Quercetin and Hydroxytyrosol As Modulators of Hepatic

Home , Hydroxytyrosol, Quercetin

Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. aul Gori Manuele a steatosis: hepatic study. of NAFLD-on-a-chip modulators as Hydroxytyrosol and Quercetin pce RS n ii eoiain r w ftemi ehnssivle nteeilg fliver El-Kader (Abd of progression etiology and the onset in the involved in mechanisms role oxygen crucial main reactive a the of lipid play of generation Intrahepatic they two of which 2004). are in terms al., NAFLD, in peroxidation, including et damaging stress, lipid Feldstein diseases, the oxidative and through 2015; and free disease (ROS) Ashmawy, content) the as The El-Den species TGs of such & development available. excessive metabolites, the El-Kader being (i.e., lipid in from (Abd accumulation other role far but lipotoxicity a still play (TGs), of may are triglycerides NAFLD effect cholesterol, as therapeutics of and stored effective (FFAs) pathogenesis acids are highly multifactorial fatty lipids and the hepatocellular understood, However, 2015; of fully Ashmawy, majority 2010). been El-Den liver Harrison, yet & end-stage & El-Kader not and (Abd has Calcagno, fibrosis (HCC) Starley, to carcinoma 2002; fatty progress hepatocellular enhanced Angulo, may and which benign inflam- cirrhosis by (ballooning), (a including characterized damage steatosis (NASH), disease, hepatocellular steatohepatitis simple nonalcoholic and from severe fibrosis more ranging NAFLD the mation, pathologies to 2015). liver) liver Ashmawy, the occurs El-Den of within which & infiltration spectrum worldwide, El-Kader disease broad (Abd liver consumption a chronic alcohol encompasses common significant most deny who the individuals is in (NAFLD) disease liver fatty Nonalcoholic the contrast Introduction to strategies novel guide their seem could to polyphenols work due both present effect the that cytoprotective no of observed a Besides, NAFLD. showing insights we of steatosis, resulting clear. experiments, progression hepatocellular the being and our acid-induced conclusion, onset from In fatty far In free is far. the properties. pathogenesis so of antioxidant complex namely NAFLD progression whose polyphenols, the for disease, restrain NAFLD occurring established liver to analysis. naturally been chronic high-content culture two of has of 2D of form method treatment conventional a effects common definitive of therapeu- using most the accuracy (NAFLD) potential the investigate predictive disease the currently liver to limited test fatty is model the non-alcoholic and overtaking liver-on-a-chip on diseases Hydroxytyrosol, limitations, a and human their present Quercetin model as we well to Here, as opportunities ini- drugs precision systems. new 3Rs different unparalleled the offer at of with pathologically platforms effect line and these in tic physiologically behave models, Indeed, cells animal which to complexity. in alternative microenvironments and valuable tissue and 3D promising create a to as tiative, on catching increasingly are Organs-on-chip Abstract 2020 5, May 5 4 3 2 1 Nicolò Merendino nvri` el td el Tuscia della Studi Università Anne” degli “St. Hospital University Tre Roma Studi Ingegneria Università Facoltà degli di Roma di Bio-Medico Roma Università Campus di Bio-Medico Campus Universita 1 aaGiannitelli Sara , 5 n let Rainer Alberto and , 2 nraZancla Andrea , 1 1 3 aeaMozetic Pamela , 4 aclaTrombetta Marcella , 1 , Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. h emtyo h irflii eiewsdsge sn A ut Lyu dtr upro G Un- Midland, UG, Corning, Juspertor Dow Editor, 184, (Layout Sylgard suite (PDMS, CAD polydimethylsiloxane a in using fabricated designed was and Germany) device terhaching, microfluidic the of geometry The was (ENZ-51010) Kit USA). NY, Detection Thermo (Farmingdale, Microfabrication from ROS/Superoxide Inc. were purchased Total Sciences, (Que) were Life ROS-ID Enzo (EthD-1) Quercetin FBS, USA); from homodimer-1 charcoal-stripped and MA, purchased ethidium Gibco), (Waltham, (HT) and (FBS, Scientific Hydroxytyrosol dye Serum Fisher nuclear V, Bovine C18:1 fluorescent Fetal (OA; fraction 33342 Germany); acid Cohn Hoechst oleic (Darmstadt, C16:0), (BSA) Merck (PA; L-glutamine, from acid albumin palmitic purchased Italy); serum M7145, (MI, solution bovine acid Rutherford, Euroclone (East amino cis-9), Lonza non-essential from from MEM purchased purchased USA); were Reagent NJ, was Assay AdipoRed (DMEM) and Medium mixture, penicillin-streptomycin Eagle’s Modified Dulbecco’s Chemicals aforementioned two development methods the the and of Materials on treatment. Finally, effects Hydroxytyrosol, level. therapeutic protective and potential cell the NAFLD, Quercetin as the study NAFLD namely of at of to polyphenols, steatosis pathogenesis plant-derived exploited liver the of occurring been investigate sinusoid parameters naturally has to liver different model us a of allowed NAFLD-on-a-chip (HCA) of microenvironment, the analysis interface dynamic culture high-content under endothelial-parenchymal cell the device, the 3D through microfluidic culture mimicking liver-on-a-chip optimized (3D) by a an three-dimensional within platform, in advanced cells) This HepG2 an conditions. perfused disclose culture (using et we Tan NAFLD 2019). here 2019; of Meer, approach/technology, al., model der et in this van Peel also leveraging & 2015; discovery, Passier, Thus, Khetani, Mummery, drug Berg, & for den Ballinger, van screening (Lin, 2019; systems high-throughput al., imaging to high-content microfluidic adapted with a being combination in canonical steatosis the are hepatic of organs-on-chips study of limitations Furthermore, models to and complex chip bottlenecks more transparent the and in overcome alternative network to proposed 2017; “Organ chip have al., 2019; 2018; 2018) et group al., Sung, al., Chang our et Recently, & 2018; et n.d.). Lee al., (Liu Lu et spread,” diseases 2018; (Bovard dynamic cells Taylor, human level cancer represent organ & studying how and platforms Gough, and tissue Vernetti, simulating these regard, at George, this NAFLD, for changer: Li, including In promise game 2019), 2009). great al., real al., offer et the Rothbauer et that Ricchi be systems 2014; can as culture al., technology cell tissue; et organ-on-chip (Chavez-Tapia, hepatic culture Gori NAFLD emerging cell of as 2007a; Gómez-Lechón complex 2D the microenvironment al., 2012; as on disease physiological et Tiribelli, multifactorial rely 3D a & they of Rosso, the chronicity as the reproduce recapitulate NAFLD, cannot accurately of they such, not pathogenesis the do investigating that thus monolayers and morphology liver 2013). traditional entire by al., the et caused findings, (Vidyashankar production these by cells Despite cytokine symptoms HepG2 NAFLD inflammatory in known contrast steatosis and with and Priore hepatic stress and attenuate acid-induced 2012; oxidative origin to oleic al., accumulation, dietary mouse shown et TG from using been (Hur intrahepatic compound synthesis, has flavonoid hepatocytes reducing lipid in activities) polyphenolic rat hepatic present hypolipidemic (a primary-cultured and and these polyphenols Quercetin as antioxidant lipogenesis Similarly, main of well on 2014). Two (the as oil) al., HT lines olive 2013). et metabolite cell virgin Patki, hepatoblastoma its extra & human and and Varma, et and Oleuropein leaves (Hur Sandeep of olive lipogenesis Vidyashankar, olives, effect intrahepatic 2014; green of beneficial Gnoni, reduction a used & the observed Hydroxytyrosol which in Siculella, namely reports studies, and polyphenols, Priore, NAFLD recent natural 2012; of two some development of al., Interestingly, the effect against protective 2002). Quercetin, the showed Ishii, and steatosis, & hepatic Adachi of models 2015; Ashmawy, El-Den & nvitro in oeso eai taoi r o utbefrrcetn the recreating for suitable not are steatosis hepatic of models 4 n tes(uuol ta. 09 hlc ta. 2018; al., et Ehrlich 2019; al., et (Bulutoglu others and 2 nvitro in elcluemodels. culture cell nvitro in Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. fe eltetet o 8,ciswr isdwt B n nuae tR o hwt blue-fluorescent with 1h for RT at incubated and PBS (5 with μ rinsed 33342 were Hoechst chips dye 48h, nuclear for treatments cell After viability/cytotoxicity cell of control scanning the Analysis laser to compared a increase automated using fold with measured as Japan) plotted Tokyo, was only). 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FFAs treatments described DMEM without 100 above of medium the penicillin, composed at steatosis FFAs IU/mL medium mM by 100 steatosis 1 method and in L-glutamine, FBS, incubated described mM charcoal-stripped were 10% previously 2 cells with the HepG2 of Briefly, Campos, supplemented components modifying & 2016). and glucose) 48 by Siles, 2014, diets for Kabagambe, induced al., western 2004) (Baylin, in et was NAFLD al., FFAs (Gori with abundant Steatosis et patients most (Feldstein either 2007b). and the Gómez-Lechón 0.2-2mM) medium, subjects al., are (i.e. 2002; normal the concentration, they et plasma both final as to human in 1mM chosen added triglycerides in at were liver FFAs was OA respectively), FFAs of and 2:1, PA two and concentration (h). 1:2 range the hours (i.e., the of ratios in Each molar is different (vehicle). which at methanol combination in in or dissolved alone was OA and PA steatosis. medium following. of the steatosis Induction in fresh in described with (o.n.) as replaced overnight treatments and different cultured treatments the discarded were Cell to was cells negligible according medium with loading, polyphenols day, chip products, and next After waste FFAs containing the barrier. metabolic to medium; chip microchannel of differential removal the culture the height of the standard to a microarchitecture and thanks sinusoid-like applying nutrients stress The by of reservoirs. shear obtained diffusion inlet/outlet continuous perfusion the the microfluidic in allowed under contained fashion, medium culture 3D the in chip, on cultured mM 2 cells/mL with 37 loading. supplemented humidified Chip glucose a low in incubated DMEM and in FBS, expanded 10% 100 were penicillin, U/mL Cells 100 (ATCC). L-glutamine, Collection Culture Type culture. Cell loading chip and culture endothelial-like the Cell and channels transport soft-lithographic two-layer the same with the together respectively. through microchamber 2016), culture barrier, cell al., the et pattern (Gori to model process published previously the on based MI), nPS hti eetv once fda el.Tenme fdead of number The cells. dead of nuclei to selective is that PBS) in M 2013) al., et Vidyashankar 2002; Wolfram, & Linseisen, Radtke, 2012; al., et Hur 2015; al., (Catalán et M via h eta ellaigcanla rvosydsrbd(oie l,21) hratr el were cells Thereafter, 2016). al., et (Gori described previously as channel loading cell central the h ua eaoacl ieHp2CA(R-04)wsprhsdfo h American the from purchased was (CRL-10741) HepG2/C3A line cell hepatoma human The eG el eedtce tsboflec,adlae nocisa est f1.0 of density a at chips into loaded and subconfluence, at detached were cells HepG2 via ofclmcocp-ae ihcnetsreig(C) sn fluorescence-based using (HCS), screening high-content microscopy-based confocal o h nuto fhptcsetss obnto fln-hi Fs namely FFAs, long-chain of combination a steatosis, hepatic of induction the For μ /Li B)ta tisalnce,adterdfloecn y tD1(4 EthD-1 dye red-fluorescent the and nuclei, all stains that PBS) in g/mL μ U n X eebt sda hsooia ocnrto f10 of concentration physiological a at used both were HXT and QUE /Lsrpoyi,MMnnesnilaioai ouin(100x), solution acid amino non-essential MEM streptomycin, g/mL ° topeecnann %CO 5% containing atmosphere C 3 vs. μ /Lsrpoyi,1 BSA, 1% streptomycin, g/mL 2 oa el aigi ahcell each in laying cells total . × 10 6 Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. 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Therefore, with promote of radicals. along to treatment cells, free seems the hepatic accumulation, scavenge of fat defenses to antioxidant excessive oxidative the of able Hydroxytyrosol and boosting are and and lipotoxicity generation Quercetin they activity), ROS/Superoxide of lipid-lowering which effect their in comparable (confirming and stress, steatosis protective hepatic despite a improved. 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Fki, Pérez-Jiménez, Lopez-Segura, in Bouaziz, M.-I. al., Perez-Martinez, 2011; Jemai, Que, 2003; et Ruano, 2008; al., al., al., Priore et et et Park 2007; Fki, Carluccio 2008; Jemai, Miranda, 2012; 2007; Sayadi, al., al., & et Feki, et Hur El 2001; (Bendini Almeida, ability & De lipid-lowering oleuropein-derivative Paiva-Martins, anti-inflammatory Gordon, the & their and 2008; anti-oxidant as for Scoditti, known (such also their Massaro, components for but (Carluccio, phenolic only properties, not acid the However, emerging, by oleic increasingly 2010). played been of Giugliano, role has content extra Hydroxytyrosol) important & of high the health Esposito its also human Fitó, 2009; recently, in to & more effect et ascribed Konstantinidou, beneficial Mar´ıa-Isabel (Marcolin Covas, the long activity 2007; Instead, been hypolipidemic Caterina, 2013). has al., and oil et anti-inflammatory Vidyashankar olive polyphenols antioxidant, 2000; virgin effect al., its natural hepatoprotective et to two and Peres related attributed 2012; potential of its al., far therapeutic concentrations and its thus accumulation dietary fat Quercetin, and been intrahepatic of Regarding steatosis, has the effects particular stress. hepatic in oxidative its protective of disease, and to the the condition lipotoxicity of closely the level features more important model cell as some pathophysiology to the against well technology liver at disease-on-a-chip as imitate functionality investigate such some to models to on recapitulating us culture leveraged and allowing cells we cell the tissue, Hence, 2D of hepatic this microenvironment conventional revolutionize the 3D may of natural of platforms the Liver-on-a-chip inability reproducing responses. the by human scenario by predict accurately hindered to experiments currently animal is discovery Drug Discussion compared stress oxidative against Quercetin 4] of [Figure concentrations. significance, chosen statistical the of Que at terms Hydroxytyrosol both in by to compounds p effect, lowered with stronger significantly polyphenolic e was slightly both and was production a 4d of Superoxide downregulation (Fig. effect and the HT ROS/RNS powerful which and the most in treatment, the 2:1 species PA:OA particular, the Superoxide the polyphenols in In and c both ROS/RNS (p and for PA-induced 1Sc-f). significant b statistically analysis the 4a, change Fig. against (Fig. log2-fold in observed alone the FFAs F.C. was the by (log2 (Fig. to proved Hydroxytyrosol the compared also and micrographs) in micrographs) as confocal confocal micrographs), representative representative the the confocal in in representative f i and and e h 4d, 4g, (Fig. Quercetin of addition swl as well as nvivo in nvitro in < .1i i.4 n o u,adp and Que, for e and 4d Fig. in 0.01 aafo h ieaue(u ta. 02 Ie l,21;Przie l,2016; al., et Pirozzi 2013; al., et LI 2012; al., et (Hur literature the from data < eut nteeet fQeadH ntefaeoko AL confirmed NAFLD of framework the in HT and Que of effects the on results .0 o u;Fg gadhwt p with h and 4g Fig. Que; for 0.001 nvivo in iuto.I at u irflii oe fNFD providing NAFLD, of model microfluidic our fact, In situation. 6 < .5i i.4 n o T.I diin in addition, In HT). for h and 4g Fig. in 0.05 < .1frH,rsetvl) highlighting respectively), HT, for 0.01 nvivo in counterpart. Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. b lKdr .M,&E-e smw,E .S 21) o-looi at ie ies:Tediagnosis The disease: liver fatty Non-alcoholic (2015). S. M. E. Ashmawy, El-Den management. & and M., S. El-Kader, Abd per # Italiana grant Fondazione Lazio, - FIRE Regione References Onlus. from by Fellowship – (co-funded Research Epatologia project a in by Ricerca INTESE Transfer Technology la supported of was Roma: framework MG di the Bio-Medico FILAS-RU-2014-1193). a in Campus into “ITHACa”) Universita organs from (009-2017 even support Grant financial or the tissues acknowledges different AR challenges connect disease. the to and face development future, human to next of tool the Acknowledgements study in promising the goal, a for final system providing the model the research, pave complex with drug also screening of may drug advancement approach IL-8, of technological NAFLD-on-a-chip IL-6, our the (e.g., conclusion, to cytokines will In fibrogenic way and that microenvironment. inflammatory cells) microarchitecture hepatic of stellate levels multicellular liver hepatic expression and complex the iPSC-derived cells of more Kupffer or analysis IL-1 cells, a hepatocytes the endothelial human also of (e.g., enable primary use cells to (i.e., liver parenchymal the non-parenchymal other and ii) including hepatocytes) compounds; types, two cell different the involve of effect synergistic uuol,B,RyBdo,C,Kn,Y .(baa) et . ams,M . sa .B (2019). and B. progression O. disease Usta, to & applications L., disease: liver M. 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(5), oiooyin Toxicology 2016 reRadical Free ora of Journal , 33 1–12. , , (5), 51 Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. iue1.Lg-odcag lg .. nlsso nrclua ii cuuainadoiaiestress oxidative and accumulation lipid intracellular of analysis F.C.) (log2 change Log2-fold 1S. Figure (Hydroxytyrosol). itgassoigMIvle xrse si a ) irgah n(,fadi r aiu intensity maximum are i) and mean f with as (c, graph reported each in in are Micrographs bars) Values (white b). p alone (a, Z-axis. 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HT (Quercetin); Que (Control); a sda oiiecnrl h ffc fQectn(,e /Qe e as n yrxtrsl(,h, (g, Hydroxytyrosol and bars) red Que, w/ e, (d, Quercetin of effect The control. positive a as used was M E;n=3a es;*p * least; at 3 = n SEM; vs. < .0.Saebr n(,fadj:50 j): and f (c, in bars Scale 0.001. oa uli(ocs 35 y nbu) h ffc fQectn( nrd n yrxtrsl( in (e Hydroxytyrosol and red) in (c Quercetin of effect The blue). in dye 33258 (Hoechst nuclei total irflii eiecaatrzto.()Shmtcsoigtegoer fteliver-on-a-chip the of geometry the showing Schematic (a) characterization. device Microfluidic μ < m. .5 *p ** 0.05, < .1 * p *** 0.01, < .5 *p ** 0.05, < < .0,** p **** 0.001, .1 cl as 50 bars: Scale 0.01. μ naliae.Lgn:Cr Cnrl;Qe(urei) HT (Quercetin); Que (Control); Ctrl Legend: images. all in m 11 < μ o 8,t h aecmiain fFA shown FFAs of combinations same the to 48h, for M .01 cl as 50 bars: Scale 0.0001. μ o 8.Rpeettv ofclmcorpsof micrographs confocal Representative 48h. for M ± μ naliae.Lgn:Cr Cnrl;Que (Control); Ctrl Legend: images. all in m E;n=4a es;*p * least; at 4 = n SEM; μ naliae.Lgn:Ctrl Legend: images. all in m < .5 *p ** 0.05, ± E;n= n SEM; μ < for M 0.01, Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. epciey u oQe( n )adH dadf diitaini eotd l ausaerpre as reported are values f, All and normalized p e reported. alone * in is ANOVA, FFAs species one-way administration the by Superoxide and f) evaluated and HT and d, or (d and Que HT w/ and c in- e) in in and (ROS/RNS (c decrease stress mean Que oxidative fold to in The due decrease respectively) fold mixtures. the FFA different reported; is the ( to accumulation triglyceride addition tracellular Hydroxytyrosol and Quercetin following u Qectn;H (Hydroxytyrosol). HT (Quercetin); Que ± E,n=3a es,adpotda h o2o h ai ewe h ieetmxue fFFAs of mixtures different the between ratio the of log2 the as plotted and least, at 3 = n SEM, a < .5 *p ** 0.05, via h dpRdasy u oQe()adH b administration (b) HT and (a) Que to due assay) AdipoRed the < vs. b .1 * p *** 0.01, ln oto ie,n ffc) ttsia infiac was significance statistical effect); no (i.e., control blank 12 < .0,** p **** 0.001, microchannel microchannel barrier < .01 eed tl(Control); Ctrl Legend: 0.0001. cell inlet perfusion channel perfusion Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. c a e

HT AdipoRed Que AdipoRed (MFI fold increase) (MFI fold increase) AdipoRed (MFI fold increase) 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Ctrl Ctrl Ctrl * OA OA ** OA *

PA PA PA PA:OA 1:2 PA:OA 1:2 PA:OA 1:2

PA:OA 2:1 PA:OA 2:1 PA:OA 2:1 w/ HT w/ w/o HT w/o w/ Que w/ w/o Que w/o 13 d b f

Ctrl Ctrl Ctrl

HT Que OA

OA + HT OA + Que PA PA + HT PA + Que PA:OA 1:2 PA:OA 1:2 + HT PA:OA 1:2 + Que PA:OA 2:1 PA:OA 2:1 + HT PA:OA 2:1 + Que Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary.

HT Viability (%) e Que Viability (%) c Cell Viability (%) a 100 120 100 120 100 120 20 40 60 80 20 40 60 80 20 40 60 80 0 0 0

Ctrl Ctrl Ctrl *

OA OA OA **** **

PA PA PA ** PA:OA 1:2 PA:OA 1:2 PA:OA 1:2 ** ** PA:OA 2:1 PA:OA 2:1 PA:OA 2:1 w/ HT w/ HT w/o w/ Que w/ Que w/o 14 d b f

HT Que Ctrl

OA + HT OA + Que OA

PA + HT PA + Que PA

PA:OA 1:2 + Que PA:OA 1:2 + HT PA:OA 1:2

PA:OA 2:1 + Que PA:OA 2:1 + HT PA:OA 2:1 Posted on Authorea 28 Jan 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158023274.43192215 — This a preprint and has not been peer reviewed. Data may be preliminary. g d a ROS/RNS MFI fold increase MFI fold increase (MFI Fold Increase) 2 3 4 5 6 7 8 2 3 4 5 6 7 8 0 1 0 1 10 0 2 4 6 8 Ctrl Ctrl Ctrl Pyocyanin **

OA OA ROS/RNS * ROS/RNS

OA ROS/RNS PA PA ** * PA:OA1:2 PA:OA 1:2 *

PA PA:OA 1:2

PA:OA 2:1 PA:OA 2:1 w/ Que w/ w/o Que w/o w/ HT w/ w/o HT HT w/o PA:OA 2:1 *** ** e b h Superox MFI fold increase MFI fold increase (MFI Fold Increase) 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0 1 2 3 4

Ctrl Ctrl Ctrl Pyocyanin *

OA OA ** Superox Superox

OA Superox 15 PA PA ** * PA:OA 1:2 PA:OA 1:2 PA PA:OA 1:2

PA:OA 2:1 PA:OA 2:1 PA:OA 2:1 w/o HT w/o w/ HT w/ w/ Que w/ w/o Que w/o *** ** f c i

HT Que Ctrl

OA + HT OA + Que Pyocyanin

PA + HT PA + Que OA

PA:OA 1:2 + HT PA:OA 1:2 + Que PA

PA:OA 2:1 + HT PA:OA 2:1 + Que PA:OA 1:2

PA:OA 2:1