Frequency Dependent Alterations of S. Cerevisiae Proliferation Due to LF EMF Exposure

BIOMEDICAL ENGINEERING VOLUME: 18 |NUMBER: 2 |2020 |JUNE F equency Dependen Al e a ions o S. Ce e isiae P oli e a ion Due o LF EMF Exposu e Roman RADIL1, Jan BARABAS 1, Ladisla JANOUSEK 1, Ma in BERETA2,3 1Depa men o Elec omagne ic and Biomedical Enginee ing, Facul y o Elec ical Enginee ing and In o ma ion Technology, Uni e si y o Zilina, Uni e zi na 1, 010 26 Zilina, Slo ak Republic 2Ins i u e o Measu emen Science, Slo ak Academy o Sciences, Dub a ska ces a 9, 841 04 B a isla a, Slo ak Republic 3Depa men o Radiologic Technology, Facul y o Heal h, Ca holic Uni e si y in Ruzombe ok, Names ie And eja Hlinku 1159, 034 01 Ruzombe ok, Slo ak Republic [email p o ec ed], [email p o ec ed], ladisla[email p o ec ed], ume be e@sa ba.sk DOI: 10.15598/aeee. 18i2.3461 Abs ac . The p esen ed pape deals wi h low e- quency elec omagne ic ield applica ion on Saccha- omyces ce e isiae cells. Expe imen s pe o med h ough wide equency ange ha e shown selec i e e- quency dependen biological esponse, which could be success ully p edic ed by ion pa ame ic esonance he- o y p oposed by V. V. Ledne . Al hough obse ed e- sul s gi e sa is ying answe o ques ions whe he o no elec omagne ic ields could a ec cell cul u es e en a non- he mal le els, he esea ch p esen ed he ein opens a mul i ude o ques ions abou he exac physical mech- anisms unde lying he obse ed mic oo ganism beha - io , as he heo y discussed wi hin he scope o his a - icle is s ill no comple ely unambiguous. Keywo ds Elec omagne ism, low equency elec omag- ne ic ields, p oli e a ion, cell memb ane, ion pa ame ic esonance. 1. In oduc ion I is well known ha he elec omagne ic condi ions in which we li e nowadays a e di e en om hose in which people used o li e in he pas . The majo i y o li ing beings a e con on ed wi h a i icial Elec o- Magne ic Fields (EMFs), which complemen he na u- ally occu ing EMF o he Ea h, and ques ions pe - aining o in luence he eo on biological sys ems a e mo e equen han e e . The numbe o expe imen al s udies in es iga ing EMF e ec s on li ing cells, issues o on human heal h in gene al, has g own apidly o e he las ew decades. Much a en ion is cu en ly being paid o esea ch e- ga ding po en ial connec ion be ween EMF exposu e and cance o o he diseases, which has been dis- cussed in a ious s udies [1], [2], [3], [4], [5], [6] and [7]. Nume ous wo ks ocusing on expe imen s wi h li ing cells ha e shown di e ences in he p oli e a ion p o- cess be ween cells exposed o EMF and con ol (non- exposed) cells [8], [9], [10], [11], [12], [13] and [14]. O he wo ks in es iga e animal beha iou when ex- posed o EMF [15] and [16] o possible genomic ins a- bili ies due o EMF exposu e [17], [18], [19] and [20]. The esul s o hese wo ks a e o en inconsis en and, as s a ed in Buchachenko [21], he biological elec o- magne ic e ec s some imes seem o be i ep oducible and con adic o y. As poin ed ou in Ma ko [22], he p oblem is o en discussed wi hin he scope o he mal e ec s wi h mi- no in e es on possible non- he mal mechanisms, e en when conside ing cu en ly accep ed s anda ds and ecommenda ions (e.g. om In e na ional Commi ee o Nonionizing Radia ion P o ec ion - ICNIRP, 1998). Ac ually, he e alua ion o EMF e ec s on biological objec s is much mo e complica ed due o p incipal di - e ences in each app oach, and no pa o he p ob- lem should be neglec ed, especially when Low F e- quency EMF (LF EMF) and weak signals, ha a e a below he ICNIRP ecommenda ions, a e conside ed. The complexi y o his p oblem is documen ed by many au ho s [23], [24], [25] and [26], who ied o p opose physical mechanism o biological LF EMF impac , bu he e is s ill lack o gene al accep ance o any p oposed heo y ac oss he scien i ic communi y. This ac is a c 2020 ADVANCES IN ELECTRICAL AND ELECTRONIC ENGINEERING 99 BIOMEDICAL ENGINEERING VOLUME: 18 |NUMBER: 2 |2020 |JUNE he same ime a s ong mo i a ional ac o o u he in es iga ion in his ield o esea ch. The Ion Pa ame ic Resonance (IPR) heo y, p o- posed by Ledne [25], [26] and [27], is one o he mos discussed models o LF EMF in e ac ion wi h bi- ological objec s. The assump ions o his heo y a e based on he Ion Cyclo on Resonance heo y (ICR), i s desc ibed by Libo [24], specula ing ha he physiological ac i i y o ce ain impo an ions can be al e ed when he equency o applied ime- a ying magne ic ield is equal o he equency o ion mo ion in a s a ic magne ic ield. Ions in IPR model a e ep esen ed by a ha monic oscilla o , bound o a speci ic loca ion a he su ace o he cell mem- b ane, and applica ion o combined magne ic ield should al e i s oscilla ions. The men ioned physical model included se e al impe ec ions, pa icula ly due o he p oblem o he mal noise, and was c i icized by Adai [28] and [29]. The heo y was de ended by Engs om [30] and men ioned impe ec ions we e ad- d essed by Ledne [26] and [31]. Despi e c i icism, he e emains an imp essi e body o expe imen al e i- dence ha can be aken as an empi ical basis o ICR and IPR hypo heses [8], [9], [10], [11], [12] and [13]. In gene al, as Halgamuge e al. concluded [33]: “. . . models based on elec ic in e ac ions ha e di icul- ies o ob ain a high enough signal- o-noise a io a low ield s eng hs. The ields ha a e needed o explain he opening o a memb ane channel p o ein a e un e- alis ically high. On he o he hand, models based on magne ic in e ac ions can easily ob ain a la ge enough signal- o-noise a io, because he le el o magne ic he - mal noise in issue is low. The eason ha hese models wo k, is he same eason ha makes he MRI echnique possible.” Ano he a emp o shed mo e ligh in o his e- sea ch a ea is p esen ed wi hin his a icle. The au- ho s op ed o expe imen ally in es iga e non- he mal e ec s o LF EMF on Saccha omyces Ce e isiae cells, a a ious uncommon equencies, o en neglec ed in o he esea ch wo ks. To gain be e ep oducibili y o conduc ed expe imen s, au ho s used hei inno a- i e exposi ion sys em desc ibed in [33], p oposed wi h he aim o achie ing he highes possible homogene- i y o magne ic lux densi y wi hin he exposed ol- ume, aking in o accoun sample dimensions and in- cuba o p opo ions. This sys em enables expe imen s wi h 3 pe i dishes (diame e o 9 cm, heigh o 2 cm - each), each i adia ed by a homogeneous magne ic ield. Ob ained esul s and obse ed biological eac- ions a e hen discussed wi hin he scope o he IPR heo y. 2. Ma e ials and Me hods Saccha omyces ce e isiae cells (b and VIVO – com- me cially a ailable om Lesa e Slo akia, Inc.) we e selec ed as he a ge biological objec o i adia ion by LF EMF. The eason o his choice is he g ow h analogy wi h cance cells [34], as well as p e iously published wo ks ega ding LF EMF e ec s on yeas cells [35] and [36]. 2.1. Expe imen al P o ocol Chemical labo a o y es ing p o ocol o he men ioned VIVO s and o S. Ce e isiae is publicly a ailable om company Mik olab, LLC. This means ha he mic obi- ological, physical and chemical p ope ies and pa am- e e s o in es iga ed mic oo ganisms we e known p io o he expe imen s. Cells we e cul i a ed on GKCH aga , in acco - dance wi h STN EN ISO 7218 (560104) and STN ISO 7954 (560087) s anda ds and speci ica ions. Cul i- a ion was pe o med unde iden ical ambien condi- ions o 72 hou s, wi h empe a u e o bo h exposed and con ol samples se sligh ly abo e oom empe - a u e, speci ically o 27 ◦C. Con inuous empe a u e moni o ing was pe o med using h ee Nega i e Tem- pe a u e Coe icien (NTC) he mis o s and p ocessed and logged in eal- ime. Two o he NTC he mis o s we e placed wi hin he incuba o chambe s o moni o he empe a u e o bo h exposed and con ol samples, and he hi d he mis o was placed ou side he in- cuba o as a e e ence senso . To a oid any e ec s om ai low, humidi y, o changes o ligh condi ions, he exposed samples we e held in a solid plas ic holde , ma ching he diame e o he coil ca i y. 2.2. Expe imen al Se up and E alua ion Me hods The expe imen al se up, used o all o he p esen ed expe imen s and de ailed in [37], is schema ically shown in Fig. 1. The se up was speci ically designed o pai ed sample expe imen s, conduc ed on wo se s o h ee Pe i dishes inocula ed by S. Ce e isiae. One se was placed wi hin one chambe o incu- ba o , housing he mu-me al magne ic shielding box. This se was he con ol, o e e ence sample - being shielded om he gene a ed LF EMF. The second se o samples, ep esen ing he exposed cells, was placed a speci ic posi ion wi hin he al eady men ioned de- eloped coil sys em, depic ed wi hin he eal scheme o expe imen al se up on Fig. 2. The coil sys em was d i en by ha monic cu en (gene a ed by he Agilen 33220A signal gene a o , manu ac u e Agilen Tech- c 2020 ADVANCES IN ELECTRICAL AND ELECTRONIC ENGINEERING 100 BIOMEDICAL ENGINEERING VOLUME: 18 |NUMBER: 2 |2020 |JUNE nologies, Inc.), and ampli ied using a linea ampli ie (Hube A1110-05, manu ac u e D . Hube GmbH) - he esul ing ampli ude Imbeing in he 0.96–1.09 A ange. The exac alue o exci a ion cu en depended on he impedance ma ching and equency used, so as o achie e he equi ed magni ude o magne ic lux den- si y. The expe imen was epea ed o 5 imes a said equency. PC NI El is AmG MS CS ES Incuba o A Fig. 1: Expe imen al se up: G - signal gene a o Agilen 33220A; Am - ampli ie ; A - ampe e me e ; Incuba o wi h wo chambe s: ES - exposed samples placed wi hin he exci a ion coil, CS - con ol samples placed wi hin he MS - magne ic shielding box; NI El is II - de el- opmen boa d o empe a u e da a acquisi ion; PC - empe a u e moni o ing and isualiza ion. Fig. 2: P opopsed and cons uc ed expe imen al se up show- ing he applica o used in p elimina y expe imen s and he newly designed applica o . F om he a o e men ioned i is e iden ha samples we e i adia ed by LF EMF wi h magne ic lux densi y ~ B, which a ies spa ially wi hin he exposed olume. The exac alue o ~ B- ield p oduced by he coil sys em was alida ed by measu emen s, using sensi i e lux- ga e senso s. Since his me hod is desc ibed in mo e de ail in [33] and [38], o he pu pose o his a icle, i is su icien o men ion he conclusion ha eal-li e measu emen s e-con i med alues ob ained om nu- me ical simula ions, documen ing minimum magne ic lux a ia ions (max. 5 %) wi hin he i adia ed ol- ume, when using he p oposed exci a ion coil sys em. The alue o magne ic lux densi y a ied be ween ~ B=2.33–2.45 mT, and was equency independen . A e con inuous i adia ion, he g ow h dynam- ics pa ame e o bo h con ol and exposed samples was quan i ied. Speci ically, g ow h dynamics we e compu ed ia semi-au oma ed so wa e called Pe i- Coun e , which was de eloped as a eplacemen o comme cially a ailable coun e s. Algo i hm he eo is de ailed in [39]. P incipally, i allows de ec ion and coun ing o yeas g ow h a eas, using me hods o im- age p ocessing, analysis, and ea u e ex ac ion, o en- able expe imen al esul s quan i ica ion and compa i- son o obse ed e ec s. S a is ical signi icance o ob- se ed esul s was inally e alua ed using S uden ’s T- es o pai ed samples, wi h signi icance le el α= 0.05. 3. Resul s and Discussion As was men ioned in p e ious sec ion o his a icle, expe imen s we e ocused on he beha iou o Sac- cha omyces ce e isiae cells in luenced by exogenous LF EMF o di e en equencies p oduced by he de- signed coil sys em. Ou wo k ollows p e iously con- duc ed expe imen s published in [40], [41] and [42], bu he equency ange di e s, and is wide in compa - ison wi h p elimina y expe imen s. A i s , we e- e alua ed he base equency o 1.6 kHz, which was ex- pe imen ally p o en as s a is ically ele an o he in- hibi o y e ec on S. Ce e isiae p oli e a ion. The i s se o expe imen s was hen conduc ed wi h equen- cies o 1.6, 0.8, and 0.4 kHz o e i y hypo hesis ega d- ing e ec s o subha monic equencies and co ela ion he eo wi h IPR heo y, p esen ed in [30]. Resul s o hese expe imen s a e shown in Tab. 1. Tab. 1: Compa ison be ween expe imen al esul s obse ed a i adia ion signal equencies and IPR p edic ed e- quencies a speci ied magne ic lux densi y a ge ing Ca2+ ion. Magne ic lux 2.39 2.39 2.39 2.39 densi y (mT) F equency 1.600 0.800 0.400 0.200 gene a ed (kHz) F equency 1.596 0.798 0.398 0.204 p edic ed (kHz) G ow h a ea a io 0.59 0.54 0.64 0.80 (exposed/con ol) F om he expe imen al esul s i is clea ha all sub- ha monic equencies ha e shown inhibi o y e ec s and s a is ically signi ican biological esponses a e p esen a equencies 0.8 and 0.4 kHz, as well as he base e- quency. These equencies co espond o he ion pa a- me ic esonance equency o Ca2+ ions, and ha monic componen s he eo in acco dance wi h Ledne ’s IPR heo y om [25], [26] and [31] and he Eq. (1): = c n=1 n · q 2πm ·Bgen,(1) whe e c ep esen s he cyclo on esonance equency o he ion, qis he elemen a y cha ge o he ion, mis c 2020 ADVANCES IN ELECTRICAL AND ELECTRONIC ENGINEERING 101 BIOMEDICAL ENGINEERING VOLUME: 18 |NUMBER: 2 |2020 |JUNE 0.850 0.800 0.890 0.640 0.899 0.690 0.540 0.750 0.864 0.590 0.720 0.650 0.100 0.200 0.300 0.400 0.700 0.750 0.800 0.850 0.900 1.600 1.800 2.000 F equency (kHz) 0 0.2 0.4 0.6 0.8 1 1.2 G ow h a ea a io (exposed/con ol) Fig. 3: G aphical in e p e a ion o p oli e a ion esponse o i adia ed samples wi hin he in es iga ed equency ange o applied ime- a ying LF EMF. mass o he ion and ndeno es he co esponding ha - monic componen . Bgen is he mean alue o gene a ed magne ic lux densi y, which in ou case was 2.39 mT. P edic ed equencies o he biological esponse based on his model a e also shown in Tab. 1. I is e iden ha expe imen al equencies a which s a is- ically signi ican biological e ec s we e obse ed a e closely ela ed o hei heo e ically p edic ed coun e - pa s. Also appa en om he p esen ed se o expe i- men s is ha he biological e ec ends o disappea a lowe equencies, as was he case in expe imen s using 0.4 kHz. To e i y his obse a ion, we op ed o in es- iga e a wide equency ange, wi h ocus on low e- quencies and also on equencies a ound he expe imen- ally p o en biologically ac i e equency o 0.8 kHz. This se o expe imen s is p esen ed g aphically in Fig. 3, o be e illus a ion and in e p e a ion he eo . Obse ed esul s once again show inhibi o y e ec s. Mo e in e es ingly, he e a e signs o pe iodici y wi hin he expe imen al da a, wi h s ong peaks a p e iously desc ibed esonance equencies o Ca2+ ha could be p edic ed by IPR model. Fu he mo e, he p e ious as- sump ion, ega ding he biological e ec ex inc ion a lowe equencies, seemed o occu a equencies below 0.4 kHz. Ano he in e es ing obse a ion conce ns he neigh- bou hood o he equency 0.8 kHz, whe e he p oli e a- i e esponse is weak in compa ison wi h he esonance (base) equency, albei s ill p esen . This obse a ion poin s o he window heo y, men ioned by Ma ko in [22], bu could be also explained as a cumula i e e - ec o o he ions, which play an impo an pa in cell p oli e a ion p ocesses. Since equency dependen biological esponse is e - iden om he p esen ed esul s, we op ed o also in es iga e he in luence o magne ic lux densi y ~ B o he gene a ed LF EMF. Fo his pu pose, we chose he equency o 0.8 kHz, whe e he biological esponse seemed o be mos signi ican , and al e ed he alue o ~ B o 50 %, 25 % and 10 % o i s o iginal alue. These al e a ions we e echnically pe o med by am- pli ude changes o e ec i e alue o d i ing cu en , which di ec ly in luences he ampli ude o ~ Bin he coil ca i y and hus he exposed olume. Resul s p esen ed in Fig. 4 show ha signi ican ly s ong p oli e a i e esponse is p esen only wi h o igi- nal magne ic lux densi y o 2.39 mT. The biological e- sponse is weak o no longe obse ed a gi en equency due o modi ied magne ic lux densi y o he applied LF EMF. F equency 800 Hz 2.39 1.195 0.5975 0.239 Bgen (mT) 0 0.2 0.4 0.6 0.8 1 1.2 G ow h a ea a io (exposed/con ol) 0.56 0.79 0.94 0.90 Fig. 4: G aph o p oli e a ion dependence o S. Ce e isiae on Bgen, a he equency o 800 Hz. c 2020 ADVANCES IN ELECTRICAL AND ELECTRONIC ENGINEERING 102 BIOMEDICAL ENGINEERING VOLUME: 18 |NUMBER: 2 |2020 |JUNE 4. Conclusion The p esen ed a icle discusses chosen heo ies ela ed o LF EMF mechanisms o in e ac ion wi h biologi- cal objec s, ep esen ed he ein by Saccha omyces ce e- isiae cells. Repe i ion o p e iously conduc ed expe - imen s published in [8], [38], [40], [41] and [42] con- i med inhibi o y e ec on he p oli e a ion esponse a he equency o 1.6 kHz. Fu he mo e, he expe - imen al esul s clea ly demons a ed non- he mal e - ec s o ex e nally applied ime- a ying LF EMF on he p oli e a ion p ocess o cul i a ed cells. Ou ind- ings ega ding he Ca2+ a e also in co ela ion wi h esea ch p esen ed in [43]. The obse ed biological e- sponses could be e e ed o as equency selec i e be- cause s a is ically signi ican e ec s occu only a spe- ci ic equencies, which poin s o he esonance cha ac- e o he obse ed bio-e ec s. The mechanism o ac ion in luencing he beha iou o he i adia ed biological samples could be heo e i- cally explained by he IPR heo y. This model success- ully p edic s obse ed biologically ac i e equencies, conside ed esonance equencies o Ca2+ ions and ha - monic componen s he eo . F om his poin o iew, we can d aw he conclusion ha Ca2+ ions play an im- po an ole in cellula p oli e a ion p ocesses, and could be exploi ed as a ge s o i adia ion by ex e - nal LF EMF uned o pa ame ic esonance equency he eo , which co esponds wi h indings p esen ed by Belo a [27]. The ele ance o V. V. Ledne ’s heo y can be also con i med by esul s o ou in es iga ion o magne ic lux densi y changes wi h espec o p oli - e a ion esponse a unchanged equency. This means ha when expec ing biological esponse in acco dance wi h he IPR heo y and Eq. (1), any change o ~ Bnec- essa ily leads o change o he ion pa ame ic esonance equency, bu no e e y change o equency mus lead o change o magne ic lux densi y. Despi e he indings p esen ed wi hin his a icle, an unambiguous explana ion o EMF in luence on li - ing s uc u es is s ill lacking. 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DOI: 10.1007/s10867-018-9500-0. c 2020 ADVANCES IN ELECTRICAL AND ELECTRONIC ENGINEERING 105 BIOMEDICAL ENGINEERING VOLUME: 18 |NUMBER: 2 |2020 |JUNE Abou Au ho s Roman RADIL was bo n in T encin, Slo ak Republic, in 1984. G adua ed om he Facul y o Elec ical Enginee ing, Uni e si y o Zilina, in 2008 om Biomedical Enginee ing and ecei ed he Ph.D. deg ee in Theo e ical Elec omagne ics a he same uni e si y, in 2012. A p esen he wo ks as assis an p o esso a he Dep . o Elec omagne ic and Biomedical Enginee ing, Facul y o Elec ical Enginee ing and In o ma ion Technology, Uni e si y o Zilina. His esea ch ac i i ies a e ocused on in es- iga ion o LF EMF e ec s on biological samples and biomedical image p ocessing. Resul s o his esea ch ha e been published in se e al jou nals and p esen ed in na ional and in e na ional con e ences. Jan BARABAS was bo n in Ba dejo , Slo ak Republic, in 1982. G adua ed om he Facul y o Elec ical Enginee ing, Uni e si y o Zilina, in 2008 om Biomedical Enginee ing and ecei ed he Ph.D. deg ee in Theo e ical Elec omagne ics a he same uni e si y, in 2011. A p esen he wo ks as esea che a he Depa men o Elec omagne ic and Biomedical Enginee ing, Facul y o Elec ical Enginee ing and In o ma ion Technology, Uni e si y o Zilina. His cu en esea ch endea ou s include ongoing in es- iga ion o elec omagne ic ields and in e ac ion he eo wi h biological objec s, he indings o which ha e been published in se e al jou nals and in e na- ional con e ences. O he ac i i ies include biomedical image p ocessing, signal p ocessing and p og amming. Ladisla JANOUSEK was bo n in Michalo ce, Slo ak Republic, in 1974. G adua ed om he Facul y o Elec ical Enginee ing, Uni e si y o Zilina, in 1997 om Elec ic Powe Sys ems and ecei ed he Ph.D. deg ee in Elec ic Machines and Appa a uses a he same uni e si y, in 2002. A p esen he is head o he Depa men o Elec omagne ic and Biomedical Enginee ing, Facul y o Elec ical Enginee ing and In- o ma ion Technology, Uni e si y o Zilina. The main ields o his esea ch ac i i ies a e elec omagne ic me hods o ma e ials’ non-des uc i e e alua ion and non- he mal e ec s o EMF on li ing o ganisms. His achie emen s ha e been acknowledged by se e al p es igious awa ds. Ma in BERETA was bo n in Ruzombe ok, Slo ak Republic, in 1990. G adua ed om he Facul y o Elec ical Enginee ing, Uni e si y o Zilina, in 2014 om Biomedical Enginee ing and ecei ed he Ph.D. deg ee in Theo e ical Elec omagne ics a he same uni e si y, in 2017. A p esen he wo ks as esea che a he Ins i u e o Measu emen Science, Slo ak Academy o Sciences in B a isla a. His esea ch ac i i ies a e ocused on LF EMF biological e ec s and moni o ing o ul a-weak pho on emission om biological samples. Resul s o his esea ch ha e been published in se e al jou nals and p esen ed in na ional and in e na ional con e ences. c 2020 ADVANCES IN ELECTRICAL AND ELECTRONIC ENGINEERING 106