SummarySinglet oxygen (1O

2)-catalyzed oxidation of water leads to a variety

of oxidants (e.g. ozone, O3) in host–parasite relationships as well. It

can be detected indirectly in TLC or OPLC zones by use of the sim-

ple BioArena system and O3-eliminating molecules (e.g. d-limonene

and indigo carmine) in the culture medium. It follows from these

new findings that not only formaldehyde but also O3

and related

bioreactive compounds may play a crucial role in the mechanism of

antibacterial activity of antibiotic-like compounds. The toxic poten-

tial of a molecule, however, originates from the ratio of the oxidants

produced in the chromatographic spots.

1 Introduction

The current central dogma is that antibodies, classical flexibleadaptor molecules of the immune system, do not have destruc-tive properties but mark foreign antigens (e.g. pathogens) forremoval by complement cascade and/or phagocytosis [1].

Recently, however, it has been established that all immunoglob-ulins (Ig), regardless of source or antigenic specificity, can cat-alyze the reaction between singlet oxygen (1O2) and water, lead-ing to the formation of several oxidants, for example dihydrogentrioxide (H2O3), from which O3 and hydrogen peroxide (H2O2)can be formed [2–5]. These oxidants are highly bioreactive andcytotoxic compounds, resulting in the direct destruction of for-eign antigens (e.g. pathogens). This is a newly discovered prop-erty of antibody molecules and it suggests a previously unex-plored effector function of the immune system. These findingsalso suggest that production of O3, rather than H2O2, might havepotential pathophysiological effects (e.g. atherosclerosis) in thisantibody-catalyzed water-oxidation pathway [6].

The authors of some papers, however, have concluded that evi-dence of O3 production, e.g. by neutrophils, is not convincing

and that studies using the same probes to implicate O3 in otherbiological processes should also be interpreted with caution[7, 8].

Because it follows from these results that endogenous formationof O3 in the biological world may open a totally new horizon,e.g. in the topics of disease resistance and cell proliferation, spe-cial efforts are needed to determine, by use of efficient analyti-cal methods, where O3 is generated endogenously from differentbiochemical reactions in diverse biological systems (human,animal, plant, and microbial).

Because it is supposed that this very reactive molecule can alsobe formed in thin-layer chromatographic spots after inoculationwith microbes, it follows from our preliminary results [9, 10]that BioArena, which is, in practice, a complex bioautographicsystem [11, 12], may be suitable for indirect detection of O3molecules.

2 Chemical and Biochemical Backgroundof the Experiments

O3 is a gas in the stratosphere that protects biological organismson Earth from the most harmful wavelengths of solar radiation.This exogenous O3 also is a significant pollutant near the Earth’ssurface (in the troposphere), where it damages lung function andinitiates autooxidation of unsaturated fatty acids, leading tomembrane destruction and severe ailments [13, 14]. ExogenousO3 is not always toxic, however, and can be used as a drug [13,14].

Recent discoveries show that antibodies, human neutrophils,and inflammatory lesions catalyze the formation of O3 in vivo,that is, there is endogenous O3 in the human organism [2–5].By use of the efficient O3 scavengers indigo carmine andd-limonene the bean variety ‘Inka’ resistant to bean rust(Uromyces phaseoli) was converted into a sensitive variety. Itfollows from this unique observation that O3 is also a funda-mental resistance factor in plants [15, 16]. From investigationswith BioArena, in which microbial detection is used, it followsthat the microbes may also generate O3 molecules [9, 10].

E. Tyihák and P.G. Ott, Plant Protection Institute, Hungarian Academy ofSciences, Herman Ottó Str. 15, P.O.B. 102, H-1525, Budapest, Hungary; and Á.M.Móricz, Department of Chemical Technology and Environmental Chemistry,Lóránd Eötvös University, H-1518, Budapest 112, Hungary.E-mail: etyih@nki.hu

Use of the BioArena System for Indirect Detectionof Endogenous Ozone in Spots after TLCor OPLC Separation

Ernô Tyihák*, Ágnes M. Móricz, and Péter G. Ott

Key Words

Aflatoxin B1BioArenad-LimoneneEndogenous ozoneFormaldehydeIndigo carminetrans-Resveratrol

Journal of Planar Chromatography 21 (2008) 2, 77–82 DOI: 10.1556/JPC.21.2008.2.1 770933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

Presented at the7th Balaton Symposium,September 5–7, 2007,

Siófok, Hungary

Original Research Papers

Indirect Detection of Ozone with the BioArena System

78 Journal of Planar Chromatography 21 (2008) 2

From these results it is clear that the occurrence of the high-energy 1O2 molecule is crucial for initiation of the O3-formationpathway. A specific and sensitive method has been developedfor intracellular detection of 1O2 and it has been demonstratedthat 1O2 is produced during phagocytosis [17], The myeloperox-idase (MPO)/H2O2/Cl system is a source of 1O2 produced duringphagocytosis. It is especially interesting that generation of 1O2by direct interaction of two molecules of O·

2– is also possi-

ble [18]. Although lipid hydroperoxides can generate 1O2, otherbiological hydroperoxides, including hydroperoxides formed inproteins and nucleic acids, may also participate in specialendogenous reactions leading to the generation of 1O2 [19];this may then lead to endogenous O3.

1O2 may also be generatedby decomposition of α-linolenic acid hydroperoxide bycytochrome C and lactoperoxidase [20]. 1O2 production isobservable in photosynthesis also [21]. The occurrence of 1O2 inplants, its significance, and its possible detection with double(fluorescent and spin) indicator reagents have been illustrat-ed [22].

It must also be taken into account that in the biochemistry ofendogenous O3 in extracellular and/or intracellular events,formaldehyde (HCHO), as an endogenous, indispensable com-ponent of all biological systems, may always be present, mainlyas labile hydroxymethyl groups [23, 24] and so there is a possi-bility of its interaction with – among other compounds – H2O2,another universal small endogenous molecule. In this interaction1O2 and excited HCHO (H*CHO) can be formed [25, 26]. It hasbecome increasingly evident there is a primary HCHO cycle inbiological systems in which the S-methyl group of L-methioninecan be formed from HCHO originating from natural HCHOgenerators and that the HCHO-yielding function of S-adenosyl-L-methionine (SAM) is an essential component of this funda-mental biological pathway [23, 24]. The term ‘formaldehy-dome’ means the complete set of HCHO-cycle-mediated andnon-mediated HCHO pathways of a given biological unit. Thenature of the distribution of HCHO molecules as methyl and/orhydroxymethyl or formyl groups (e.g. hypo and hypermethylat-ed regions in DNA or methyl groups in histones) and, at thesame time, the demethylation of different methylated and/orhydroxymethylated compounds (e.g. demethylation of N-methylated L-lysine molecules in histones [27]) fundamentally

affect the nature of the formaldehydome and have a characteris-tic profile for any biological unit.

It is supposed that HCHO , originating from the formaldehy-dome system, is also present in the process of activation of anti-bodies and may, in general, be one basis of the formation of 1O2in this complicated system. Figure 1 illustrates this supposedseries of reactions, taking into account both early and morerecent observations [2–4, 25, 26]. It is probable that the mecha-nism by which antibodies and antibiotics kill bacteria is basedon the same or similar principles. These very reactive small mol-ecules – from HCHO to O3 – may be crucial aspects of innate(natural) resistance and form a common bridge between theinnate and adaptive (induced) resistance of plants and animalorganisms alike [15].

3 Materials and Methods

3.1 Chemicals

The test substances were purchased from Sigma–Aldrich(Budapest, Hungary). The dye reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma–Aldrich)was used to reveal antimicrobial activity. MTT was dissolved indistilled water and the solution was used immediately afterpreparation.

Indigo carmine and d-limonene were also from Sigma–Aldrich.All solvents and other chemicals were of analytical grade andwere purchased from Merck (Darmstadt, Germany) and Reanal(Budapest, Hungary).

3.2 Chromatography

TLC and OPLC were performed on silica gel 60 F254 plates(Merck). Before use the plates were conditioned at 120°C for3 h [28]. For OPLC all the edges of the plate were sealed. Thetest substances were applied to the layers by use of a microsy-ringe or a Camag (Muttenz, Switzerland) automatic sampleapplicator. TLC plates were developed in a Desaga glass cham-ber with a variety of mobile phases (e.g. chloroform–methanol80:8 v/v). The plates were dried and were then ready for bioau-tographic detection.

OPLC was performed with an automatic instrument (OPLC-NIT, Budapest, Hungary) consisting of a liquid-delivery systemand the separation chamber. A cassette containing the plate withapplied samples can be inserted into the chamber [29, 30]. After

Figure 1

Simplified reaction scheme showing the interactions of HCHO andH2O2 molecules produced by the action of antibodies and antibiotics.

Figure 2

The chemical structure of d-limonene.

the separation process the cassette is withdrawn and the plate isdried and can be used for bioautographic detection. The condi-tions used for OPLC were: mobile phase (typical) chloro-form–methanol 80:4 (v/v), external pressure 5.0 MPa, flow rate

250 μL min–1, mobile phase volume 8700 μL. The separationtime was 2680 s.

3.3 Bioautography

The phytopathogen Pseudomonas savastanoi pv. phaseolicolarace 6, which causes halo blight on beans, was used for the bac-terial biotest,. Bacterial cells were grown in a rich medium(King’s B broth) at 28°C, on centrifugal shaker at 200 rpm, untilthey reached the late exponential phase (OD560 nm = 0.7), corre-sponding to 1.5 × 109 cells mL–1.

Just before use, 50% (v/v) fresh medium was added to the cul-ture and different co-factors were also added. The developedand dried TLC plates were immersed in the bacterial suspensionof Pseudomonas savastanoi for 20 s. Visualization of the chro-matograms with MTT was performed either after a short drain-ing period or after overnight incubation [9].

4 Results and Discussion

In this series of experiments we tried to detect, indirectly, accu-mulation of O3 in chromatographic spots using two O3 scav-engers of different chemical structure [31]. Figure 2 illustratesthe structure of d-limonene and Figure 3 shows the interactionof indigo carmine with O3.

4.1 Detection of O3 in the Spots of trans-Resveratrol

It has been established that trans-resveratrol (TR), a commoncomponent of grapes, wine, etc. [32], with almost non-toxic andextremely diverse beneficial activity, can interact with HCHO inmodel chemical reactions and in biological systems (e.g. pigheart tissue) [9, 23, 32, 33]. Mobilization of HCHO from a bio-logical unit by TR is a totally new approach to the ‘red wine’topic; its effect is twofold:

– first, elimination of HCHO from the tissue (or cells) by TRmay have a chemopreventive effect;

– second, products of the reaction between TR and HCHO mayact as toxic or inhibiting factors, e.g. against pathogens or can-cer cells [9, 32, 33].

Indirect Detection of Ozone with the BioArena System

Journal of Planar Chromatography 21 (2008) 2 79

Figure 3

Interaction of indigo carmine with O3 with formation of isatic acid.

Figure 4

Effect of d-limonene on the antibacterial activity of trans-resveratrol.50 μL d-limonene was added to 100 mL Psm (Pseudomonas savas-tanoi cells in culture medium). A. 3 h between inoculation and stain-ing (with MTT). B. 18 h between inoculation and staining (with MTT).For chromatographic conditions see text.

Figure 5

Effect of different amounts of d-limonene on the antibacterial activityof trans-resveratrol. A. Control (Psm only). B, C, D. Addition of 10, 50,and 100 μL d-limonene, respectively, to 100 mL Psm. a, basic picture;b, picture after computer-balancing of light and contrast conditions.For chromatographic conditions see text.

Figure 6

Effect of indigo carmine on the antibacterial activity of trans-resvera-trol. 20 mg indigo carmine was added to 100 mL Psm. A. 3 h betweeninoculation and staining (with MTT). B. 18 h between inoculation andstaining (with MTT). For chromatographic conditions see text.

0.1 0.5 1.0 μLtrans-resveratrol

0.1 0.5 1.0 μLtrans-resveratrol

0.1 0.5 1.0 μLtrans-resveratrol

0.1 0.5 1.0 μLtrans-resveratrol

This characteristic double effect of TR may be one basis of itsmost diverse beneficial activity.

The BioArena system is a good way of studying the microchem-ical reactions of TR with HCHO using an endogenous HCHOcapturer and mobilizing molecules in the culture medium. Whenendogenous HCHO-capturing molecules are added to the cul-ture medium with the bacterial cells the antibacterial activity ofTR decreases dose-dependently. TR behaves as an antibacterialantibiotic against Pseudomonas savastanoi pv. phaseolicola. Itis especially interesting that Cu(II) ions mobilize and coordinatethe HCHO molecules in hydroxymethyl groups [34] and so cantransport HCHO molecules to TR spots on the chromatographicplate. The antibacterial activity of TR can be increased dramati-cally by using a large amount of Cu(II) ions in the culture medi-um [34, 35].

Figure 4 illustrates the antibacterial activity of the TR (see con-trols). It was supposed that O3 could be formed in the chromato-graphic spots of TR and may be partly responsible for theantibacterial effect. d-Limonene was used to eliminate (quench)eventual O3 molecules from the TR spots and it was establishedthat in the presence of d-limonene in the short time (3 h)between inoculation and staining (with MTT) the antibacterialactivity of TR decreased, as is apparent from Figure 4A. This ismost obvious for small amounts (0.1 and 0.5 μg) of TR. Whenthe incubation time was longer, however (Figure 4B), this dif-ference disappeared completely. It follows from this that TRprobably does not generate H2O2 molecules For the short incu-bation time the decrease of activity originated from the interac-tion between originally present H2O2 and HCHO molecules.

Figure 5 shows the effect of different amounts of d-limonene onthe antibacterial activity of TR after incubation for 18 h. It isapparent that the antibacterial activity increased dose-depen-dently. This increase in the activity of TR might have originatedfrom partial decomposition of d-limonene in the course of whichHCHO can be formed (among other compounds) [36], increas-ing the activity of TR. Computer balancing of the original pic-ture did not cause the effect of the increase to disappear.

Use of indigo carmine as another O3 scavenger furnished resultsafter short and long incubation times (Figure 6) similar to thoseillustrated in Figure 4 for d-limonene. After the long incubationtime (18 h), however, there was no dose-dependent increase inactivity (Figure 7) or, more precisely, TR did not generate theH2O2 molecules which are necessary for the O3-formation path-way (Figure 1). This can be seen especially clearly after com-puter balancing (Figure 7b).

4.2 Demonstration of the Relationship Between ToxicAntibacterial Activity and O3 Accumulation

Figure 8 shows that when indigo carmine was added to the cul-ture medium (Psm) the antibacterial–toxic activity of aflatoxinB1 decreased dose-dependently. This observation is very similarto our previous results with trans-cinnamic acid [37] and withochratoxin A and other mycotoxins [10, 38, 39] .

It seems from these results that TR has the most varied benefi-cial activity, the basis of which is the HCHO-dependent doubleeffect discussed elsewhere [23, 33]; aflatoxin B1 and othermycotoxins [10, 38, 39] and, e.g., paclitaxel (Taxol) [40] arevery toxic molecules whose mechanism of action is, mainly,local accumulation of O3 molecules.

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80 Journal of Planar Chromatography 21 (2008) 2

Figure 7

Effect of different amounts of indigo carmine on the antibacterialactivity of trans-resveratrol. A. Control (Psm only). B, C, D. Additionof 10, 20, and 40 mg indigo carmine, respectively, to 100 mL Psm. a,basic picture; b, picture after computer-balancing of light and con-trast conditions. For chromatographic conditions see text.

Figure 8

Effect of indigo carmine on the antibacterial activity of aflatoxin B1.A. Control. B, C. Addition of 20 and 40 mg indigo carmine, respec-tively, to 100 mL Psm. The upper traces were obtained with aShimadzu CS 930 densitometer at 590 nm; for other chromatograph-ic conditions see text.

5 Conclusion

It has been established indirectly in a microbiological system(i.e. by use of BioArena) that O3 is involved in the antibacterialactivity of some compounds. Recent results, and resultsobtained in this work, suggest that O3, an indispensable, endoge-nous molecule, can be detected in almost all biological sys-tems [2–5, 9, 10, 15, 16]. It is probable that endogenous O3 hasextremely diverse, mainly unknown, positive and negativeeffects, e.g. it is a resistance factor in plants [15, 16] and mightbe involved in the pathogenesis of atherosclerosis via the anti-body-catalyzed water-oxidation pathway [6].

These new findings support the general idea that natural preven-tive systems in different biological units include the productionof highly bioreactive and cytotoxic compounds, e.g. H2O2,HCHO, 1O2, H2O3, and O3, which participate in the directdestruction of foreign antigens, e.g. pathogens (in this case abacterial cell, Figure 9) and it is also valid for interactions inchromatographic spots. It should be pointed out that amongthese oxidants O3 is the determining factor, as can be seen in thisschematic diagram of a bacterial cell. The discovery of this fun-damental biochemical pathway shows that the final killingprocess in the prevention of different types of biological activi-ty is similar or identical. The killing potential of these differentbioreactive compounds may be basically different, however.

The BioArena system is an effective means of gaining deeperinsight into problematic biochemical pathways and for search-ing for new ways of preventing and treating different diseases.Results obtained so far from BioArena investigations help us togroup and distinguish compounds on the basis of the mechanismof their toxicity. At two ends of the toxicity chain compounds ofsimilar activity are present. At one end are almost nontoxic butvery effective compounds, e.g. trans-resveratrol; at the otherend are very toxic compounds, e.g. mycotoxins and moleculeswith most diverse transient toxicity are between the two ends ofthe toxicity chain. It seems that the ratio of bioreactive and cyto-toxic compounds formed determines the potential toxicity of asubstance.

Further study is necessary to discover positive and negative evi-dence of endogenous production of O3 and other bioreactivemolecules via different preventive systems (e.g. the antibody-catalyzed water-oxidation pathway).

Aknowledgement

The authors are grateful for the financial support from OTKA T048919.

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Ms received: February 19, 2008Accepted: February 21, 2008

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