Indian Journal of Experimental Biology Vol. 41 , May 2003, pp. 483-493

Biophotons from stressed and dying organisms: Toxicological aspects

Janusz Slawinski

Department of Radio and Photochemistry, Institute of Chemistry and Tec hni cal Electrochemistry , Faculty of Chemical Technology . Poznall Uni versity of Technology , PL-60-965 Poznan, Piotrowo 3 str. Poland

Cc ll s and orga ni sms exposed to de trimental and tox ic substances show different responses in photon emi ssion dependent on amou nt, kind and ex posure time of tox in as well as on the organism in ves tigated. Radical reaction-generati ng substances and dehydra ting, lipid di ssolving and protei n denaturating tox ins which do not induce direct chemiluminescence resulting from reactive oxygen species were applied. Lethal doses of tox ins and stress factors such as osmotics and temperature evoke increase in the intensity of photon emission resulting from a rapid and irreversible perturbation of homeostasis. Bacteri al and fun gal tox ins that elicit hypersensitive death of plant cells or deFense response correlated with photon emiss ion are also brieny discussed. Co llective molecul ar interacti ons contribute to the photon-generating degrada ti ve processes in stressed and dy ing organi sms. The measurement s of biophoton signals and analysis of their parameters arc used to elucida te the possible mechani sms of the toxi n-organism interacti on and the resistance of organi sms. Tox icological perspecti ves of the use of these sensiti ve and rapid measurements as a part of direct tox icity assessment are di sc ussed.

Keywords: Biophoton, Defence response, 2- D imaging, Homeostas is, Rad ical reacti on, Toxicity assess ment , Toxin , Yeast cell s

An intrinsic association of biophoton emission with fundamental biological processes such as cell divi sion and differentiation , egg fertilization, oxidative metabolism, photosynthesis has been the subject of study fo r last four decennia t

-3

. However, the question whether the death of a cell or a multicellular organi sm is al ways accompanied by enhanced photon emission is still a matter of uncertainty. Only sporadic experiments were performed on biophoton emission from plants and still less from animals treated with a variety of detrimental or toxic (lethal) factors4

-to . This

paper presents results of further experiments on enhanced emission from animal and plant organisms subjected to stress and lethal factors, maily chemical tox ins. It also discusses relevant recent data of other authors in terms of cooperative biochemical and biophysical processes underl ying an irreversible perturbation of homeostasis t

Lt2 . The poss ibility to apply a holi stic integrative biophotonic response to detrimental fac tors for the evaluation of lethal doses and the resistance of organisms in toxicology is considered.

Materials and Methods The strain TF-29 abd TF-32 of Saccharomyces

ccrevlszae in the late stationary phase (not synchroni zed) in the form of suspension in di stilled water or PBS-buffer after triplicate centrifugation was

For correspondence : E-mail: slawillsk@sol.put.poznan.pl

used. Moreover, Sachcaromyces boulardi (5. Cerevisiae Hansen CBS 5926) containing 109 vital cells per sample (2 ml suspension) was used in other ex periments. Spermatozoa cells were from Balicel Cracow National Institute of Animal Production . The cells were incubated with the peroxidation-generating system of ascorbate + Fe(I1). Detai Is are given elsewhere t3

-t7

• Paramecium sp. and Chlorella vulgaris were cultivated in standard medi a. Water flea (magna, Entomostraca, Phtllopoda) SO individuals of parthenogenetic females in standard water were tested in each sample. Worms and insects tested were healthy organisms freshly caught in the Cracow, Poland, Kaiserslautern, Germany and Sendai, Japan region s. Details on experiments with algae Characeae, Nitelloplis obtusa are described elsewhere to

.t8

.

The objects were placed in a quartz cuvette withi n a light-tight, thermostated camera and allowed to relax ca 20 min in order to extingui sh photoinduced delay luminescence and transport-induced stress emi ssion. Then a photocount time series {n(t) } (t = 1,2, ... N = 100-200) reflecting a spontaneous quasi­stationary emi ss ion was regi stered. The sampling time (the time interval of photocount acqu isition L1t) was fro m 0.2 to 10 s depending on the photocount rate. Next, an aliquot of detrimental or toxic substances was rapidly introduced into cuvette by light-tight tubings and photocount senes (N = 300-2000)

484 INDIAN J EXP BIOl, MA Y 2003

coresponding to non-stationary elTiiSSlOn was recorded . After the ca 80% extinction of the maximum signal, the sample was taken off the camera and inspected or the number of alive/dead cells es timated. Photocount-time series were recorded Llsing the SPC technique. Hi gh-sensitivity, low-noise PMT 9558QB EMI , FEU-79, Hamamatsu R 1333 and R 375, sensiti ve in the UV-near IR spectra l region were employed as dedectors of biophotons.The data acqu isit ion , the storage and kinetic/spectral and stat istical analyses were perfo rmed by an on-line Pc. Details are given elsewhere9.'4. ,s.

Results

Biophotonic response to detrimental conditions and toxins in cell suspension cultures

Yeast cells- Ultraweak biophoton emission from yeast cell s has been used as a conveni ent model for the chemi cal stress agent- li ving cell interaction . Forma ldehyde (HCHO) - a protei n-denaturat i ng low molecu lar substance easi Iy penetrati ng biological membranes was chosen as a non-speci fic tox i n. Yeast cultures Saccharomyces cerevi.liae strain s SPA (wild), DSCD-I (SO D-deficient), Hansen CBS 5926 and Harvest Gold bakery treated with lethal concentrJtions (0.0 I-I 0%) of HCHO increased the intensity by a fac tor of LIp to 2500. The spectral changes were observed in the native and HCHO­perturbed cells that have been described elsewhere IS.

These emissions were not correlated with minor changes in nuorescence exc itation and emission spectra originat ing from tryptophan , navins and unidentified emitters. ]n all cases the emi ss ion intensity 1= <1> dn/dt (where <1> is the efficiency of photon counting alld dn/dt the photocount rate) and total intensity (light sum 2.1) strongly depends on the presence of oxygen. This is va lid for both the SOD­deficient DSCD-I mutant and SP-4 (wild) strain . The data obtai ned indicate that the emission is connected with HCHO-deri ved peroxy radi cals and their lethal effects on the constitutional and functional proteins in the cell membrane. The cristae membrane of mitochondria in yeast cells contains fo rmaldehyde dehydrogenase f: N A 0 + ox idoreductase (g I utath ione formylati ng) (EC 1.2.1.1 ); an enzyme cata lys ing the

AD-dependen t formation of S-formylglutathione from GS H and HCHO I6

. This may be the :'irst stage in the conversion of HCHO to fo rmate. The second stage would be catalysed by S- formylglutathi one hydrcbse:

GSH+ HCO- EC 1.2. I . I -)GS-CHo-hydrolasc-) GSH+ Ho-CHO

t \ t NAD+ NADH+ H+ l-hO

Hydrogen abstraction from lipid hydroperoxides ROOH by HCHO can also be a probable reaction producing reacti ve radicals and electron ic excited species: carbonyl s R = 0 and singlet molecular oxygen 102* : ROO - H +O=C=H r -7 RO l " + HO-C= H1"

2 HO-C=H2" -7 CH 30H + CH20 *

2 R O2" -7 R 0 H + R = 0 + 102 *

The mathematical model of formation and recombination of radical reactions initiated by formaldehyde was tes ted I 1.1 3- 15.

In new ex periments trichloracetic acid (TCA) denaturating proteins by an ionic interaction were used . An enhanced emission accompanying the death of ycast cells, their aglomeration and precipitat ion was also observed, but its intensity w' s much lower than that from HCHO. Emission spectra of the TCA­yeast ce ll s and HCHO -yeast cell suspe nsions show small differences in A max but not in the spectral range they cover (380-870 nm).

Spermatozoa - ROS produced in pathological internal processes or in contaminated environment are hi ghl y toxi c to generati ve cells. In our ex perimcnts spermatozoa cells fro m bull, boar and ram were exposed in vitro to the ascorbate-Fe( lI ) redox -cycling

d h . . t' F' fll )17-19 system an t en to tOXI C concentrations 0 e, . Some crucial results are exemplified in Figs 1 and 2. Fig. I shows two separated phases of biophoton kinetics: I) the process of membrane-lipid peroxidation 12-4l and hydroperox ide decomposition followed by a rapid recombination of peroxy radicals 14l after inj ection of hi gher concentrations of Fe(JI) are exemplified for fresh spermatozoa of bu ll in Fi g. I . The shape of kinetic curves of photon emission is different and species-characteri sti c. Total intensity of photon emission during the fi rst phase 13l is proportional to the rate of fo rmation and decomposition of hydroperoxides in the ascorbate­Fe(lI) -assisted perox idat ion . During the second phase 14l a sharp increase in the intensity results from the rapid recombi nation of ROO. radicals. For the purpose of 4uanlitati ve comparison of these tWI) phases, the X coefficient was introduced 19:

X = I [l (t) - IS]FjI[I (t) - Is l Ase 1= 0 I = ()

SLAWINSKI: BIOPHOTONS FROM STRESSED & DYING ORGAN ISMS 485

1800

b\ A 3

"°°1 1400 I~ ' (\: 1200 ./

"5 1000 / \ n;

800 a J :: )L2 200f - -.-.-... 1

4 0,

0 500 1000 1500 2000 brne [sl

Fig. I - Time-cou rse of photon emission (k ineti c cu rves 1= f(t ) fro m bovi ne spermatozoa cells ( I ± 0.1 cells) free of se min al plasma in 0.9% NaCI incubated at 44°C. The regions arc 1-backgrou nd emission, 2-spontaneous emission fro m cells, 3,4-regions of summati on for y coefficient :!nd a indicates inj ec ti on of ascorbate-Fe ll redox cycl ing system promoting the perox id:llionof membrane lipids (0.22 mM and 0.045 mM final cOll centration~.

respectively), b - injection of 1.5 ml 0.96 mM FeS04 solution (final concentration)

Here I(t) is time-dependent intensity after addition of ascorbate-Fe( lI ) system or FeS04 at the toxic concentration (mmolar) to the sample, Is denotes the spontaneous photon intensity of the cell s that is constant during the measurement, and the last term denotes the intensity of the ascorbate-Fe(1 I)-enh ancecl emission minus the intensity of spontaneous emi ss ion, summed up fo r the first 500 s from the moment of ascorbate-Fe(lI ) or FeS04 add ition to the sample. Fig. 2 shows the dependencies of X coefficient on the incubat ion time of the sample. An increase of incubat ion time as a rule decreases X coefficien t. In all these experiments viabi lity and motility of the cell s decreased significantly with the inccrease of incubation time. Add ition of the toxic amount of FeS04 caused the death of all cells I8

. '9 . The total number of photocounts from the two phases of emission correlates wel l with the ce ll vitali ty. Correlati on cocfficients between thc integrated photon intensity L [I (t) and the life/dead indices fo r domestic ani mals spermatozoa are fol lowing: for bull -0.82, fo r boar -0.93 and for ram -0.95 (P = 0.05 ).

The spectral distribution of photon emission fro m spermatozoa cells tested so far by means of the S PC techn ique and cut-off opt ical filte rs covers the wavelength region fro m 350 to 800 nm and is not a species characteristic 1fi

.17

. The main drowbacks are extremely low intensity and necess ity to use broad band optical filters.

36

34

32

6

4

' .

,

-~~ -- . -- 81 .. . * .. C

...... 'Y 0

, ....• : : : : ~.:: :~~~:.~:~ .... : .... , o ----,-~

o 100 200 300 400

incubation t ime , min

Fig. 2 - Dependence of photon emission-y coefficient on incuha­tion time. The results arc given fo r four sampl es bull (A), ram (13 ), b0:1r (e) and bull spermatozoa equilibrated with glycerol Gnd cryopreservcd (-79°C) (D)

Algae- These simple unicellular organisms abu ndant in water ecosystems appear to be a versatile mode l for studying the effect of environmental chemicals such as ac id ra ins, herbi cides, mineral nutrients and heavy metals as well as pharmacody namical agents, e.g . anaes thetics. In this d irection fi rst works on the effect of environtmentally important humic substances and toxic diphenols on ultraweak photon em ission from Characeae cel ls were initiated2o. Recent extended research on C/wrace{/e species have revealed several important aspects concerning the effect of environmental conditions and xenobiotics on photon emi ssion, electri cal and physiological characteristi cs and their interconnectedness2o

-23

. In these studies the 1, 1= f (t) and 1= f (I-) were measured si multaneously with the transmembrane potential, electrical resistance, the rate of cyc losis (cytop lasmic streaming) and oxygen consumption in Nifelopsis obfllsa (Oesv, in Lois) cel ls. The results obtained have proved that the sample consist ing of e.g. 30 cells exposed to anaestheti cs , osmotics or increased temperature behave as one holi st ic system giving a synchronized oscillatory photon ic response. The kineti cs of thi s response reveals two important feat ures: ( I) The enhanced emIssIon with a dis tinct maximum correl ated with the rate of cell death . This result co nfirms previou ~ reports on "degradative" radiation for dying organi sms and seems to be important for possible futu re app lications to toxicology for the eva luation of lethal closes of xenobiotics4

.6

-8

.9

; and (2) an oscillatory pattern with damped amplitude. -Figure 3 presents a typica l pattern of the biophotonic response

486 INDIAN J EXP BIOL, MA Y 2003

fro m N. obtusa to procaineJ(). A similar kinetic pattern of delayed luminescence was also observed from higher plants and a frequency-stab le damped oscill ator has been proposed as a model of biophoton emission24

-26

.

Several experiments on the effect of herbicide atrazi ne (2-ch loro-4-eth y lami no-6- isopropy lami no-s­tri az ine) on the de layed (photosynthetic) luminescence from Acetabularia acetabulurn revealed an increased emission from poisoned samples and fas ter decay of photon emi ssion. A good fit of luminescence decay to hyperbolic decay from intact organ isms (non poisoned) and multiexponenti al one from the atrazine- treated cells suggests that the toxin uncouples cooperative phys iological processes27

.28

.

Biophotonic response to stress and toxins in plants The primary process initiated during pathogen or

toxin recognition is locali zed in pl asmolemma. Transmembrane signalling and information transfer is

cpl0s

121 '100,

10 1·

' ':-". ',. ' . . ' .. -::; .. ~

:/ _ '100 . 0k--1:":0~::':20~-='3-0 ~4~'0~5"'0~-60-'7080 s

cpl0s '1 00

40

30

2° b,--': o 10 ~, ~20-~ 30

cp10 s

t '100 16

1

121

. .,

' .. " ~; .... ', ..

'100

40 50 s

8/ .,' 4 ( .. . :,~ ... , .... , ...... J '::<' ·'Y:""/ ·

: , _ , __ ~ ~ __ • _, -r , __ , __ , _ . ' ~ _-'.20_0_0 __ o 2 4 6 8 10 12 14

Fig. 3 - Kinet ics of photoni c response from 30 algae Nitel/opsis obtllsa cci ls to IS mM anacstheti c procai nc at different tcmpcra­tures. V is thc percentage of a li ve cel ls as determined aftc r the expcrimcnt by the rate of cylosis, cp 10 s is the countin g rate pcr 10 scc, s is the timc of cell s inc ubati on in seconds after the injec­tion o f procaine. (By the courtesy of Dr A. Jaskowska).

a complex process leading to the immunological defense, resistance and adaptation . Cellular responses to infection-generated toxins are often correlated with locali zed cell death "the hypersensiti ve response". Thi s is a programmed cell death (apoptosis)29.

It is well estab li shed that pl ants react to microorganism tox ins or to hyphal wall components of pathogeni c fungi (e licitors) by changing their metabo li sm to produce reacti ve oxygen species (ROS ), i.e. an oxidative burst. This burst is accompanied with photon emission and can be noninvas ively monitored using several photon emi ss ion measuring techniques. Three methodological approaches to the monitoring pathogen/tox in -p lant response have been so fa r appli ed .

Luminol-enhanced chemiluminescence combined with a two dimensional (2D) imaging - Enhancers of CL such as luminol, lucigenin and certa in luciferins react ing with ROS hi ghly increase the sensitivity and decrease the registrati on (sampling) time. Chemiluminescence spectrophotometers, CCO camera or autoradiographic system with X-ray film were used to obtai n 20 images of the el icitor/toxin­treated area. This technique was appli ed to so lanaceous plants such as sweet pepper, tomato, potato and tabacco infected by Phytophthora sp. Each of the Phytophthora sp. penetrated into epidermal cells of a host pl ant, causing in non-resistant hosts hypersensitive cell death with cell necrosis. It was shown that elicitor treatment may cause not only a local but also a sub-systemic oxidative burst in tissues. The last one may be related to systemic

. d ' f h h . 30-)? acquire resistance o ' t e ost organism -However, the use of chemiluminescence enhancers does not allow measuring an intrinsic spectral di stribution of the direct photon emission coupled to metabolic processes . The spectral distribution is assumed to refl ect the energetics of metabo lic exergonic reactions (Gibbs free energy L1G), its distribution over the reaction products and the nature of emitters.

Direct SPC im.aging oj photon emission - This approach does not use CL enhancers and requi res ultra-high sensitivity of a slow-scan CCO cameras for photon counting imaging and special analysi s of the acqui red 20 charge di stribution . Biophotons generated by sweet potato Ipomoea batalas and nonpathogenic Fusarium OXYSpOrlllll interactions associated wi th a defense response were recorded. Production of ipomeamarone as a phytoalex in­substance produced by plants in response to stimul ation

SLAWINSKI: BIOPHOTONS FROM STRESSED & DYING ORGAN ISMS 487

was also shown33. Photon emission in the development

of gray mould Botryotinia fu cke/iana (B. cinerea) on poinsettia (Euphorbia pulcherrima Willd) leaves susceptible and moderately resistant to the mould and participation of -0-2 and -OH radicals as well as lipoxygenase activity were investi gated34

. A postinfection increase of emi ss ion in the zone surrounding di seased areas and correlated with enhanced production of ROS was observed. In thi s study an improved software eliminating optical artifacts and improving the quality of SPC images appeared to be very useful 35

.

Spectral distribulion meaSllrel1l ents-A multi sample SPC system with 7 band-pass filters (6.A = 50 nm) covering the wavelength region 280-630 nm was used for measurements of photon emission kinetics after inocul ation a sweet potato (I. batatas) with F. oxysporul11. After 2-10 hI' of inoculation the spectrum changed dramatically, with an increase of the total emission. A new major component of the spectral distribution appeared in the short wavelength region 430-480 nm. The rati o of spectral components showed sharp varI atIons correlated with the inoculation time and the synthesis time of phytoalexins. Moreover, a sy ntheti c auxin 2,4-dichlorophenoxyacetic ac id (2-D) that induces sweet potato to form embryogeni c calli was applied. Transient variations in the rat io 480-530/530-580 nm and a decrease in 580-630 nm were observed. A change in the physiological state connected with the sy nthesis of defense-related substances, e.g. sa licy lic acid was suggested36

.37

. In another experiment the time-dependent intensity, the spectral analys is and 20 imaging of biophoton emission were done using a set of sophi sticated ultrahigh sensiti vity SPC and CCD­camera systems. The resu lts have shown "maps" of emission in the zone of inocu lation of the host pl ant I . balatas with F. oxysporwll and Ceratocystis fimbriafa. The intensity of emiss ion depended on the concentration of the conidi a inoculation, condition of the sweet potato and was correlated wi th the germination time of coni dia and production of j pomeamarone. No emi ssion was recorded from dead

. 3337 F I potato samples or treated wtth water . rom t lese results, it is concluded that th e generation of biophotons and thei r spectral characteristi cs are related to a defense response to infecti on. As the spectral analysis is a noninvasive and rea l-t ime method, it can be used as a new parameter for identyfying the physiological state of an organism.

Biophotons frol11 the interaction of ROS, NO, reactive qllinones and oxalates with plant and animal cells-Cellul ar responses to infections and toxins another th an those prev iously di scussed involve several specific mechani sms. Plants produce a reactive radical NO during R-gene-dependen t resistance responses; NO acts sy nergistica ll y with ROS and sa licy lic ac id to increase death of cell s, e.g:

-NO + -0-2 ) ONOO- peroxynitrite

This reaction is a di ffusion-controlled and proceeds under pathophysiological conditions. ONOOH is an extremely destructive ROS in volved in shock, inflamation and ischemia-reperfusion. Necrotrophic plant pathogens probably use the NO-ROS to kil l the

11 I . d d f t ' t 29.38.39 ce s t ley tn va e an use or nu rI en s Reacti ve qui nones Q in monovalent oxygen

reduction fo rm semiquinone rad ica ls SQ and -0-2 :

Q+02 ) -SQ + -0-2

Q and -SQ compri se chemi cal weapons and the establi shment of mechani cal barriers aga inst toxins or pathogens. As an example one can mention galla or zoocecidia containing gallic ac id and tannins or hydroxybenzotropolones (purpurogallin ; a "purple pea") on oak or willow leaves. Naturally occurri ng p­naphthoquinones like juglone (5-hydroxy-l,4-naphthoquinone) and its derivatives reveals a wide spectrum of cytotox ic and pharmacody namic properties. Interac tion of toxic cati ons of transition and heavy metals (Fe, Cu, Mn, Ni , Ti) with ROS may be the one ultimate tox ic principle unco ntrolabl e by

• 'J'J the endogeneous cellular defense equtpmenr- .

Oxalic acid (HOOCCOOH) and oxalates are natura l, but very powerful toxi ns to mammalian cell s. It is a product of human metaboli sm and its high level in body fluids is considered to be pathogenic. However, there is no enzyme-catalysed oxalate metaboli sm known in human metabolism. A highly sensit ive and specific chemiluminometric method was developed to determine oxalates in body tl uids4o. It appeared that the concenrrat ion of oxalate in various body fluids is very high. This implies the questions: is oxalate an end­product of human metabolism? What might be purpose of high intracellular concentration of this metabolite? It cannot be excluded that mechanism shoud exi st by which this toxic compound can be enzymatically metaboli zed as it is ill cettain bactetia and plant cell s~1.42:

HOOCCOOH + O2 - oxalate oxidase --7 2 CO2 + H20 2

HOOCCOOH - oxalate decarboxylase --7 HCOOH +C02

488 INDIAN J EXP BIOL, MA Y 2003

Oxalate esters have a poor quantum yield of CL but react readily as energy transfer donors in a sensitized CL. Oxalate ester CL energy transfer is used in commercially available light sticks43

. There exists possi ibility of ultraweak CL from oxalates products as shown in the above reactions The problem may be pertinent to the stimulating effect of carbonates and formates on ultraweak CL invol ving ROS redox reactions with possible formation and di ssociation of transient dioxetanes44

•

Photonic response to the toxin-lower animal interactions

The basic data of experiments- The literature on this subject is very scarce. The resu lts of our experiments with intact (i), alive but stressed (s) or dying (d) lower animals exposed to stress and lethal agents are summarized in Table l.The effect of the stress/toxin ac ti on used is expressed as the relati ve max imum intensity (Is 11;)max or (l d II; )max. Moreover, Table 1 includes data on the decay kinetics of

Table I - Enhanced photon e mi ssion from the interac tion between the organism(s) and tox ins

Organi sm tested Stress factor

Protozoa, ParameciulIl aurelia , 4% HCHO -5000rga ni sms/ml

Paramecilllll bursa ria 4% HC HO

PartJlIlecilllll caudallllll 4% HC HO

Crustacea, Daphllia lIlaglla Strauss 0.0 1 HCI, 0.01 N NaOH

SOOt 4 organisms/mL

Snail, Helix pomalia mL I % heparin I mL IN HCI

mL (CH3)2CO I mL 1% NaCi

hea t, I min SO°C

Earth worm, Lumbricus lerreslris mL C2HsOH

mL (CH3h CO

Caterpill ar, Van essa alalallla mL (CH3h CO

0.5mL (C H )3CO

heat, ISO s, 62°C

Fruit bug, Chlorochroa sp 0.5 mL (CH3)2CO

Grasshopper, Telligonia viridis mL C2HsOH 0.1 mL 35% HCHO

Fly, Musca dom eslica 0.1 mL (C H3)2CO

mL35 %HCHO

Ladybird, Coccinella seplempunclata mL (CH3h CO mL 35%HCHO

Spider, Araneus sp. mL (C Hj)2CO 0.2 mL C2HsO H

mL35 %HCHO

Ant , Formica TUfa mL (CH3h CO

Bee, Apis melliflca mL (CH3hCO mL C2HsOH

mL 35% HCHO

Cockroach, Blalla orientalis I mL (CH3hCO 2 mL 20% TCA

(Is / Ii),"" Sampling time /j, t,

1.9 t 0.5

5.7 to.6

< 1.0 tO.6

< 1.OtO.6 10

4.2± 1.1 10

to.2

1.5 to.2

to.3

to.3

IAtO.2

to.2

5 .St l. l 0.1

to.2 0.2

to.3 0.2

4.2tO.6 10

2.3 to.3 0. 1

tOA 0.1

5.2tO.6 0. 1

to .3 0.1

13. lt1.7 0.2

to.S I

31.3±4.2 0.2

to.3 0 .2

tOA 0.2

3.3±OA 0.2

3.5tOA 0.1

to.6 0.2

tOA 0.2

7.5tO.7 0.2

tOA 5

2.0tO.3 5

Effect

d

d

d

30% d 50% d

a d

d

?

a

a

d

a

d

d

d

cl d

d

d

d

d

a

d d

d

d

d d

d d

Decay kinetics

ic

h

ic

ic

h ic

h

ic

h ic

Ie

2 ex h

h

h

h h

2 ex ic

h

h

2ex h

icc

(Is lI i)max - the ratio of the max imum emission intensity (amplitude of the signal) from the specimen stressed by a toxin to the stationary emiss ion intensity of the intac t spec imen (organi sm) /j, t - sampling time, a - alive after the ex periment , d - dead, Dec - decay kinet ics, 2 ex-biexponential, h - hyperbolic, ic­inconclusive, Ef - effect of the interaction of a toxin with an or- organism.

-1

•

SLAWINSKI: BIOPHOTONS FROM STRESSED & DYING ORGANIS MS 489

photonic signal and on physiological state of a specimen. The toxic compounds used are dehydrating, lipid-di ssolving solvents (acetone, eth anol ), protein­denaturating (formaldehyde, trichloroacetic ac id), ionic compounds (hydrochl ori c acid, sodium trichloroacetate, sodium chloride or hydrox ide). Elevated temperature wa~ sporadica lly applied. As can be seen from Table I almost all organ isms tested give enhanced emission (l j I;)max or (I ,til; ) max > I when exposed to toxins. Only protozoan Paramecilll1l cauda/ul1I and crustacean Daphnia magna treated with HCHO and HCl? respecti vely, do not show enhanced emiss ion (Is!I;) max or Oi l; )m"x ~ I .

A relati ve l y strong enh ancement of biophotons from ParameciulIl bursa ria is probably due to the presence of symbioti c algae inside the organism. Chlorophyll in algae acts as an efficient acceptor of the electroni c excitation energy and reemits sensiti zed secondary emission with higher quantum yield . This interpretati on is supported by relatively high values of (I s!I; )max for Chlorella 1'lIlgaris . In the case of Daphnia magna, a calciferous crust reabsorbs and scatters emitted photons that leads to the decrease of the Daphnia magna (I , II ,)m<lx va lue. However, thi s does not account for the 4-fold higher signal eli cited by 0.0 I N NaO H in comparison to th at meas ured for 0.01 HC!. Ev iden tl y, higher pH vaiues facilitate ox idat ive reacti ons with the part icipati on of - 0 -2 which produce a "tri vial" CL. Thus, a logist ic problem arises concerning both the methodology of ex periments and correctnes of interpretation of this kind of ex peri men ts.

Me/hodological cOllsiderations -- Previous research on yeast cul tures poi soned with TCA, TCNa and NaOH confirms thi s interpretati on II. They also lead to the important conc lusion th at the "tri via l" ox idat ive

CL can contribute significantly to photon emission at pH values>7. In li ght of these findin gs a new methodological req,uirement regarding the correctness of experiments on "degradati ve" or "necroti c" photon emission has to be formul ated: in order to measure a "pure" photon emission, max imally free of direct CL from ox idative processes, the set of experimental cond itions shou ld eliminate those stress/toxin agents wh ich induce direct exergonic luminescent reacti ons. The above requirement was not taken in to consideration in the majority of previous studies. Therefore, in these research "soft" chemicals, wh ich do not induce a direct exergoni c CL, reactions are used. Moreover, it was checked whether the chemica l used give CL when reacting with aminoacids, proteins, nucleic acids , phospholip ids, fatty acids and sugars in aerated solutions at pH 4-7 at 290-300 Kin lIi/ro. In all cases studi ed no stati sti cally signi ficant emission was recorded . Therefore, one can conclude that the measured photonic signals originate predominantly from intrinsic biochemical and physiological degradative processes underlying stress/death event rather than from "tri vial" CL.

Kin elics of ph%nic response from slressed/dying organisl1ls- As can be seen from Tables 1-3, the kinetic pattern of the stress/poison-organism interaction contains an irreversib le increase of the (Is /I ;) max value, i. e. a maxi mum on the ] = f (t) curve, announcing the irreversible perturbation of biohomeostasis of the tested organi sm. Thi s kinetics also provides in fo rmation about the moment and the strength of the action of stress/tox in. Low doses perturb homeostasis weak ly and revers ibl y. This is manifested by a <; Iow increase in the intensity of emi ss ion, which reaches a new quas i-stationary level several times hi gher than that of the unperturbed (intact) organ ism. A rapid and

Table 2 - Degradal ive photon e miss ion from a s ing le bee (Apos lIIeliiflca) lrealed Wilh 0 .2 m! acelone (A).

sampli ng lime 6 1= 200 ms, lhe 10lulnllmber o f counlS N = 16 10

Ali ve Dy ing (J. A) Died Died (d ri~d body + A)

< I > ± SD. cps 7.Y5 ± 4 .35 10AO ± 4.64 9.27 ± 7. 13

± 4 .76 (lA) n . counlS 350 200 200 250

Tab le 3 - Degrad ali ve phoio n e mi ss ion rrom a , ingle eale rpill a r ( Vallessa atalanla ) lrealed eonsec uli ve ly Wilh 0. 2 ml and 0 .3 ml of aeelone (A). The 10la l number of counlS N = 261 0, sampl ing lime .0.1=200 ms

MJgnilude Ali ve S I ressed (J. A) Dy ing Died aftc r 480 s 0.2ml A 0.3 ml A

< I > ± SD, cps 8.08 ± 4.89 11 .87 ± 6.92 17.70 ± 6.37 i 2.09 ± .8.29 n , counls 200 200 200 200

11.02

490 INDIAN J EXP SIOL, MAY 2003

irreversibl e perturbat ion of homeostas is that leads to the death of the organ ism results in a rapid , stronger increase of I and integrated intensity (light sum) of photon emi ss ion . The intensity I and duration of the emission depend on the type of toxi n or stress agent, its dose and tile type of organi sm. For example, in sects intoxicated with acetone, alcohol or fo rmaldehyde die rapid ly and the va lue (Is IJ j)max is greater than that of an earthworm or a snail (Table I ) since the rate of toxin penetrati on through hydrated thick ti ssue is much slower. The decay kinetics - <dn 1 dt> = J (t) of the descending quasi-relaxation phase obeys the hyperbolic fun ctions in the majority of cases presented in Tables 1-3.

I = A (t- tor f3 or I (t) = 101 (t+ to)'"

where I < ~ < 3 , to is the time between the moment of the ac tion of stress fac tor and the first stati stica ll y signi ficant change in the Is value (a delay time), A is a constant (amplitude), and ~ and m are decay constants. A good fit to the hyperbolic decay suggests that the observed emission cannot be the sum of uncorrelated excitation-emission processes on the molecul ar leve l. Rather it seems to support the hypothesis that there are cooperative phenomena correlated with physiological processes and states of the stressed organi sm. Many kinetics of irreversibly perturbed organisms (Table I) fi t to the hyperbolic function . Unambigous assignment of the decay requires, however, the change of I and t-values over at least two decades of the 1 and T-scale. This requirement is hardly attainable because of extremely weak photonic signal s.

Comparison of the photonic response from intact, ali ve and dead organisms previously treated with the same lethal doses of tox in shows that the dead body responds to the second (postmortem) treatment with a much lower (Is Ilj)max value than that of the alive one (during the first treatment). Moreover, the kinetic pattern of the photon count dn/dt = f (t) is significantly di fferent. Thus, the destructi on of intact structures and metaboli sm of the li ving state underlines enhanced photonic response.

Spectral anolysis- Spectral anal ys is of photon emission from poisoned dying animal organisms has been app li ed on ly in few cases because of low 1-va lues and short time of signals and extreme experimental difficulties. These analyses are mostly limited to ultraweak luminescence frolll the interact ion of ROS with animal cell cultures6

.11

.45.46A7.

Attempts to measure spectral distribution can be

illustrated with data on cockroach (Table I and Fig.4). Cockroach, Blatta orientalis, treated with acetone or TCA emits photons in a very broad spectral range 180-860 nm. This spectral range is ach ieved due to the use of a UV- and a red-sensiti ve photollluitiplier tubes. When the UV-sensiti ve PMT is used, a small fl at maximum around 380 Illn can be recorded from the insect exposed to acetone or TCA. However, thi s UV emiss ion is transient and has a short duration time of several minutes. Spectral distribution in the visible part of the spectrum can be measured more reliably due to a higher intensity and longer time. The spectra cover 450-860 nm wavelength range and have different maxIma for acetone and TCA-treated cockroaches, 550 nm and 660± 10 nm, respecti vely. The small spectral resolution > ± 10 nm and large experimental errors make it difficul t to extract a reliable information about the nature of emitters. A continuous di stribution of the emi ssion and a very broad spectral range of the observed photonic responses suggest that there are multiple excitation energy levels or a broadband and cooperativity between excitations and emission processes . In conclusion, photon emission responses obtained from the interaction between a living intact organism and stress/lethal chemicals provide the set of parameters that could find applicati on in toxicology for better characterization and prediction of the toxi n effects.

Perturbation of homeostasis and hypersensitive photonic response

In prev ious papers a semiquantitati ve model fo r the evaluation of biohomeostas is perturbations

TCA

Fig. 4 - Spectra l distribution of photon emi ss ion from a s ingle cockroach Blatta oriel/ta/is to trichloracetic acid (2 ml 10%) and acetone (2 ml ). The spectra were measured with a S-20 photo­C:llhode photomultiplier tube and a set of cU l-o ff filte rs. The widths of I'ectangles correspo nd to the short wavelength limi ts be tween two consec uti ve cut -off fitl e rs. The vert ical bars repre­sent average experimental e rrors.

"

SLA WINSKI: BIOPHOTONS FROM STRESSED & DYING ORGANISMS 491

has been elaborated on the basis of experimental data and far-from equilibrium (open systems) thermo-d . 9 II i? 48 Th ' d I f h h . . ynamlcs' . -'. IS mo e 0 t e omeostasls III a stable regul atory living system has a geometrical representation in the form of paraboloid I 1.43. It predicts that a small stimulus, e.g. toxin or phys ical stress causes reversible perturbations the amplitude of which is damped. However, when the bioregulatory system is already perturbed, it becomes more sensi­tive to any additional stimulus. This results from the fact that the system is nonlinear with respect to the stimulus-response value and amplifies perturbations stimulated by large doses of toxins or hi gh intensities of physical stresses. The same chemicals used at lower concentrations perturb homeostasis more weakly and reversibly than used at hi gher concen­trations . Experiments on photon emission from cucumber seedlings (Cucumis sati vus) perturbed with increasing concentrations of HCHO-a protein­denaturating toxin, confirmed these predictions49

. For the function 1= f ([HCHO]) the ratio J..l = Ig 111/ Ig 11 c is O. I I in the HCHO concentration (c) range 0.00 I 70.06 %, whi le J..l = Ig 11 I / Ig 11 c = 1.0 for the concentration range 0.17 1.0%. Thus, in the reversibly perturbed state of homeostas is, the biological system may become hypersensitive to external stimuli and thi s behaviour is reflected in the values of certain parameters of photon emission. This nonlinear sy nergetic behaviour of the perturbed biohomeostasis enabled us to evaluate the effect of homoeophatic drugs on photon emission of cucumber seedlings sen­siti zed with low concentrations (c < 1%) of HCH049

.

Another feature of biophoton response to st ress factors and particularly to toxins is the so-called "necrotic radiation" , accompanying irreversible perturbation of homeostasis and the death of organism. Since the photon emission is a holistic response of the whole organism to external stimuli, it may be useful in evaluation of the capacity of homeostasis, i.e. ability of a biological system to adaptation and resistance to drugs, toxins and xenobiotics. Therefore measurements of photon emission from organisms in the reversibly perturbed homeostas is i.e. in a hypersensitive state, may serve as a very sensitive noninvasive multiparametric method. Its potential utility to toxicolog ical problems has not yet been recognized.

Conclusion There is now growing interest in the development

of in vivo and in vitro methodology by which to

eval uate the toxicological potential of drugs, environmental contaminants, substances producing occupational hazard , poisons and xenobiotics. Toxicological perspective of the use of CL as a probe to investigate chemical-cell interactions, covering interactions with phagicytic cells, oxidant-mediated cel l injury, bioluminescent bacteria as an assay system for genotoxic agents and toxins for reproductive cells was the subject of a chapter in the extensive elaboration, Cellular Chemiluminescence. This article attempts to show possibly wide horizon of various aspects pertinent to the interaction of these substances with living organism as expressed by changes in biophoton emiss ion . It discusses not onl y the toxin-organism interactions, but also phenomena induced by pathogenic microorgani sms. Moreover, a new methodological approach is proposed, name ly hypersensitization of a biological system by a weak perturbation of homeostasis that results in the amplification of photonic response to stress factors. For all types of enzymatic and non-enzy matic reactions as well as physiological states occurring in biological systems, in which electronically excited states are generated and radiatively deacti vated producing biophotons, the monitoring and single photoelectron imaging of the emitted radi ation can be used to study alterations in molecular and cellular biochemistry , morphology and bioenergetics. Therefore the application of ultraweak photon emission as a holistic multiparametric probe for monitoring and evaluation of the perturbation of homeostasis, resistance to drugs, toxins and xenobiotics and prediction of lethal doses of toxins should be put into the subject of serious consideration.

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