable leachate treatment processes and the concomitantimpact of leachate.

Acknowledgements

This study was supported by the University of Patras-GreeceResearch Project “KARATHEODORIS 2011–2014. Code: D179”. Theauthors express their thanks to Dr. Demopoulos N.A. and Dr.Stephanou, G. (Department of Genetics, Cell and DevelopmentalBiology, Department of Biology, University of Patras) for kindly pro-viding the laboratory equipment for conducting the Comet assayexperiments.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2013.05.054.

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