Layout 1 INTRODUCTION Despite intensive research identifying causes, conse- quences and possible preventive measures of cyanobac- terial mass proliferations, cyanobacterial blooms represent a major problem in fresh waters throughout the world. The adverse consequences include water quality degradation, accumulation and microbial decay of bloom biomass fol- lowed by lowering of oxygen content in water (Wiegand and Pflugmacher, 2005). In addition, a wide spectrum of toxins and secondary metabolites produced by cyanobac- teria have been shown to adversely affect aquatic organ- isms (Codd et al., 2005; Zanchett and Oliveira-Filho, 2013), livestock (McGorum et al., 2015) and human health (Kuiper-Goodman et al., 1999; Zanchett and Oliveira-Filho, 2013). While different cyanobacterial taxons can contribute to the formation of dense cyanobacterial water blooms de- pending on geographical and ecological conditions, Micro- cystis sp. represents cosmopolitan and pervasive cyanobacterial genera, which is frequently reported to dom- inate water blooms in freshwaters of all continents except Antarctica (Harke et al., 2016). Microcystis species (such as Microcystis aeruginosa) are also among the prominent producers of the most broadly studied cyanobacterial toxins microcystins (MCs) (Bláha et al., 2009). MCs are known liver tumour promoters (Nishiwaki-Matsushima et al., 1992), with the most common structural variant micro- cystin-LR (MC-LR) being classified by the International Agency for Research on Cancer as a possible human car- cinogen (IARC2B) (Grosse et al., 2006). MCs have been shown to act via inhibition of serine/threonine protein phos- Advances in Oceanography and Limnology, 2017; 8(1): 107-120 ARTICLE DOI: 10.4081/aiol.2017.6342 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). Chlorination and ozonation differentially reduced the microcystin content and tumour promoting activity of a complex cyanobacterial extract Iva Sovadinová,1,2* Pavel Babica,1,2,3 Ondřej Adamovský,1,2 Alla Alpatova,4,5 Volodymyr Tarabara,4 Brad Luther Upham,2 Luděk Bláha1 1RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 753/5, 62500 Brno, Czech Republic; 2Department of Pediatrics and Human Development, and Institute for Integrative Toxicology, Michigan State University, 1129 Farm Lane, East Lansing, MI 48824, USA; 3Department of Experimental Phycology and Ecotoxicology, Insti- tute of Botany, Czech Academy of Sciences, Lidicka 25/27, 60200 Brno, Czech Republic; 4Department of Civil and Environmental Engineering, Michigan State University, 428 S. Shaw Lane, East Lansing, MI 48824, USA; 5Civil and Environmental Engineering, University of Alberta, AB T6G 2E1 Edmonton, Canada *Corresponding author: sovadinova@recetox.muni.cz ABSTRACT Despite intensive research and management efforts in the past decades, cyanobacterial blooms and their toxins, such as microcystins (MCs), continue to represent a major ecological and health problem in fresh waters throughout the world. Our objective was to compare the efficacy of two commonly used drinking water treatment technologies, chlorination and ozonation, in removing MCs and in reducing tumour promotion-related effects of cyanobacteria, such as inhibition of gap junctional intercellular communication (GJIC) and activation of mitogen activated protein kinases (MAPKs) in a rat liver epithelial stem-like cell line (WB-F344). This combined chemical and bioassay approach demonstrated that ozone effectively removed all MCs from an extract of a globally important bloom-forming cyanobacterium, Microcystis sp. Ozone also significantly reduced the overall tumour promotional potency of the cyanobacterial extract, as indicated by a substantial reduction in the ability of the extract to inhibit GJIC and activate extracellular receptor kinase 1/2 (ERK1/2). Although comparable reduction of total organic carbon was achieved by ozone and chlorine treatment, chlorination was much less ef- fective in removing MCs and reducing the effects on GJIC. Chlorination had a biphasic effect with an observed decrease of extract-in- duced activation of ERK1/2 at the lower chlorine doses; whereas at high doses of chlorine the by-products of chlorination actually induced the activation of ERK1/2. The extracts induced p38 activation, and chlorination was not effective in reversing this effect, while ozone did reverse this effect, albeit not as much as the activation of ERK1/2. Thus, ozone was effective in reducing the toxicity of cyanobacterial extracts while chlorination was not only lacking efficacy, but at high doses of chlorination further produced by-products that were equally toxic as the untreated samples. Our study indicates the value of using an effect-based approach to assess the efficacy of water treatment systems in removing toxins, and more specifically demonstrates that ozone was more effective at reducing the toxic potential of cyanobacterial-contaminated water. Key words: Cyanobacteria; water treatment; toxicity; chlorination; ozonation; microcystin. Received: 11 October 2016. Accepted: 14 February 2017.Non -co mmerc ial us e o nly I. Sovadinová et al.108 phatases (PPs) (Campos and Vasconcelos, 2010) and in- duction of oxidative stress followed by damage to cellular macromolecules (Mathe et al., 2016). Chronic exposures to cyanobacteria and their toxins – e.g., via contaminated drinking water – have been associated with increased oc- currence of liver and colorectal cancer (Yu, 1995; Zhou et al., 2002; Svircev et al., 2009). Other (often yet unidenti- fied) compounds produced by complex cyanobacterial blooms can also induce or contribute to different adverse effects (Oberemm et al., 1997; Berry et al., 2009) including tumour promotion (Bláha et al., 2010). Occurrence of toxic cyanobacteria in surface water presents a challenge to drinking water treatment facilities. Preventive measures as well as various water treatment technologies used to minimize human health risks caused by cyanobacteria and their toxins have been recently sum- marized and critically discussed (Westrick et al., 2010; Merel et al., 2013; Roegner et al., 2014; Hiskia et al., 2016; Ibelings et al., 2016). The available approaches able to remove MCs to different extents include coagulation- flocculation-sedimentation, standard oxidation and disin- fection by chlorine or permanganate, ozonation and UV disinfection, sorption by activated carbon, nano- and ul- trafiltration, and advanced oxidation processes (AOP) (Westrick et al., 2010; Merel et al., 2013; Roegner et al., 2014). Different drinking water treatment technologies are applied in different countries and contexts, and eval- uation of their treatment efficiency should focus on i) re- moval of targeted priority pollutants (e.g., MCs in case of cyanobacterial toxins) to comply with current treatment goals; as well as ii) removal of other potentially harmful and toxic components of the complex material which may not be fully chemically characterized; and iii) formation of new harmful metabolites/toxic by-products during the application of the water treatment technology (Upham et al., 1994; Upham et al., 1995; Prasse et al., 2015). To address these different aspects of drinking water treatment, complementary chemical and biological tools (i.e. instrumental analyses and bioassays) should be in- cluded in the monitoring plans (Maier et al., 2015). This ongoing effort can be highlighted by current implemen- tation of the effect-based tools into the monitoring guide- lines for water quality assessment (Wernersson et al., 2014), which combines several bioassays targeting differ- ent toxic modes of action (MoAs) to provide additional information to classical chemical analyses and thus a more integrative view. Gap junctional intercellular communication (GJIC) plays a fundamental role in maintaining tissue homeosta- sis, and provides an excellent biological endpoint to assess potential adverse health effects of many anthropogenic toxicants and natural toxins (Vinken et al., 2009). GJIC is a critical cellular process for the coordination of different intra-, extra-, and inter-cellular signalling pathways re- quired for proper cell behaviour, tissue development, tissue function and maintenance of tissue homeostasis. Most chemical carcinogens and tumour promoters inhibit GJIC in in vitro assays, and demonstrated to be a representative marker of tumour promoting potency (Rosenkranz et al., 1997). Recently cyanobacterial extracts and exudates were determined to be potent in vitro inhibitors of GJIC (Bláha et al., 2010; Novakova et al., 2011). Inhibition of GJIC by these extracts were independent of the well-recognized tu- mour promoting cyanotoxins, MCs or cylindrospermopsin, indicating the existence of not-yet-identified toxic com- pounds (Novakova et al., 2013). Aquatic contaminants, such as polycyclic aromatic hydrocarbons (PAHs) or poly- chlorinated biphenyls synergistically potentiated the in- hibitory effects of cyanobacterial extracts on GJIC (Novakova et al., 2012), which further highlights the need to bioassay these mixtures for adverse effects in the com- plex assessment of drinking water quality, and efficacy of treatment technologies. In fact, in vitro assessment of GJIC has been success- fully used along with chemical analysis as a principal bioassay to study tumour promoting activity of water chlorination by-products (Hakulinen et al., 2004; Nishikawa et al., 2006). Similarly, GJIC assay has been applied to evaluate the efficiency of removing different anthropogenic contaminants and their toxic by-products with ozone, such as polycyclic aromatic hydrocarbons (Upham et al., 1994; Upham et al., 1995; Herner et al., 2001; Luster-Teasley et al., 2002; Luster-Teasley et al., 2005) and various pesticides (Upham et al., 1997; Masten et al., 2001; Wu et al., 2007). The in vitro bioassays used in these studies to assess GJIC were based on the scrape- load dye transfer (SL-DT) technique (El-Fouly et al., 1987). This SL-DT assay provides fast (minutes of expo- sure) and integrative responses, which reflect dysregula- tions of different cell processes and multiple signalling pathways controlling GJIC (Upham et al., 2016). Dysreg- ulation of GJIC is an epigenetic, phenotypic marker for determining tumour promotional activity, which contrasts and compliments the more commonly used genotoxic and specific nuclear receptor transactivation assays (Leusch and Snyder, 2015). GJIC can be assessed in vitro using diverse non-tumorigenic cells, with a rat liver epithelial cell line, WB-F344, being one of the most widely used GJIC model for the assessment of tumour promoting ac- tivity, as well as determining chemopreventive effects of chemicals (Upham et al., 1998; Sovadinova et al., 2015; Babica et al., 2016b). To further validate tumorigenic ac- tivity, compounds that dysregulate GJIC are often tested for effects on signal transduction pathways implicated in neoplastic transformation, such as mitogen-activated pro- tein kinases (MAPK-ERK1/2 and MAPK-p38) (Upham et al., 2008; Osgood et al., 2014; Babica et al., 2016a). Our objective was to evaluate and compare the effi- Non -co mmerc ial us e o nly Chlorination and ozonation of cyanobacterial extract 109 cacy of two broadly used drinking water treatment oxida- tion technologies, namely chlorination and ozonation, on the removal of known cyanotoxin MC concentrations as well as on changes in biological effects that are independ- ent of the MC content (i.e., removal of overall cytotoxicity and tumour promotional potency). We used a natural bloom sample that was dominated by the cosmopolitan and environmentally relevant bloom-forming cyanobac- teria M. aeruginosa. Extracts were prepared and charac- terized for content of MC, total organic carbon (TOC) concentration, initial cytotoxicity, effect on GJIC and modulation of signalling kinases (MAPKs). These cyanobacterial extracts were treated by chlorine or ozone, and evaluated for the changes in the toxin content, TOC, and in vitro cytotoxicity and tumour promoting activity. METHODS Cyanobacterial sample The sample of toxic cyanobacterial water bloom was collected from a lake located within the campus of Michi- gan State University (East Lansing, MI, USA; 42°40’50.09”N, 84°29’14.27”W) in September 2008 using a 20 μm plankton net. The bloom was dominated by Microcystis species: M. aeruginosa (>50% of the cell counts) accompanied by M. flos-aquae (~20%) and M. ichthyoblabe (~20%). The biomass was freeze-dried and 38 g of dry weight (DW) was extracted by 20 min sonica- tion (Fisher Sonic Dismembrator Model 300; Fisher Sci- entific, Pittsburgh, PA, USA) while stirring on ice with 566 mL of 50% methanol (i.e., 66.7 g DW L–1 equivalent). The samples were centrifuged at 31,000 × g and the supernatant fraction was collected and dried using a vacuum evaporator. The dry extract was dissolved in 47.2 mL of MilliQ water (MilliQ Synthesis A10; Millipore, Billerica, MA, USA) to obtain the final concentrated extract corresponding to 800 g DW of original biomass per one litter of water. Chlorination and ozonation The extract was aliquoted into 4 mL fractions to be treated by chlorination or ozonation as summarized in Tab. 1. Chlorination was carried out with sodium hypochlorite (NaOCl) in a phosphate buffer (0.5 M K2HPO4-0.293 M NaOH, pH 7.0±0.2) according to the Method 5710C (Clesceri et al., 1998). The sample was treated for 30 min to 24 h with NaOCl at different concentrations of free chlo- rine (7 to 1000 mg L–1) corresponding to contact time (CT) values 0.21×103, 7×103, 50×103 and 1440×103 mg min L–1. The oxidation reaction was stopped by addition of 10% w/v of NaHSO3. Vehicle controls were prepared from MilliQ water equally treated with chlorine and quenched. No resid- ual chlorine was present in the vehicle controls after the quenching. To increase the weight ratio of chlorine to dry weight of the extract (or TOC or MC concentration), the concentration of the original biomass was also diluted four- and eight times (i.e., “¼” or “⅛”) before the chlorination step with 500 mg L–1 of chlorine for 100 min. The original extract dissolved in MilliQ water was ozonated for 30 min using a commercial ozone generator (OZ2PCS-V; Ozotech, Yreka, CA, USA) at the concen- tration of O3 (gas)=5.0 mg L–1 with a gas flow rate of one L min–1 and temperature of 20.0±0.5°C to maximize ozone dissolution. Microcystin analysis The concentrations of MCs in the original biomass as well as in the samples after the chlorination and ozonation treatment were analysed by HPLC-UV/DAD following the procedure described earlier (Babica et al., 2006). Tox- ins were identified based on their retention times and characteristic UV absorption spectra and quantified using the calibration curves of standards of MC-RR, -YR, -LR, -LW, -LF, and nodularin. An example of the HPLC chro- matograph of the cyanobacterial extract recorded at 238 nm is in Supplementary Fig. 1. TOC analysis TOC of the extract before and after chlorination and ozonation was determined using LiquiTOC analyzer (El- ementar Analysensysteme, Hanau, Germany) where measurements were made by high temperature oxidation of the carbon (850-900°C) and detection of CO2 by an NDIR photometer. Bioassays WB-F344 rat liver non-tumorigenic stem-like cells (Tsao et al., 1984) were cultured in D-media (Kao et al., 1997) with 5% v/v of fetal bovine serum (Gibco, Life Technologies, Grand Island, NY, USA) at 37°C and 5% CO2. Cells were cultured to full confluence for 48 h in 35 mm tissue culture dishes (Costar; Cambridge, MA, USA). These confluent cells were used for the various time and dose related experiments. The vehicle controls were water chlorinated or ozonated using the same conditions as ap- plied for studied samples. A sample or vehicle was added directly to the cell culture medium in the dish and gently mixed. Non-treated cells were used as negative controls. The final concentrations of extracts in the bioassays were expressed as the original weight of dry biomass used for extract preparation per unit volume (g DW L–1). Viabil- ity/cytotoxicity was tested after 30-min and 24-h expo- sures using the neutral red assay as reported before (Babica et al., 2016b). The method determines viable cells capable of neutral red inclusion into lysosomes (Boren- freund and Puerner, 1985). Viability was expressed as the fraction of negative (non-treated) control (FOC). Non -co mmerc ial us e o nly I. Sovadinová et al.110 Ta b. 1 .C on di tio ns o f c hl or in at io n an d oz on at io n of th e st ud ie d cy an ob ac te ria l e xt ra ct . A bb re vi at io n Tr ea tm en t t yp e D os e of C l o r O 3 Tr ea tm en t d ur at io n To ta l M C M C -L R T O C 2 4- h C el l v ia bi lit y 3 0- m in 3 0- m in 3 0- m in a nd C T va lu e ( m g L –1 ) (µ g g–1 D W )° (µ g g–1 D W )° (µ g g–1 D W )° IC 50 (9 5% C I) G JI C E R K 1/ 2 p 38 ( m g m in L –1 ) L O E C I C 50 (9 5% C I) L O E C L O E C (g D W L –1 ) L O E C (g D W L –1 ) ( g D W L –1 ) ( g D W L –1 ) N T no tr ea tm en t -- -- 51 7 4 11 1 04 ,7 61 1 1. 6# 7. 8# ( 0. 52 -1 2. 41 )§ (0 .4 1- 9. 86 )§ (1 05 -2 51 4) § (1 0. 2- 13 .1 ) (7 .0 to 8 .7 ) 1 2 8 8 4 C l(7 -3 0) C hl or in at io n 7 3 0 m in 5 59 4 43 1 14 ,7 61 1 4. 8 10 .0 0 .2 1× 10 3 (0 .5 6- 13 .4 2) (0 .4 4- 10 .6 3) ( 11 4- 27 39 ) (1 2. 6- 17 .3 ) (9 .3 -1 0. 7) 1 2 8 8 4 C l(7 0- 10 0) C hl or in at io n 70 10 0 m in 44 7 3 52 88 ,3 55 1 4. 3 1 0. 7** *^ 7 ×1 03 (0 .4 5- 10 .7 4) (0 .3 5- 8. 44 ) (8 8- 21 21 ) ( 12 .2 -1 6. 5) (9 .7 -1 1. 8) 1 2 8 1 6 4 C l(5 00 -1 00 ) C hl or in at io n 5 00 1 00 m in 46 7 3 71 86 ,8 95 9. 1 1 2. 6** * 5 0× 10 3 (0 .4 7- 11 .2 0) (0 .3 7- 8. 91 ) (8 7- 20 85 ) (6 .9 -1 2. 2) (1 1. 2- 14 .1 ) 1 2 8 1 6 4 ¼ C l(5 00 -1 00 )$ C hl or in at io n 5 00 1 00 m in 34 6 2 75 77 ,2 29 1 0. 4 9 .6 5 0× 10 3 (0 .3 5- 8. 31 ) (0 .2 8- 6. 60 ) (7 7- 18 53 ) (7 .1 -1 5. 4) (8 .4 -1 1. 0) 1 2 8 8 4 ⅛ C l(5 00 -1 00 )$ C hl or in at io n 5 00 1 00 m in 41 2 3 31 81 ,1 93 7. 0 8 .1 5 0× 10 3 (0 .4 1- 9. 88 ) (0 .3 3- 7. 94 ) (8 1- 19 49 ) (7 .0 -1 5. 5) ( 6. 7- 9. 7) 1 2 8 n. a. n .a . C l(1 00 0- 24 h) C hl or in at io n 1 00 0 24 h 3 67 2 95 78 ,7 54 1 2. 3 1 2. 7** * 1 44 0× 10 3 ( 0. 37 -8 .8 1) ( 0. 29 -7 .0 7) (7 9- 18 90 ) (9 .7 -1 5. 7) (1 0. 7- 15 .0 ) 1 2 12 16 4 O 3 O zo na tio n 5 °° 3 0 m in n .d . n. d. 70 ,6 22 >2 4 > 24 (7 1- 16 95 ) > 24 2 4 > 16 4 C T va lu es : t he c on ce nt ra tio n of fr ee c hl or in e m ul tip lie d by th e co nt ac t t im e w ith th e sa m pl e be in g di si nf ec te d; it is e xp re ss ed in u ni ts o f m g m in L –1 . ° Th e co nc en tra tio n is c al cu la te d pe r g D W o f o rig in al bi om as s be fo re m et ha no l e xt ra ct io n. # Pl ai n te xt re pr es en ts IC 50 va lu es w ith 9 5% c on fid en ce in te rv al s in p ar en th es es , i ta lic iz ed n um be rs s ho w L O EC v al ue s. § V al ue s in p ar en th es es in di ca te th e ra ng e of ac tu al M C a nd T O C c on ce nt ra tio ns (m g L–1 ) i n th e ex pe rim en ts (c al cu la te d fo r t he ra ng e of te st ed c on ce nt ra tio ns o f 1 -2 4 g D W L –1 ). ^ S ig ni fic an tly d iff er en t t ha n IC 50 va lu e of N T ex tra ct (S tu de nt ’s t- te st , P< 0. 05 ). $ B ef or e ch lo rin at io n, th e co nc en tra tio n of o rig in al b io m as s w as d ilu te d fo ur (¼ ) o r e ig ht ti m es (⅛ ). °° G as fl ow ra te o f 1 L m in –1 fo r 3 0 m in ; n. d. , n ot d et ec te d: m in im al d et ec tio n lim it (M D L) fo r an in di vi du al M C v ar ia nt =1 .3 µ g g–1 D W ; n .a ., no t a na ly se d. Non -co mmerc ial us e o nly Chlorination and ozonation of cyanobacterial extract 111 Tumour promotion assay based on GJIC assessment used only the concentrations that were determined to be noncytotoxic using the neutral red assay. Cells were ex- posed for 30 min to the tested samples or corresponding vehicle. Treatment with 1-methylanthracene (70 µM, 30 min) was used as a positive control inducing complete in- hibition of GJIC. GJIC was assessed using modified SL- DT technique (El-Fouly et al., 1987; Babica et al., 2016c; Upham et al., 2016) . The migration of the dye through gap junctions was visualized with a Nikon Eclipse TE3000 phase contrast/fluorescent microscope and the images dig- itally captured with Nikon EZ Cool Snap CCD camera (Nikon Instruments, Melville, NY, USA), where three rep- resentative images were acquired from each dish. The area of dye transfer was measured for each image using ImageJ (https://imagej.nih.gov/ij/). The measured areas were ad- justed by subtracting an area of the dye transfer in the pos- itive control with completely inhibited GJIC. Adjusted areas from each image were compared with an averaged adjusted area of the negative control and expressed as FOC. Activation of regulatory kinases MAPK ERK1/2 and p38 after a 30 min exposure to the sample was determined by Western blotting. Western blot analyses were done as reported previously (Babica et al., 2016a). Briefly, the pro- teins were extracted with 20% SDS solution containing in- hibitors of proteases and phosphatases, and the protein concentration of the cell lysates was determined with DC assay kit (Bio-Rad, Hercules, CA, USA). The proteins (20 µg per lane) were separated on 12.5% SDS-PAGE (Laemmli, 1970) and then electrophoretically transferred to a 0.45 µm PVDF membrane (Millipore). To visualize activated, i.e. phosphorylated ERKs and p38, we used rab- bit phospho-specific polyclonal antibodies directed to ERK-1 phosphorylated at Thr 202/Tyr204, and ERK-2 phosphorylated at Thr185/Tyr187 (Cell Signaling #9101S, Danvers, MA, USA) and directed to p38 phosporylated at Thr180/Tyr182 (Zymed #36-8500, San Francisco, CA, USA), and secondary donkey anti-rabbit IgG conjugated with horse radish peroxidase (Amersham Bioscience # NA934V, Life Science, Denver, CO, USA). Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping protein, and determined with mouse anti-GAPDH antibodies (Chemicon #MAB374, Millipore) and secondary sheep anti-mouse IgG conju- gated with horse radish peroxidase (Amersham Bioscience #NA931V; Life Science). The ERK, p38 and GAPDH pro- tein bands were detected using the ECL SuperSignal West Pico Chemiluminesence detection kit (Pierce, Arlington Heights, IL, USA) and Bio-Rad Image Analyzer. Data analyses and statistics At least three independent experiments were done for each treatment (except for Western blot analyses), and the mean ± standard deviations from independent experi- ments were calculated. IC50 concentrations causing 50% inhibition of the studied effects and their 95% confidence intervals were derived using non-linear regression in GraphPad (GraphPad software Inc., La Jolla, CA, USA). One-way Analysis of Variance (ANOVA) followed by Dunnett’s post-hoc test was used to identify treatments significantly different from the control. Differences be- tween IC50 values were assessed by Student’s t-test. P-val- ues less than 0.05 were considered statistically significant. RESULTS The original biomass contained 517 μg of total MC per g of DW with MC-LR being the dominant variant (411 μg g–1 DW, 79%, Tab. 1). The other variants present included MC-RR (2%), MC-LW (2%), MC-LF (3%) and two other structurally unidentified MC variants (14%). The total MC concentration in the original extract (corre- sponding to 800 g biomass DW L–1) was 414 mg L–1 (MC- LR concentration: 329 mg L–1). TOC concentration in the non-treated extract of 800 g biomass DW L–1 was 83.81 g L–1, which represents 105 mg of extractable total organic carbon per g of biomass DW. Ozonation of cyanobacterial samples had direct effects on the concentrations of organic matter in the biomass. As shown in Tab. 1, the 30-min treatment with ozone caused complete degradation of MCs and 33% reduction of TOC compared to non-treated extract. The chlorination with low dose of chlorine (7 mg L–1) for short time (30 min) had no effect on MC and TOC concentrations. The higher doses (70 to 1000 mg L–1) of chlorine and longer treat- ments (100 min to 24 h) were more effective in reducing MC levels (by 10 to 33%), and this reduction was depend- ent on chlorine concentrations and treatment times. There was no observed shift in the proportion of individual MC congeners with the MC-LR being the dominant variant. TOC degradation by chlorine (by 16 to 25%) correlated well with MC degradation. The increase of the weight ratio of chlorine to original biomass by diluting non- treated extract before chlorination (Tab. 1, variants ¼ or ⅛ Cl(500-100)) slightly improved reduction of MC and TOC amount, when the results were adjusted by the cor- responding dilution factor and then compared to the non- diluted extract chlorinated under the same conditions (Cl(500-100)). The original extract (non-treated, NT) showed no sig- nificant cytotoxic effects on WB-F344 during 30-min ex- posure within the range of concentrations tested (up to 24 g DW L–1; negative data not shown). After longer expo- sure time (24 h), the sample significantly decreased via- bility of WB-F344 cells (Fig. 1A) with the calculated IC50=11.6 g DW L–1 (Tab. 1). The lowest observed effect concentration (LOEC) inducing statistically significant reduction of the cell viability was 12 g DW L–1 (Fig. 1A). Non -co mmerc ial us e o nly I. Sovadinová et al.112 The effects of the NT extract on GJIC in WB-F344 cells after 30-min exposure are shown in Fig. 2A. The sample had pronounced inhibitory effects on GJIC, which were concentration-dependent with the calculated IC50=7.8 g DW L–1 (Tab. 1). LOEC concentration of NT extract for inhibition of intercellular communication was 8 g DW L–1 (containing 4.1 mg L–1 MCs) (Fig. 2A). The tumour promotional activity of the NT sample indicated by its ef- fect on GJIC was also confirmed by additional experi- ments assessing phosphorylation of signalling protein Fig. 1. The effect of chlorination (B-E) and ozonation (F) of the tested cyanobacterial extract on viability in WB-F344 cells after 24-h exposure to 6 different concentrations (4-24 g DW L–1). Data are fractions of controls (FOC) as means ± standard deviations of inde- pendent repetitions of the experiment (n≥3). Significant differences from the vehicle control (A) are indicated by asterisks (one-way ANOVA followed by Dunnet’s post-hoc test; *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001). NT, original non-treated extract; Cl, chlorinated extracts (in parentheses - free Cl concentrations 7, 70, 500 and 1000 mg L–1; treatment durations 30 min, 100 min, 24 h); O3, ozonated extract (30 min, the flow rate of 1 L min–1 with 5 g O3 m–3). Non -co mmerc ial us e o nly Chlorination and ozonation of cyanobacterial extract 113 kinases in WB-F344 cells (MAPK-ERK 1/2 and p38, Fig. 3A). Rapid and clear concentration-dependent activation of both MAPKs was observed after 30-min exposure. The LOEC values for hyperphosphorylation of MAPK- ERK 1/2 and p38 were 8 or 4 g DW L–1 of the NT extract, re- spectively, with maximal effects observed at the highest tested concentration of 16 g DW L–1 (Fig. 3A). Ozonation and chlorination of the original sample had a pronounced effect on the biological activity. The cyto- toxic effect was completely eliminated by the ozonation of the extract (Fig. 1F). At the same time, none of the chlorination experimental protocols had any significant effect on cytotoxicity of the original NT extract (Figs. 1B- E), with the estimated IC50 values ranging between 9.1 and 14.8 g DW L–1 and not being significantly different (P<0.05) from the IC50 value for the NT extract (Tab. 1). Also, the LOEC values for viability of cells exposed to chlorinated extracts remained at 12 g DW L–1. Interest- ingly, slight but non-significant increase of IC50 values was associated with lower rather than higher chlorine doses. The increase of weight ratio of chlorine to the orig- inal biomass by diluting non-treated extract before chlo- rination did not decrease the cytotoxicity with the IC50 values of 10.4 g DW L–1 for ¼ Cl(500-100) and 7.0 g DW L–1 for ⅛ Cl(500-100) (Tab. 1, Supplementary Fig. 2). The corresponding vehicle controls for these chlorination con- ditions caused 20 to 30% significant decrease in WB- F344 cell viability when compared to non-treated control (Supplementary Fig. 2). The ozone application was also highly potent in elim- inating tumour promotional activity of the original NT ex- tract, as shown in Fig. 2F. After 30-min ozonation, the LOEC value for GJIC inhibition was 24 g DW L–1, i.e. three times higher than in NT extract. GJIC was reduced only by 30% at 24 g DW L–1 concentration (Fig. 2F), while nearly 100% inhibition of GJIC was observed at 16 to 24 g DW L–1 of NT extract (Fig. 2A). In contrast, the chlorination treatments had much less pronounced effects on the inhibition of GJIC, with the IC50 values of the chlorinated extracts ranging between 10.0-12.7 g DW L–1. Although these IC50 values were rel- atively similar to the IC50 estimated for NT extract, they were significantly higher except for the lowest free chlo- rine doses and shortest treatment (P<0.05), and their in- crease depended on the chlorine dose- and treatment duration (Tab. 1). When the weight-ratio of chlorine to the original biomass was increased by diluting the non-treated extract before chlorination (Supplementary Fig. 3), there was no observable decrease in the dysregulation of GJIC induced as compared to the original NT extract, with the IC50 values after chlorination being 8.1 to 9.6 g DW L–1 (Tab. 1, Supplementary Fig. 2). Both ozone and chlorine treatments significantly attenuated the activation of MAPK ERK1/2 induced by NT extract (Fig. 3). Ozona- tion was more effective, when no activations of the ERK1/2 kinase were observed at concentrations between 4 to 16 g DW L–1. Chlorination also resulted in reduced levels of MAPK ERK1/2 activation (Fig. 3, Tab. 1). The protective effects had biphasic character, when initially increased but then became less apparent with an increase in chlorine dose and chlorination time (Fig. 3, Tab. 1). The effects of both ozone and chlorine treatments on the acti- vation of p38 MAPK were much less pronounced when compared to MAPK ERK1/2, and activation of p38 was still apparent for the ozonated extract as well as most of the chlorinated extracts even at the lowest experimental concentration of 4 g DW L–1 (Fig. 3). DISCUSSION Adverse effects of toxic cyanobacteria on human health remain a major issue for both researchers and water managers. Our study confirmed that rapid inhibitions of GJIC and activations of MAPK-ERK1/2 might be a com- mon effect induced by bloom-forming cyanobacteria. The effective concentration for 30-min GJIC inhibition (IC50=8 g DW L–1) was similar to previously reported val- ues for other extracts from natural blooms dominated by Microcystis sp. (IC50=4 or 6 g DW L–1, respectively) (Bláha et al., 2010; Novakova et al., 2011). Modulation of these cellular events by chemicals in vitro are consid- ered to be relevant biomarkers of tumour promoting po- tency in vivo, as was demonstrated e.g. for tumour promoting phorbol esters like TPA (Madhukar et al., 1996), organochlorine pesticides (Trosko et al., 1987), PCBs (Kang et al., 1996), low molecular weight PAHs (Bláha et al., 2002), clofibrate, phenobarbital, perfluo- rooctanoic acid, or organic peroxides (Upham et al., 2007; Upham et al., 2009; Vinken et al., 2009). MCs and other cyanotoxins like cylindrospermopsin have been shown to modulate development of tumours (Nishiwaki-Mat- sushima et al., 1992; Falconer and Humpage, 2005; Svircev et al., 2010; Zegura et al., 2011; de la Cruz et al., 2013). However, neither MC-LR nor cylindrospermopsin had any direct effect on rapid inhibition of GJIC or acti- vation of MAPK ERK 1/2 indicating that other metabo- lites in cyanobacteria might be responsible for their GJIC-dependent tumour promoting activity (Bláha et al., 2010; Novakova et al., 2011). However, these metabolites have not been identified yet. In addition, our present study demonstrates, for the first time, a rapid activation of another MAPK-p38 by cyanobacterial environmental extract in the cell line WB- F344, which possesses characteristics of liver progenitor cells (Babica et al., 2016a, b). MAPK-p38 is a critical par- ticipant in cellular stress responses and has a key role in in- flammation, as well as in tissue homeostasis, by controlling cell proliferation, differentiation, death, survival and the Non -co mmerc ial us e o nly I. Sovadinová et al.114 migration of specific cell types (DiDonato et al., 2012). In contrast to MAPK-ERK1/2, which are activated by mito- gens or growth factors, MAPK-p38 is activated by envi- ronmental and genotoxic stresses including hypoxia, UV, ROS, hyperosmolarity, and heat shock, and its activation has been linked to protein phosphatase 2 (PP2A) (Nebreda and Porras, 2000; Wagner and Nebreda, 2009). Indeed, MC-LR, a known PP2A inhibitor, has been previously re- ported to activate MAPKs ERK1/2, p38 or JNK in rodent liver in vivo and also in experiments with various cell lines Fig. 2. The effect of chlorination (B-E and ozonation (F) of the tested cyanobacterial extract on gap-junctional intercellular communi- cation (GJIC) in WB-F344 cells after 30-min exposure to 6 different concentrations (1-24 g DW L–1). Data are fractions of controls (FOC) as means ± standard deviations of independent repetitions of the experiment (n≥3). Significant differences from the NT extract (A) are indicated by asterisks (one-way ANOVA followed by Dunnet’s post hoc test; *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001). NT, original non-treated extract; Cl, chlorinated extracts (in parentheses - free Cl concentrations 7, 70, 500 and 1000 mg L–1; treatment durations 30 min, 100 min, 24 h); O3, ozonated extract (30 min, the flow rate of 1 L min–1 with 5 g O3 m–3). Non -co mmerc ial us e o nly Chlorination and ozonation of cyanobacterial extract 115 in vitro. However, MC-LR effects on MAPKs seem to be dependent on the kinase type, a cell type, and probably also exposure times and concentrations. Depending on the study, MC-LR was found to activate ERK1/2 but not p38 (Dias et al., 2010; Zhang et al., 2013; Adamovsky et al., 2015), p38 but not ERK1/2 (Meng et al., 2011; Lezcano et al., 2012), and both ERK1/2 and p38 (Komatsu et al., 2007; Daily et al., 2010; Sun et al., 2011; Chen et al., 2012; Liu et al., 2016; Wang et al., 2017). The effective concentra- tions of MC-LR in these studies were typically between 1 and 10 µM (~1-10 mg L–1). In the present study, cytotoxicity, GJIC and MAPKs were affected by the cyanobacterial extract diluted to 4- 12 g DW L–1 containing MCs with concentrations be- tween 2 and 6 mg L–1, which corresponds to 2 to 6 µM range and is quite comparable with the other in vitro stud- ies reporting MAPK activation by MCs. However, we previously demonstrated that tumour promoting events, such as rapid GJIC inhibition and MAPK ERK1/2 acti- vation in rat liver progenitor cells, are induced by other cyanobacterial metabolites but not MC-LR or cylindros- permopsin (Bláha et al., 2010). Our results suggest that MAPK p38 can also be activated by transformation or degradation products of MCs, other compounds of cyanobacterial origin and/or their transformation or degra- dation products, since p38 was activated not only by MC- containing non-treated cyanobacterial extract, but also by ozonated extract without detectable levels of MCs. These findings suggest that progenitor cells, in comparison with differentiated hepatocytes, might be less prone to the ef- fects of MCs, possibly due to limited expression of key proteins involved in MCs uptake and metabolism, such as organic-anion transporting polypeptides (OATPs). Nev- ertheless, progenitor cells can be apparently a target of other compounds present in cyanobacterial biomass, which are capable to induce toxic and tumour promoting effects in this specific population of liver cells (Bláha et al., 2010) known to play a critical role in the maintenance of liver tissue homeostasis, liver regeneration and hepa- tocarcinogenesis (Canovas-Jorda et al., 2014). The evidence supporting the existence of other com- ponents of cyanobacterial biomass contributing to the tu- mour promoting and toxic effects of complex cyanobacterial samples emphasize the need for effect- based evaluation of the efficacy of water treatment tech- nologies in addition to chemical analyses. Different physicochemical purification processes employed in DWTPs may have different efficacies in removing the tar- get contaminant, such as well recognized toxicants (such as MCs) vs. elimination of the overall toxicity. Our results demonstrate that ozone effectively and rapidly removes the MC fraction of the complex cyanobacterial samples. Although the highest chlorine dose resulted in a decrease of TOC that was comparable to the ozone treatment, re- active chlorine was not as effective compared to ozone in removal of MCs. Similarly, removal of cytotoxicity and overall epigenetic toxicity of the studied sample by ozona- tion appeared to be much more effective than chlorination, although ozone had a less pronounced effect in decreasing p38 activation as compared to ERK1/2 and GJIC. With regard to chlorine the literature demonstrate that its application removes MCs but the efficiency of removal decreases with increases in pH and dissolved organic material along with formation of less effective oxidant ClO– (Merel et al., 2010). Several studies agreed that 0.5 mg of residual chlorine per liter should effi- ciently remove pure MCs in distilled water during 5-30 min (depending on MC concentration) at pH lower than 8 (Nicholson et al., 1994; Newcombe and Nicholson, 2004; Acero et al., 2005). Similar scenarios are also ex- pected at DWTP at environmentally relevant concentra- tions of MCs (Merel et al., 2009). Several previous studies using PP-inhibition as a bio- marker of toxicity as well as other bioassays reported a de- crease in MC concentration and toxicity after chlorination of cyanobacterial samples (Nicholson et al., 1994; Tsuji et al., 1997; Rodriguez et al., 2008; Merel et al., 2010). For example, the chlorination of M. aeruginosa extract (16 mg DW L–1) with the dose of 1 mg L–1 chlorine and contact time of 30 min (CT value=30) effectively removed 95% of MCs (initial concentration: 192 µg L–1) and completely eliminated the acute toxicity of this extract in a mouse bioassay (Nicholson et al., 1994). Interestingly, our study showed that chlorination of cyanobacterial extract was less effective. The dose of 7 mg of chlorine per liter for 30 min at pH 7.2 did not decrease MCs nor TOC concentrations. Higher doses and longer treatment times removed up to 10- 30% of the original MCs and TOC levels, and caused only moderate reduction of toxicity (cytotoxicity, GJIC inhibi- tion and MAPK activations). Chlorination apparently re- moved some compounds responsible for the inhibition of GJIC and activation of ERK1/2 as reflected by the slight, yet statistically significant, increase in respective IC50 or LOEC values, and had only a minor effect on cytotoxicity and p38 activation. The lower effectiveness of chlorination observed in our study could be explained by the interactions of chlorine with the relatively higher concentrations of or- ganic matter, which might have reduced the effectiveness of the oxidation process due to the competition between the toxins and the dissolved organic carbon reacting with the oxidant (Rodriguez et al., 2008). An important problem associated with the chlorine ap- plication is the formation of by-products such as halo- genated organic compounds, especially in the presence of high amounts of organic matter. These by-products can have toxic or potential carcinogenic potencies (Neale et al., 2012); and also for MCs, chlorine was shown to cause substitutions and modifications of the toxic Adda moiety Non -co mmerc ial us e o nly I. Sovadinová et al.116 Fig. 3. Activation of mitogen-activated protein kinases (MAPKs) by studied samples after 30-min exposure to 3 different concentrations (4-16 g DW L–1). Phosphorylation of extracellular receptor kinases 1 and 2 (ERK1/2) and p38 was determined by Western blotting (A). The bar graphs (B) show values from the densitometric image analysis normalized to negative control (NC=1). NT, original non-treated extract; Cl, chlorinated extracts (in parentheses - free Cl concentrations 7, 70, 500 and 1000 mg L–1; treatment durations 30 min, 100 min, 24 h), O3 – ozonated extract (30 min, the flow rate of 1 L min–1 with 5 g O3 m–3); NC, negative control (no treatment of the cells); PC, positive control for ERK1/2 activation (12-O-tetradecanoyl phorbol-13-acetate, 10 nM, 30 min). Non -co mmerc ial us e o nly Chlorination and ozonation of cyanobacterial extract 117 (Tsuji et al., 1997; Merel et al., 2009). In addition, de novo formation of chlorinated by-products with potencies to af- fect GJIC and activate intracellular signalling (Hakulinen et al., 2004; Nishikawa et al., 2006) should also be con- sidered, and could be related to the weak efficiency in re- moval of GJIC inhibitions and MAPK activations during the chlorination as observed in the present study. Possible toxicity of chlorinated by-products could also explain the observed biphasic effect, when lower doses of chlorine were slightly more effective in the elimination of cytotox- icity and MAPKs activation than the higher doses, al- though concentrations of TOC and MCs were slightly but progressively reduced with increasing chlorine dose and treatment time. We demonstrated that ozonation completely removed MCs, substantial fractions of TOC and protected against cytotoxicity, GJIC inhibition or activation of ERK1/2. These findings are in agreement with similar studies, which documented complete MC removal (5 mg L–1) by 2 mg L–1 O3 within 2 min (Al Momani and Jarrah, 2010). Further improvements in kinetics could be achieved by increased O3 doses and temperature, and decreased pH (Al Momani and Jarrah, 2010; Shawwa and Smith, 2001). Naturally, organic matter negatively reduces the efficiency of ozonation, but under realistic DWTP situations of lev- els as low as 0.05 mg L–1 of residual O3 assures MC re- moval (Newcombe and Nicholson, 2004; Brooke et al., 2006). Despite a high amount of organic material in our sample that also competes with toxins for ozone, ozona- tion was highly effective in MC reduction and elimination of toxicity even after short treatment. A high efficiency of oxidation of MC is known to be mediated by hydroxyl radicals attacking conjugated diene structure in MC followed by the cleavage of the Adda side chain (responsible for PPase inhibition) and ulti- mately opening of the peptide ring (Al Momani and Jar- rah, 2010; Miao et al., 2010). Biological assessments using PP-inhibition assay or mouse test confirmed elim- ination of the toxicity along with the described structural changes of MC (Brooke et al., 2006; Miao et al., 2010). Although ozone was quite efficient in removal of cyto- toxic, GJIC inhibiting and ERK1/2 activating compounds in our study, it had only a partial effect on the removal of p38 activating components. This might indicate that p38 is not involved in GJIC inhibition and its activation was caused by metabolites with different modes of actions (Wagner and Nebreda, 2009). With respect to the critical role of p38 in cellular responses to different types of stress and also in controlling the proliferation, differen- tiation, survival, migration and inflammatory responses of specific cell types, further research should address in- teractions of cyanobacterial metabolites with this sig- nalling pathway and evaluate its relevance as a biomarker of environmental and genotoxic stress induced by cyanobacteria. Interestingly, activation of p38 was found to be the most sensitive endpoint in this study, where the increased levels of p38 phosphorylation were observed after 30-min exposure to the non-treated extract at con- centration of 4 g DW L–1, whereas significant inhibition of GJIC and activation of ERK1/2 occurred at concen- trations 8 g DW L–1 and higher, and significant reduction of cell viability required 24-h exposure to 12 g DW L–1. Inhibition of GJIC and activation of MAPKs was induced by lower concentrations and after shorter exposures than the cytotoxic effects, which indicates that these cell sig- nalling events were altered via rapid non-genotoxic and non-cytotoxic mechanisms. In vitro evaluation of GJIC and MAPKs thus represent a simple and sensitive bioas- say for assessment of ‘epigenetic toxicity’ and tumour promoting potential of complex cyanobacterial extracts, which is also suitable for effect-based studies focusing on the elimination of these hazardous properties of con- taminated water. CONCLUSIONS Ozonation of an extract of a Microcystis water bloom sample was shown to be a very effective method in the complete removal of MCs, as well as the substantial elim- ination of the overall cytotoxicity and tumour promotional potency. On the contrary, chlorination experiments, de- spite high doses and long exposures, were much less ef- fective, and potentially led to the formation of by-products, which could add to the observed toxic ef- fects. Our study also demonstrated strong activations of p38 MAPK by cyanobacterial samples, which were not effectively removed by chlorination and only partially by ozonation. With respect to the role of p38 in inflammation as well as maintenance of tissue homeostasis, further re- search should address interactions of cyanobacterial sam- ples with this biomarker of cellular stress and evaluate its environmental relevance. In agreement with several re- cent reports, the study also demonstrates the need to en- force effect-based (bioassay) tools into the assessment of water quality and monitoring the efficacy of water treat- ment systems. ACKNOWLEDGMENTS Supported by the Czech Science Foundation project GA15-12408S; the Czech Republic Ministry of Educa- tion, Youth and Sports infrastructure projects No. LO1214 and LM2015051; long-term research development project RVO 67985939; and by National Institute of Environmen- tal Health Sciences (NIEHS) grant #R01 ES013268-01A2 to Upham. We would like to thank Dr. Ondřej Mikeš for the help with TOC analysis. Non -co mmerc ial us e o nly I. Sovadinová et al.118 REFERENCES Acero JL, Rodriguez E, Meriluoto J, 2005. Kinetics of reactions between chlorine and the cyanobacterial toxins micro- cystins. Water Res. 39:1628-1638. Adamovsky O, Moosova Z, Pekarova M, Basu A, Babica P, Svi- halkova Sindlerova L, Kubala L, Blaha L, 2015. Immunomod- ulatory potency of microcystin, an important water-polluting cyanobacterial toxin. Environ. Sci. Technol. 49:12457-12464. Al Momani FA, Jarrah N, 2010. Treatment and kinetic study of cyanobacterial toxin by ozone. J. Environ. Sci. Health A Tox. 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