RESEARCH ARTICLE

Mammalian cell-line based toxicological evaluationof paper mill black liquor treated in a soil microcosmby indigenous alkalo-tolerant Bacillus sp

Monika Mishra & Mihir Tanay Das &Indu Shekhar Thakur

Received: 27 May 2013 /Accepted: 11 October 2013 /Published online: 30 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Organic pollutants present in the soil of a microcosmcontaining pulp and paper mill black liquor were extracted withhexane/acetone (1:1 v/v) to study the biodegradation anddetoxification potential of a Bacillus sp. gas chromatography-mass spectroscopic (GC-MS) analysis performed afterbiodegradation showed formation of simpler compounds likep-hydroxyhydrocinnamic acid (retention time [RT] 19.3 min),homovanillic acid methyl ester (RT 21.6 min) and 3,5-dimethoxy-p -coumaric alcohol (RT 24.7 min). Themethyltetrazolium (MTT) assay for cytotoxicity, 7-ethoxyresorufin-O-deethylase (EROD) assay for dioxin-likebehavior and alkaline comet assay for genotoxicity were carriedout in the human hepatocarcinoma cell line HuH-7 before andafter bacterial treatment. Bioremediation for 15 days reducedtoxicity, as shown by a 139-fold increase in black liquor’s LC50

value, a 343-fold reduction in benzo(a)pyrene equivalent valueand a 5-fold reduction in olive tail moment. The EROD assaypositively correlated with both the MTT and comet assays inpost biodegradation toxicity evaluation.

Keywords Bacillus . Biodegradation . Pulp and paper millblack liquor . ROS . Comet assay . EROD assay

AbbreviationsPOPs Persistent organic pollutantsPPM Pulp and paper millAhR Aromatic hydrocarbon receptorAOX Adsorbable organic halidesPAH Polycyclic aromatic hydrocarbonROS Reactive oxygen speciesRT Retention timeGC-MS Gas chromatography-mass spectroscopyEROD 7-Ethoxyresorufin-O -deethylaseDH DihydroethidiumDCFH-DA 2′,7′-Dichlorodihydrofluorescin diacetate

Introduction

Microbial degradation of persistent organic pollutants (POPs)has the potential to be a useful tool for removing the pollutant ina cost effective manner. However, the microbes recruited forbioremediation may produce intermediate metabolites that aremore toxic than the parent compound (Van deWiele et al. 2005).The evaluation for toxicity of intermediary compounds ofpollutant is thus required. In vitromodels and biochemical assaysboth are well-proven tools for rapid and accurate evaluation oftoxicity at the acute, chronic and sub chronic levels.

Pulp and paper industry is one amongst the 11 mostpolluting industries in India and utilizes natural resources suchas lignocellulose, inorganic and organic materials, and largevolume of water in pulping and bleaching stages of papermanufacturing. In India, due to lack of proper implementationof environmental laws, the final discharges from the pulp andpaper mills (PPMs) contaminate the surrounding soil andsediment. In the present case, the main effluent channel ofthe mill discharges highly coloured effluent which movesthrough the villages, agricultural lands, nearby forests and

Responsible editor: Robert Duran

Electronic supplementary material The online version of this article(doi:10.1007/s11356-013-2241-5) contains supplementary material,which is available to authorized users.

M. Mishra (*) :M. T. Das : I. S. ThakurSchool of Environmental Sciences, Jawaharlal Nehru University,New Delhi 110 067, Indiae-mail: momis_biotech@yahoo.co.in

I. S. Thakure-mail: isthakur@hotmail.com

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ultimately joins the river Gola, 15 km southeast of the mill(Malaviya and Rathore 2007). The coloured compounds andadsorbable organic halogens (AOX) formed due toenvironmental factors in waste water exhibit strong mutageniceffects, physiological impairment (Ali and Sreekrishnan 2001;Mishra and Thakur 2012).

The effluents released from PPMs cause considerabledamage to the receiving waters if discharged untreated sincethey have a high biochemical oxygen demand (BOD),chemical oxygen demand (COD) and high levels ofchlorinated compounds measured as AOX, suspended solids(mainly fibers), fatty acids, tannins, resin acids, lignin and itsderivatives, sulphur and sulphur compounds, etc. While someof these pollutants are naturally occurring wood extractives(e.g., tannins, resin acids, stillbenes, lignin), others arexenobiotic compounds that are formed during the process ofpulping and paper making chlorinated lignins, resin acids andphenols, dioxins, furans, thereby turning PPM effluents into ‘aPandora’s box of waste chemicals’ (Peck and Daley 1994).Some of the pollutants listed above, notably polychlorinateddibenzodioxins and dibenzofurans (dioxins and furans), arerecalcitrant to degradation and tend to persist in nature(Mishra and Thakur 2012). They are thus known as POPsand have been classified as ‘priority pollutants’ by the UnitedStates Environmental Protection Agency (USEPA 1998) aswell as the ‘dirty dozen’ group of POPs identified by theUnited Nations Environment Program. It is well establishedthat many of these contaminants are acute and/or chronictoxins. Chlorinated organic compounds, which includedioxins and furans, have the ability to induce genetic changesin exposed organisms (Nestmann 1985; Costigan et al. 2012).In particular, DNA damaging agents have been shown toinduce inherited genetic defects and cancer (Easton et al.1997), with dioxins being named as ‘known humancarcinogens’ by the World Health Organization (WHO1997). This has resulted in a growing concern about thepotential adverse effects of genotoxicants on aquatic biotaand public health through the contamination of drinking watersupplies, recreational waters, or edible organic species (Mc-George et al. 1985). Studies with both bleached andunbleached Kraft pulp mill effluents showed evidence ofimpaired liver function in fish exposed to these effluents(Oikari and Nakari 1982; Orrego et al. 2010). There arereports of adverse health and environmental effects linked toorganochlorinated dioxin-like compounds, often related toendocrine disruption, such as, growth retardation, thyroiddysfunct ion decreased fer t i l i ty, feminizat ion ormasculinisation of biota and even tumour promotion(Mandal 2005). The genotoxic potential of PPM black liquorhas also been studied (Mishra and Thakur 2010).

Immortal human cell lines of human are able to mimic thetoxic response to in vivo with fair reproducibility (Das et al.2012). Cancerous cell lines are used for toxicity evaluation

because they retain the inherency of the organ, tissue, or cell-specific responses for a long time unlike primary cell lines.For example, HuH-7 cell line has been shown to be apromising cell line for toxicity evaluation as it has capacityto retain biosynthetic capabilities and surface receptors andcan produce metallothionein in response to challenge bymetaland aromatic compounds (Tai et al. 1994; Jahroudi et al. 1990;Miura et al. 1999). The ability to synthesize cytochrome P450monooxygenase enzymes, glucuronidation and sulphateconjugation abilities of the HuH-7 cell linemake it appropriatefor cell bio-detoxification and biotransformation studies.

The toxicity caused by many aromatic compounds likepolycyclic aromatic hydrocarbon (PAHs) can be measured.For evaluation of toxicity, there are many parameters whichprovide useful information regarding the structural andfunctional disruption caused by the toxicant, such asmorphological deformation, membrane integrity, cellviability, and mitochondrial membrane potential(Rikans and Yamano 2000).

The compounds present in PPM black liquor disrupt theendocrine system and produce stress responses like generationof reactive oxygen species (ROS), while the toxicity pathwaysof this black liquor are becoming understood. It binds toaromatic hydrocarbon receptors (AhR) and inducescytochrome P-450 monooxygenases especially CYP1A1 andCYP1A2 (Chaloupka et al. 1994; Nebert and Karp 2008);however, the exact mechanism(s) of induced toxicityremain unexplored.

Thw present investigation was designed to study thedegradation and detoxification of PPM black liquor in soilmicrocosm by indigenous Bacillus sp. The strain was earliershown to decolourize and delignify 10 % (v/v) black liquor inliquid minimal salt medium (MSM). In the present study, thepotential of the strain was evaluated to degrade and detoxifythe PPM contaminants in a solid (soil) medium so that it canbe used to reclaim the land contaminated with PPM blackliquor. The study simultaneously aimed to reveal whetherthe methyltetrazolium (MTT), 7-ethoxyresorufin-O -deethylase (EROD) and alkaline comet assays can beused to evaluate biodegradation and detoxificationefficiency in the HuH-7 cell line.

Materials and methods

All chemicals were purchased from Sigma-Aldrich (St. Louis,MO, USA) or Merck (Darmstadt, Germany) unless statedotherwise, and used without further purification.

Sample collection

PPM black liquor samples were collected from the M/sCentury Pulp and Paper Mill, Lalkuan (79°28′E longitude

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and 29°24′N latitude), Nainital, Uttarakhand, India. Theindustry mainly uses eucalyptus trunk, bamboo shoot andsugarcane bagasses for the kraft pulping process. The pulp isbleached using amultistage chlorination process leading to thegeneration of effluents that contain high amount of kraftlignin, chlorinated phenolic compounds, and other solublecomponents of the raw materials (Raj et al. 2007). Thesampling was carried out during the month of November(2005) when the COD, BOD and lignin content of theeffluent streams was comparatively higher (Malaviyaand Rathore 2007).

Microorganism and culture condition

The bacterial strain Bacillus sp. used in this study wasindigenous to the sampling site and was isolated along withthree other strains after stabilisation of the native microbialpopulation in a chemostat culture. The bacterium wasscreened out of the four strains over its potency to decolourizeand delignify the PPM black liquor in liquid MSM containing10 % (v/v) black liquor. The bacterium was regularlymaintained as a pure culture in a chemostat condition asdescribed in an earlier study (Mishra and Thakur 2010).

Preparation of soil microcosm and microbial treatmentof black liquor

The soil microcosms were prepared in 2 l (40 cm [width] ×40 cm [height]; effective size, 40 cm [width] × 20 cm [height])plastic containers (Gautam et al. 2003). Each container hadthree layers: one of gravel (400 g) at the bottom, sand (300 g)in the middle, and the soil (500 g) at the top. Each container

was connected with glass tubes to provide aeration. Soilcollected from the uncontaminated forest-land surroundingthe Century Pulp and Paper Mill, was used in the microcosmto provide an environment of identical inorganic nutrientconstituents to the indigenous bacteria. In order to eliminateunwanted microbial populations, the soil samples weresterilized by autoclaving at 121 °C for 60 min on 3consecutive days (López et al. 1999). The sterility of the soilwas checked afterwards by streaking dilutions of soilsuspensions on LB agar plates. Subsequently, vacuum filter(0.22 μM) sterilized black liquor sample was added to the soilto reach a final concentration of 40 % (volume/weight). Thesterile soil served as the inorganic nutrient source for bacteriawhereas the sterile black liquor served as the carbon source.Finally, the bacterial suspension from the MSM-chemostat culture was inoculated into the soil in themicrocosm to give final concentration of 1.0×107cellsg−1 and then the microcosms were incubated at 30 °Cin optimum light.

The moisture level (40 %) was maintained in the soil withblack liquor and was determined by measuring the waterholding capacity (Srivastava and Thakur 2006). Formeasuring water holding capacity, 12–15 cm of soil wasplaced in a percolation tube and was compacted by gentlebouncing. Black liquor was added until the column waswet down about 5 cm. Then the tube was covered andleft to stand in the same condition for 2 days, afterwhich, the top half-inch of soil was discarded and wetsoil was weighed out in a pre-weighed moisture cane.Then the dry weight was measured after placing the wetsoil samples in an oven at 105–110 °C for 24 h. Percentsoil water holding capacity was calculated as

%Water holdingcapacity ¼ Wetweight−Ovendryweightð Þ=Ovendryweight� 100

Two sets of microcosms were prepared and each set wasprepared in triplicate. The first set included two microcosms(C2, C15) and was prepared as microbe-free control, where nobacteria were inoculated. Soil samples were collected foranalysis after 2 and 15 days from the C2 and C15 microcosms,respectively. These two microcosms represented the photo-geochemical degradation of contaminants in a microbe freeenvironment. The second set of microcosms (T2, T15) wasprepared in a similar way but it was inoculated with bacterialsuspension and was sampled, as above. This second set ofmicrocosms represented the photo-biogeochemical degradationprocess in presence of the bacterium. The 0 h sample, whichwas same for both sets, represented the untreated contaminants.This sample served as the untreated control against which otherfour treatment groups were compared.

Extraction of compounds from soil microcosms and GC-MSanalysis

Soil samples (250 g) were air dried and passed through a 2-mmscreen before Soxhlet extraction (US EPA method 3500C;U.S.E.P.A 2007) with hexane/acetone (1:1, v/v) for 24 h. Thefiltered extracts were divided into two parts and both parts wereevaporated to dryness at room temperature using a vacuumrotator evaporator. The first sample was reconstituted withDMSO for toxicological analysis, while the other was dissolvedin acetone for gas chromatography-mass spectroscopic (GC-MS) analysis. The concentration of the final extract wasmaintained at 200 g sediment equivalent ml−1 of solvent (gSed Eq ml−1), which means that the total contaminants from200 g of soil were dissolved in 1 ml of solvent.

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Toxicological analysis

Cell culture

HuH-7 cells were grown as a monolayer in DMEM/Ham’s F12medium (1:1 mixture) supplemented with 10 % (v/v) foetalbovine serum and 1 % (w/v) antibiotic antimycotic solution(final concentrations: penicillin, 100 units ml−1; streptomycin,0.1 μg ml−1; amphotericin B, 2.5 ng ml−1) at 37 °C in ahumidified atmosphere of 5 % (v/v) CO2 (Das et al. 2012).

For the MTT and EROD assays, cells were seeded in 96-well plates, whereas for the alkaline comet assay, cells wereseeded in 12 well plates, both at a density of approximately2.5×105 cells ml−1. After 24 h of seeding and at 90 %confluence, cells were either only vehicle (DMSO) treated,treated with a positive control chemical [benzo(a )pyrene(BaP)] or treated with test samples for an additional 6 h (ERODassay), 18 h (Comet assay) or 24 h (MTT assay). All positivecontrol chemicals and test samples were dissolved in DMSOand the final concentration of DMSO in the medium was 0.5 %(v/v). The samples were added to the cell cultures in differentdilutions to work out the dose response relationships and allexperiments were carried out in three replicates.

EROD assay

The EROD assay was carried out as previously described (Daset al. 2012). Briefly, medium was removed after 6 h of sampleexposure and replaced by 100 μl of fresh medium containing5 μM of 7-ethoxyresorufin and 10 μM dicumarol. Cultureplates were incubated for 30 min at 37 °C and then methanol(130 μl) was added to stop the reaction. Resorufin-associatedfluorescence was measured at 530 nm excitation and 590 nmemission by using a multi-well fluorescence plate reader(SpectraMax M2, Molecular Devices). The data were fittedto a resorufin standard curve, and the results were expressed aspercentage of EROD activity induced by the positive control(BaP, 4 μM). BaP-equivalents (BaP-Eq) for each sample weredetermined as the ratio of the EC50 of BaP expressed as g l

−1 tothat of the sample expressed as g Sed Eq l−1 (Das et al. 2012;Kinani et al. 2010).

Cell viability

The effect of test compounds or samples on cellular viabilityin the cell lines was evaluated by using the MTT assay(Mosmann 1983). Cell viability was expressed as a percentageof the corresponding control (DMSO 0.5 % [v/v]).

Detection of reactive oxygen species

ROS, consisting of hydroxyl, alkoxyl, peroxyl, superoxide ornitroxyl radicals, are released in the cytoplasm of cells in the

process of xenobiotic metabolism. Thus, production of ROS,an indicator of oxidative stress to cells, can be utilized tomonitor the toxicity of individual xenobiotic compounds orcomplex industrial discharge mixtures. In present experimentproduction of ROSwasmeasured by using both the oxidation-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescindiacetate (DCFH-DA) and the superoxide specific fluorescentdye dihydroethidium (DH). After entering into the cell,DCFH-DA is hydrolyzed to form DCFH which is oxidizedby different ROS in the cytoplasm to form a fluorescent aproduct 2′,7′-dichlorofluorescein (DCF) in the cytoplasm. DHreacts with superoxidein the cytoplasm to form 7-hydroxyethidium, which in turn crosses nuclear membranesand intercalates with DNA, thus giving a fluorescent signalfrom the nucleus (Zhao et al. 2005).

HuH-7 cells were seeded on a tissue culture-treated slide at adensity 1×105 cells ml−1. After overnight growth cells wereexposed to different microcosm samples for 6 h. At the end of6 h treatment, the cells were loaded with 100 μM DCFH-DAand 2 mM DH both dissolved in 0.5 % (v/v) DMSO andincubated for 30 min at 37 °C. After dye exposure, HuH-7 cellswere washed with PBS (phosphate buffer saline) and wereobserved under a confocal laser microscope (CSLM). Thefluorescence images in false colour were recorded with oilimmersion of the objective lens (Olympus FluoView™FV1000). Both the fluorescent dyes were excited at 488 nm,and detection was carried out at 525 and 620 nm for DCF andDH, respectively. All images were processed with the inbuiltsoftware FV10-ASW version 01.07.01.00 (Jaiswal et al. 2012).

For estimation of ROS formation in these cells, FACS(fluorescent-assisted cell sorter) analysis was also performedusing a FACSort cytometer (Becton Dickinson, CA). Here,samples exposed to DH were excited at 490 nm and detectedat 620 nm. DCFH-DA treated samples were excited at 488 nmand detected at 525 nm (Jaiswal et al. 2012).

Analysis of DNA strand breaks by single-cell gelelectrophoresis (comet) assay

Alkaline comet assays were performed with HuH-7 cellstreated with either test samples, positive control (BaP,75 μM) or negative control (DMSO 0.5 % v/v) according tothe guidelines of Tice et al. (2000). At the end of exposure,cells were harvested, mixed with 1 % (w/v) low-meltingagarose and added to slides pre-coated with 1 % (w/v)agarose. Cells were then denatured with lysis buffer (2.5 MNaCl, 0.1 M Na2EDTA, 10 mMTris–HCl, pH 10) for 1 h, un-winded with alkaline electrophoresis solution (0.3 M NaOH,1 mM EDTA, pH 13) for 20 min and subjected toelectrophoresis at 25 V/300 mA for 15 min. At the end ofelectrophoresis, cells were neutralised with 0.4 M Tris–HCl,pH 7.5 for 15 min and the slides were stained with ethidiumbromide (2 μg/ml, 100 μl/slide). Cells were visualised with a

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40× lens fitted to a fluorescence microscope with an excitationand emission setting of 518/605 nm.

The percentage of the cells’ DNA in tail, tail moment andthe olive tail moment (OTM) of 40 randomly selected cellswere analyzed from each slide by using CometScore Freewaresoftware (www.tritekcorp.com). The comets were divided intofive classes on the basis of amount of the DNA in tail: Class I,less than 1 % (intact nucleus); Class II, 1–20 %, Class III, 20–50 %, Class IV, 50–75 % and Class V, more than 75 %(Miyamae et al. 1998).

Statistical analysis

All experimental data were expressed as means ± standarddeviation of three replicates. Sigmoid dose–response curves,EC50 and LC50 values were derived from the global curvefitting analysis with four parameter logistic curve equation.An F -test for dose–response parallelism was carried outconsidering both shared and un-shared parameters for four-parameter logistic curve equation.When the data failedF -test,results obtained with unshared parameters were considered forEC50 or LC50 values and further calculation. Statisticaldifferences between the control and treated cells wereexamined with the aid of ANOVA followed by multiplecomparisons (Dunnett’s method). All statistical analyses wereperformed with the Sigma Plot 11 statistical package (SystatSoftware, San Jose, CA, USA).

Results and discussion

Microbial degradation of contaminants of pulp and paper millblack liquor

The black liquor samples before treatment (i.e., crude blackliquor) and remediated black liquor (treated with bacteria,Bacillus sp.) were selected for GC-MS analysis. Thechromatographs corresponding to compounds extracted from0 h control and T15 samples are shown in Fig. 1a and b,respectively, and the identified compounds are summarized inTable 1. The degradation products listed in the table wereidentified from the available standards of the authenticcompounds documented in the NIST-05 and Wiley-8 libraries(Raj et al. 2007). The degradation of organic compounds ismay be due to the presence of the enzyme xylanase (Mishraand Thakur 2011). In Fig. 1, GC-MS shows the presence ofderivatives of pyrans and pyrazine. The compounds detectedin crude black liquor have many nitrogen and sulphur groupsattached to them. After treatment the original compounds didno t p e r s i s t , a n d s imp l e r c ompound s l i k e p -hydroxyhydrocinnamic acid (a type of monilignol),homovanillic acid methyl ester, 2,6-bis(1,1-dimethyl ethyl)4-methyl phenol and 3,5-dimethoxy-p -coumaric alcohol were

detected (Table 1). Cinnamic acids, which is involved inlinking the lignin and hemicellulose fraction of lignocellulose,was detected in extract degraded by Bacillus sp. A number ofpeaks present in the initial sample disappeared, and some newpeaks were visible in the bacterial remediated sample ofFig. 1a and b; while some compounds were present in all thesamples, their concentrations were reduced, which is evidentfrom the comparative peak area data (Fig. 1).With increases inthe duration of bacterial treatment, the total peak area in theGC-MS chromatogram also gradually reduced, indicatingsteady mineralisation of organic compounds. The lowmolecular weight aromatic compound identified in bacteriadegraded sample favours the idea of biochemical modificationof the lignin to single aromatic unit by bacteria and thesearomatic compounds have been reported to be lignindegradation intermediate.

In the present study, GC-MS was used because it has beenproven to be a suitable technique to analyse low molecularweight compounds released from lingocellulose due todegradation (Ksibi et al. 2003). Various researchers haveidentified many compounds as a result of microbialdegradation of PPM black liquor by GC-MS such as furancarboxylic acid, glyoxylic acid, 3-acetoxybutyric acid,guaiacol, valeric acid, benzene acetic acid, propanedioic acid,acetoguiacone, phenyl propionyl glycine, t-cinnamic acid, 3,4,5-tr imethoxy benzaldehyde, dibutyl phthalate ,acetophenone, gallic acid, ferulic acid, octadecanoic acid,benzyl butyl phthalate, 1-phenanthrene carboxylic acid, andbis(2-ethylhexyl)phthalate (Folke and Guerra 1992;Hernandez et al. 2001; Gupta et al. 2001; Raj et al. 2007).

EROD assay

The dose–response curves for EROD induction by differenttest and control samples are shown in Fig. 2. The 0 h controlsample elicited significant EROD activity shown in terms ofEC50 and BaP-Eq values. The highest values of BaP-Eq werefound for the 0 h control sample (3.90 μg g Sed Eq−1), and thelowest values were found for the 15-day bacterial-treated(T15) sample (0.0113 μg g Sed Eq−1). The BaP Eq valueshowed a 343-fold decrease after 15 days of bacterialtreatment (Table 2).

EROD induction by dioxin-like compounds in liver celllines has been effectively utilized to monitor such compoundsin environmental samples (Das et al. 2012; Brack et al. 2000).Results of this bioassay have been routinely compared withchemical analysis methods. EROD assay-derived BaP-Eqswere positively correlated with toxic equivalents derived fromchemical PAH analysis (Kinani et al. 2010; Louiz et al. 2008).However, Brack et al. (2000) and Kaisarevic et al. (2009)found that biologically derived toxic equivalents mostlyexceed the chemically derived toxic equivalents. To explainthis deviation, Kaisarevic et al. (2009) have suggested the

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Fig. 1 a GC-MS chromatographof untreated (0 h) pulp and papermill effluent. b GC-MSchromatograph of treated PPMblack liquor (T15)

Table 1 Identification ofmetabolites formed after bacterialdegradation

Peak R\retention time (min) Present in sample Name of the compounds

0 h C15 T2 T15

7.2 + + + − 1,2-Ethanediol diformate

9.8 − − + + Methyliminodiacetic acid

15.3 − − + + 2-Methoxy 4-vinyl phenol

15.9 + + + − 1-Hydroxy-4-methyl-2,6-di-tert-butylbenzene

17.6 − + + + 2,6-Bis(1,1-dimethyl ethyl)4-methyl phenol

19.3 − − + + p-Hydroxyhydrocinnamic acid

21.6 − − − + Homovanillic acid methyl ester

24.7 − − + + 3,5-Dimethoxy-p-coumaric alcohol

26.1 − − − + Ethyl 4-hydroxy-3-methoxyphenyl acetate

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presence of non-analysed compounds with EROD inducingpotency in the sediment extract and effluent. Similarly, Bracket al. (2000) have hypothesized the presence of heterocyclicaromatic hydrocarbons and amines, aminotoluenes, imidazoles,and other pesticides, a number of natural compounds, such aswood components, retene, and juvabione in their sample toexplain the exceeding biological equivalent value. Even thoughthe EROD assay-derived toxic equivalent lacks generalagreement with chemically derived toxic equivalents, it canbe utilized to monitor the toxicity of pre- and post-bioremediated samples qualitatively and quantitatively. In thepresent study the untreated (0 h control) sample elicitedsignificant EROD activity, indicating a general contamination

by dioxin-like and other EROD-inducing compounds at thestudied site. Further post biodegradation increases in ERODEC50 values and decreases in BaP-Eq values indicated steadymineralization of EROD-inducing chemicals.

Cell viability

The cell viability in terms of MTT assay-derived LC50 values(Table 2) and the dose–response curves (Fig. 3) showed thatthe level of toxicity in the microcosms decreased withincreasing duration of bacterial treatment. The lowest valueof LC50 was found for the 0 h control sample (10.46 g Sed Eql−1) and the highest for T15 (1457.76 g Sed Eq l−1). LC50

values thus increased about 139 times after 15 days ofbacterial treatment.

The widely usedMTTassay is based on the ability of livingcells to reduce the dissolved MTT (yellow) into the insolubleformazan (blue) in the presence of the mitochondrial enzymesuccinate dehydrogenase (Mosmann 1983). Thus, at a sub-cellular level it is an indicator of mitochondrial damage. At thesame time, it is an overall indicator of cytotoxicity asmitochondria are involved in many significant cellularpathways, including oxidative metabolism, which whendamaged leads to apoptosis. The MTT assay results thusprovide evidence of PPM black liquor induced mitochondrialdamage, leading to cellular cytotoxicity. Further, MTT assayresults were supported by the results of an LDH leakage assaywhich was performed to monitor cytotoxicity in terms of cellmembrane integrity (Fig. S3).

In the present study, EROD EC50 value showed a 343-foldincrease after 15 days of bacterial treatment whereas the MTTassay-derived LC50 values showed a 139-fold increase underthe same condition. This indicated that, the EROD-inducingcompounds were apparently degraded by the bacteria leading tothe increase of the EROD EC50 value, but at the same timesome other factors were responsible for overall cell cytotoxicity.

Concentration (g Sed Eq L-1)

1e-1

2

1e-1

1

1e-1

0

1e-9

1e-8

1e-7

1e-6

1e-5

1e-4

1e-3

1e-2

1e-1

1e+

0

1e+

1

1e+

2

1e+

3

1e+

4

ER

OD

act

ivity

(10

0% =

4µM

BaP

)

0

20

40

60

80

100

120

BaP (g L-1)

0h (g Sed Eq L-1)

C2 (g Sed Eq L-1)

C15 (g Sed Eq L-1)

T2 (g Sed Eq L-1)

T15 (g Sed Eq L-1)

Fig. 2 EROD induction measured in HuH-7 cell line after 6 h exposureto different test and control samples. Acronyms correspond to thedifferent samples to which cells were exposed; BaP Benzo(α)pyrene, 0h untreated control soil microcosm extract, C2 un-inoculated soilmicrocosm extract after 2 days, C15 un-inoculated soil microcosmextract after 15 days, T2 bacterial inoculated soil microcosm extract after2 days, T15 bacterial inoculated soil microcosm extract after 15 days.Values represent the mean ± SD, n =3. 100 % EROD induction wasconsidered for 4 μM BaP treatment. Global goodness-of-fit R2=0.9984

Table 2 EROD EC50, MTT, LC50 and comet assay OTM values of untreated and treated samples along with corresponding BaP-Eq values

Treatmentsa MTT LC50b R2 (LC50) EROD EC50

b R2 (EC50) BaP-Eqc Comet assay: OTM

BaP 1.64e−6 0.9889 1.60 e−6 0.9974 – 10.6207±3.3499

0 h Control 10.46 0.9918 0.4105 0.9990 3.90e−6 9.4887±4.0853

C2 11.64 0.9935 1.2700 0.9977 1.26e−6 6.5919±4.0776

C15 14.37 0.9968 4.1680 0.9991 3.84e−7 6.2930±3.8752

T2 907.63 0.9990 6.5590 0.9987 2.44e−7 5.4238±3.8075

T15 1,457.76 0.9951 140.9833 0.9964 1.13e−8 1.8578±1.8848

aHuH-7 cell lines were treated with different test samples (dilutions, 10−3 to 103 g Sed Eq l−1 ) or positive control BaP (dilutions, 10−11 to 10−3 g l−1 ) forMTT and EROD assay. For comet assay, only a single concentration was considered: 75 μM for BaP and 103 g Sed Eq l−1 for test samplesb LC50 was derived using global curve fitting model with four parameters logistic nonlinear regression equation, expressed in terms of g Sed Eq l−1 (testsamples) or g l−1 (BaP)c BaP-Eq (g gSed Eq−1 ) = EC50 of BaP (g l−1 )/EC50 of sample (g Sed Eq l−1 )

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In the above context, it is noteworthy that some PAHcompounds found in paper mill black liquor like phenanthrene,anthracene and pyrene have a cellular proliferative effect and

simultaneously they demonstrate a high EROD-inducingpotency (Kaisarevic et al. 2009). Machala et al. (2008) haveshown that certain PAHs and their methylated derivativesdisrupt the cell cycle and enhance cell proliferation, in rat liverepithelialWB-F344 cells via the AhR-mediated pathway. Thus,degradation of these PAH compounds might have led to thedecrease in proliferative effect which is reflected in MTT assayderived LC50 values. Overall results of the MTT assaysuggested that the PPM black liquor contained toxicants thatdisturbed cell growth due to observed effects on cellproliferation or induction of cytotoxicity.

Reactive oxygen species

Figure 4a shows a CSLM-based image in which ROS weredetected inside the HuH-7 cells with the help of oxidationsensitive dyes DH and DCFH-DA. The blue colour is theindicator of the superoxide generation and was due to theformation of DNA staining fluorescent agent 7-hydroxyethidium, as a product of DH and the superoxidereaction (Zhao et al. 2005). The green colour is the indicator ofgeneral degree of oxidative status (due to total ROS) andwas due

Concentration (g Sed Eq L-1)

1e-1

2

1e-1

1

1e-1

0

1e-9

1e-8

1e-7

1e-6

1e-5

1e-4

1e-3

1e-2

1e-1

1e+

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1e+

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1e+

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% C

ell V

iabi

lity

(100

% =

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%)

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80

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120

BaP (g L-1)

0h (g Sed Eq L-1)

C2 (g Sed Eq L-1)

C15 (g Sed Eq L-1)

T2 (g Sed Eq L-1)

T15 (g Sed Eq L-1)

Fig. 3 Cell viability measured after 24-h exposure period. Acronyms areas given in Fig. 2. 100 % cell viability was considered for 0.5 % DMSOtreatment. Values represent the mean ± SD, n =3. Global goodness-of-fitR2=0.9940

DMSO Crude effluent Remediated effluent

Rel

ativ

e va

riatio

n in

RO

S g

ener

atio

n

0

100

200

300

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(0 h) (T15)

*

a

b

Fig. 4 a DCFH-DA and DHbased detection of ROSgeneration inside the HuH-7 cellsby confocal laser microscopy.Blue colour indicates amount ofsuperoxide in the cell,fluorescence light generated byDH and green colour indicatestotal cytoplasmic ROS,fluorescence light generated byDCFH-DA (refer toMaterials andmethods for detail). A is DMSOcontrol, B is T15 remediated andC is 0 h control. b DH-baseddetection of ROS inside HuH-7cells by flow cytometric analysis.ROS generation was monitored inHuH-7 cells exposed to differentmicrocosm extracted samples for6 h followed by incubation withDH for 30 min. Values representthemean ± SD, n =3. The asteriskdenotes the significant differencefrom the DMSO control group(Dunnett’s method, p< 0.05)

Environ Sci Pollut Res (2014) 21:2966–2976 2973

to the formation of fluorescent agent DCF as a product of ROS-induced DCFH oxidation (Duranteau et al. 1998). Themicroscopic image indicates that the 0 h sample induced moreROS in HuH-7 cells than the T15 microcosm sample.

Relative ROS generation was measured quantitatively byflow-cytometric analysis. Relative ROS generation by the 0 hand T15 samples are shown in Fig. 4b. Levels of ROS weresignificantly higher in the 0 h sample.

It is evident from earlier studies that PPM effluent can induceoxidative stress (Oakes et al. 2004; Ahmad et al. 2000). Thepresent study also confirms that PPM black liquor inducesoxidative stress in the HuH-7 cells. Increased oxidative stresscould lead to apoptosis, carcinogenesis, genetic damage and othersignalling dysfunctions (Bartsch and Nair 2004). The ROSproduction may be due to the presence of a wide array ofxenobiotic compounds in PPMblack liquorwhich induce damageto different cellular signalling pathway (Maria et al. 2003).

Comet assay

The results of single cell gel electrophoresis (Comet assay)with biodegraded (T2, T15) and 0 h control samples areshown in Fig. 5a and b. HuH-7 cells treated with the 0 h

sample resulted in 95 % comets that fell under either of theclasses III, IVor V; whereas only 25 % comets fell under thesame classes with T15 sample. Tail moments of 40 randomlyselected comets are presented as quartile box plots. The plotshows that distribution of comets became more homogenouswith lower tail moment (2.4±3.6) by the T15 sample incomparison to 0 h control sample (tail moment = 19.4±10.6). This indicated that the comet class shifted towards alower value of tail moment after the bacterial treatment. TheOTM data showed a decreasing trend with increasing durationof bacterial treatment. The T15 sample had a 5-fold decreasein DNA migration (OTM=1.9±1.9) in comparison to that ofthe 0 h control sample (OTM=9.5±4.1). Statisticallysignificant DNA damage (Dunnett’s method, p< 0.05) wasfound in 0 h, C2, C15 and T2 samples with respect to thenegative control group (DMSO 0.05% v/v) and confirmed thegenotoxic nature of the black liquor. The comet assay can beperformed with different eukaryotic cells includingmammalian cell lines. The assay has been carried out in yeastcells earlier to demonstrate post biodegradation toxicityreduction of PPM black liquor (Mishra and Thakur 2010;Singhal and Thakur 2009), but HuH-7 cell line was a betteroption here due to its sensitivity in the EROD assay.

Fig. 5 Genotoxicity of thecontaminants before and aftertreatment with Bacillus sp. a Thetail moment and the olive tailmoment plotted against differentsamples. Tail moments of 40randomly selected comets arepresented as quartile box plots.The edges of the box representthe 25th and the 75th percentiles;a solid line in the box presents themedian value while dotted linerepresents mean value. Errorbars indicate 90th and 10thpercentiles and the black circlesindicate outlying points beyond5th and 95th percentiles. OTMs ofthe same 40 comets are shown asthe mean ± standard deviation.The asterisk denotes thesignificant difference from theDMSO control group (Dunnett’smethod, p< 0.05) for both datasets. b Images of different classesof comets seen under fluorescentmicroscope after stained withethidium bromide. Romannumerical indicates class ofcomet

2974 Environ Sci Pollut Res (2014) 21:2966–2976

Conclusion

Microbial biodegradation of toxic and recalcitrant moleculefor bioremediation has commercial potential. In the presentstudy we showed, using in vitro models and biochemicalassays, the effectiveness of our bacterial strain Bacillus sp.in detoxifying PPM black liquor within 15 days in themicrocosm. The results of bioassays viz. MTT, EROD andROS production corresponded well with each other, showingreduced toxicity of PPM black liquor. GC-MS also confirmsthe degradation of toxic compounds. The bioassays bothquantitatively and qualitatively describe different aspects oftoxicity including overall cytotoxicity byMTTassay and ROSproduction by DCFH-DA and DH assay, dioxin likebehaviour by EROD assay and genotoxicity by comet assayin a cost- and time-effective manner.

Acknowledgments This paper was supported by the research grants ofDepartment of Biotechnology, Government of India, and New Delhi,India. The author (MM) thanks Council for Scientific and IndustrialResearch, Government of India, New Delhi, India, for providingResearch Fellowship. We thank the Century Pulp and Paper Mill,Uttarakhand, India for providing black liquor during the course ofinvestigation and Mr. Ajai Kumar of the Advanced InstrumentationResearch Facility (AIRF) Jawaharlal Nehru University, New Delhi forGC-MS analysis. We also thank Dr. Vijay Kumar of the InternationalCentre for Genetic Engineering and Biotechnology, New Delhi, for kinddonation of the HuH-7 cell line.

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