Cytogenetical effects of sonication in mice and their modulations by actinomycin D and a homeopathic drug, Arnica 30

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1 Indi an Journal of Experimental Biology Vol. 39, December 2001, pp Cytogenetical effects of sonication in mice and their modulations by actinomycin D and a homeopathic drug, Arnica 30 Jayati Chakrabarti, Surjyo Jyoti Biswas & Anisur Rahman Khuda-Bukhsh* Department of Zoology, University of Kalyani, Kalyani, Nadia , India Received 11 April 2001; revised 17 August 2001 Experiments were designed to examine if Actinomycin D, an antibiotic, and Arnica 30, a homeopathic drug used against shock and injury, can ameliorate cytogenetic damage induced by single or multiple exposures to ultrasonication. Separate sets of healthy mice were directly exposed to sonication for two minutes either once or they received multiple exposures at an interval of 20 days. The mice were then assessed at different intervals, against suitable controls, using parameters like chromosome aberrations (CA), mitotic index (MI), sperm head anomaly (SHA) and micronucleated erythrocytes (MNE). Separate groups of sonicated mice were ei ther orally administered with Arnica 30 (alcohol 30 in control) or injected intramuscularly with Actinomycin-D (AMD). Elevated frequencies of CA, MI, MNE and SHA were noted in sonicated series. AMD had genotoxic effects of its own and also had additive effects on sonication induced genotoxicity. Sonicated mice fed with Arnica 30 showed appreciably reduced genotoxicity as against alcohol 30 and distilled water fed controls, thereby showing ameliorating effect which may have human application. The effect of sonication on mammalian genetic system appears to be inadequately studied and the results have been rather inconclusive. While some authors suggested no significant effects in in vitro system 1-3, others claimed some positive genotoxic changes in various in vivo test 4-9. However since recent work also demonstrated some positive cytogenetical changes in vivo in three test models, grasshoppers, fish and mice, subjected to whole-body ultrasonications 10, it was of interest to search for any agent that would have no cytogenetical ill effects of its own, but could protect the ultrasound induced cytogenetical damages. This seemed important because the use of ultrasonic sound waves as a tool in medicine can not be objected to for its superiority and several advantages over other methodologies like X-rays in pin-pointing location/site of abnormalities in internal anatomy of human beings without causing any apparent injury to them. Of various antibiotics, Actinomycin-D has been reported to have anti-radiation activity 11, but it was also reported to have a great deal of genotoxic effect of its own On the other hand, the homeopathic drugs, which are used in ultra-low doses and are known to have no toxic side-effects, are becoming increasingly popular in both developing Asian countries and developed European L:Ountries 14 after their clinical efficacy was supported by many wellconducted research publications including two major *Correspondent author: arkb@klyuniv.ernet.in Phone : (033) Extn. Zoology (313/315) meta-analysis Keeping the above in view the homeopathic drug, Arnica Montana, commonly used against shock and injury has been examined for its possible protective role against ultrasonication. Incidentally, the potentized form of the homeopathic drug, Arnica Montana 30, had earlier been reported to have anti-clastogenic activities against X-ray induced cytogenetical damage Materials and Methods Healthy Swiss albino mice (Mus musculus) of both sexes weighing between g, reared and maintained under the supervision of the Animal Welfare Committee, Department of Zoology, Kalyani University, were used. Experimental design Control series Unsonicated healthy mice were examined for their baseline chromosome aberrations and other protocols used (S 1 ). Another set of unsonicated healthy mice were fed with Arnica 30 (S 2 ) alone in doses described below and were scanned for the data at the same fixation intervals as in sonicated lot. Exposure to sonication Single exposure series For homeopathic drug series, 9 batches of 5 mice each were subjected to whole-body ultrasonic sound waves from an ultrasonic cell disrupter machine (LSL, SECFROID, Switzerland) operating at a

2 1236 INDIAN 1 EXP BIOL, DECEMB ER frequency wave of 23 KHz, and at an energy output percentage of 70 for a total peri od of 2 min (twice for I min each with an interval of I min in between). Mice receiving this two min exposure conformed the materi als for the single exposure study. Mice were immobilised during sonicati on in a thin wet cloth bag and the lower part of the body submerged in water in glass beaker. The beaker was surrounded with ice before start of sonication to avoid any possible rise in temperature of water inside beaker during sonicati on. Out of the 9 batches, 3 were sacri ficed at 2, 3 at 24 and the other 3 at 48 hr. Among these 3 batches, I batch of mice was orally ad ministered w ith pote nti zed homeopathic drug, A r ni ca 30 [HAPCO, Kolkata,(0.06 ml of liquid Arnica 30 diluted with 20 ml double di still ed water (ddw) to make the stock solution of the drug, from whi ch l drop, i.e ml was fed thrice at an interval of 30 min to mi ce whi ch were sacri ficed at 2 hr and th rice a day at an interval of 8 hr to mice sacrificed at 24 and 48 hr)]. Another batch of mice was fed with dilute succussed alcohol (alcohol 30) (positi ve control I) prepared as per homeopathic potenti zati on procedure similarl y at corresponding intervals of time and the third batch of mice were neither fed with the homeopathi c drug, nor with the diluted alcohol measured as per homeopathic principle (i.e. alcohol 30), but was allowed to take only dd w at corresponding intervals (positive control 2). Another set of unsonicated healthy mice served as negati ve control. Similarl y, in Actinomycin D (AMD) treated series, 5 batches of mi ce, unsoni cated and soni cated, were intramuscul arl y inj ected with % Actinomycin lml/100 g body weight and were sacrificed at 2, 24 and 48 hr. Contro ls were maintained fo r sonicated mice injected with distilled water (AMD dissolves in water). In another set of experiments, soni cated and AMD injected mice were either fed with the homeopathic drug, Arnica 30, or with alcohol 30 (as control of homeopathic drug, Arnica, the "vehi cle" of the drug being eth yl alcohol). As AMD itself showed a good amount of clastogeni c effect, whi ch could onl y be partiall y reduced by the feedi ng of the homeopathic drug (Table 1), further experimentation with AMD was not continued. Multiple exposure series Four sets of mice (each set compnsmg 5 mi ce) were subjected to repeated exposures as mentioned above for 2 min each at an interval of 20 days, so that mice sacrificed at 30, 60, 75 and 90 days after the initi al exposure actually received 2, 3, 4 and 5 such exposures to sonicati on, respecti vely. In the repeated exposure seri es, 1 batch of sonicated mice was orall y administered with th e stock solution of potenti zed homeopathic drug, Arnica 30 in the arne way, but at an interval of 12 hr till sacrificed. Onl y one control was maintained in this series, that of th e alcohol 30 fed control, as there was no signi fica nt di fference between the succussed alcohol 30 fed control and ddw-fed controls. Observers were " blinded" during observati on and scoring of data on various cytogenetical parameters like chromosome aberrati ons (CA), mitotic index (Ml), mi cronucleated erythrocytes (MNE) and sperm head anomaly (SHA). MI was not scored in the repeated exposure seri es. Chromosome aberration study-mice at all fixation intervals were inj ected intraperitoneall y with 0.03% colchicine l mill 00 g body weight 1 'h hr prior to sacrifi ce. The conventi onal citratefl ame drying-giemsa technique was followed for the bone marrow chromosome preparati on. Micronuclei testing and mitotic index studies-a part of suspension of bone marrow -::e ll s in 1 % sodium citrate solution was smeared on clean grease free slides. The slides were briefl y fi xed in methanol and subsequently stained with May-Grunwald soluti on followed by Giemsa staining 20. Sperm head anomaly study-epididy mis of each side of the control and treated male mice was di s sected and taken out separately into 10 ml of 0.87% normal saline. The inner contents were taken out and thoroughly shaken to make the sperm suspend in saline solution. The suspension was filtered th ro ugh a silken cloth to remove debris and was dropped on clean grease-free slides uni fo rml y. The slides were allowed to air-dry and then stained in dilute Giemsa h. d 2 1 as per t e routme proce ure. Results The frequencies of chromosome aberrati ons in the sonicated mi ce, sonicated and unsonicated mi ce injected with AMD, soni cated mice inj ected with Actinomycin D and also fed with the homeopathic drug, Arnica 30, have been presented along with the controls in Table 1. The frequencies of CA in the AMD treated unsonicated mice at different fixation intervals were 23.6%, 20.8%, and 19.4%, at 2 br, 24 hr and 48 hr, respecti vely. However, when compared against the only sonicated lot, the frequencies in the soni cated and AMD treated mice were found to be enhanced,

3 CHAKRABARTI eta/.: SONICATION EFFECTS IN MICE & THEIR MODULATIONS 1237 Table!-Frequencies of chromosome aberrations in 500 bone marrow cells examined ( 100 cells from each 5 individuals) at different fixation intervals in single exposure series: Unsonicated, AMD treated, Sonicated+ AMD treated, Sonicated+ AMD treated + Arnica-30 fed and Sonicated + AMD treated+ alcohol 30 fed Fixation Series Chromosome aberrations Intervals % ±SE % of Qrotection Unsonicated Sonicated+ AMD Sonicated+ AMD + Arnica-30 vs vsamd vsamd Sonicated+ AMD + alcohol 30 2 hr 24 hr 48 hr Unsonicated 0.40 ± 0.25 AMD 23.6 ± 0.75 Sonicated + AMD ± 1.30 Sonicated+ AMD + Arnica ± 0.51 Sonicated + AMD + alcohol ±0.86 AMD ± 0.66 Sonicated + AMD ± 1.41 Sonicated + AMD + Arnica ± Sonicated + AMD + alcohol ±0.93 AMD ± 1.60 Sonicated + AMD ± 0.97 Sonicated + AMD + Arnica ± 0.98 Sonicated + AMD + alcohol ± c b 20.40c b *Chromosome aberrations include stickiness, precocious centromeric separation, erosion, condensed, crumpled, c-mitotic effect, polyploidy, aneuploidy etc. SE= Standard Error, bp < 0.001, cp < 0.01 % level of significance at t-test. showing apparently additive action of AMD on sonication-induced genotoxicity. The oral administration of Arnica 30 to these AMD treated sonicated mice only marginally reduced the frequency of aberrations, while the Alcohol 30 in sonicated mice tended to increase the damage in some cases. The frequencies of various chromosome aberrations at different fix ation intervals encountered in unsonicated mice (S 1 ), unsonicated fed with Arnica 30 (S 2 ), only sonicated mice (S 3 ), in sonicated mice fed with the homeopathic drug Arnica 30 (S 4 ) and sonicated mice fed with alcohol 30 (S 5, positive control) have been summarized in Tables 2 and 3 for single exposure and Tables 4 and 5 for multiple exposures to sonication. Representative photomicrographs of various types of chromosome abnormalities (Figs 1-9), micronucleated erythrocytes (Figs I 0 and 11 ) and sperm head anomalies (Figs 12-14) have been provided. The chromosome complements in both the experimental and control sets of mice were critically studied for possible abnormalities. In the normal healthy unsonicated mice (negative control), out of some 500 bone marrow cells examined, normal complements (Fig. I) were obtained in all but two cell s, one of which showed an achromatic lesion and another which contained a chromosome wi th a constriction, that made the spontaneous aberration baseline as only 0.04%. Similarly, the baseline for micronucleated erythrocytes examined from 2000 cells and sperm head anomaly examined from some 2000 sperm in normal healthy unsonicated controls were extremely low, the mean being % for MN and 0.002% for SHA. In unsonicated healthy mice fed with Arnica 30 alone, no statistically appreciable difference in any of the protocols used was noticed. Therefore, the oral administration of Arnica 30 did not itself bring any apparent clastogenic/genotoxic effects in mice. On the other hand, in the sonicated mice the percentages of chromosome aberrations, mostly of the physiological and numerical types (Figs 2-6), were at 2 hr, at 24 hr and at 48 hr which were all statistically significant at various levels (Table 1 ). Similarly the incidence of micronuclei induction was 0.44% in the sonicated mice at 24 and 48 hr. The same kind of increase in the frequencies of sperm with abnormal head shape was noticed in the sonicated mice, being 1.84 and 1.48% at 24 and 48 hr, respectively. There w a~: also some increase in the mitotic index in the sonicated mice, being 2. 70% at 24 hr and 2.06% at 48 hr as compared to about 0.69% in unsonicated normal mice. Therefore, there was a positi ve change in these cytogenetical parameters even for the single exposure to ultrasonication. However. no break type or other more seri ous type of aberrations

4 1238 INDIAN J EXP BIOL, DECEMBER 2001 '/... ',.,,,,'"" \',_. \ \ ~ v "' '. \-.....""'='!?. ~,..--,, ml _., ill - - _rn Fig Photomicrographs of normal (I) and aberrated (2-9) metaphase complements of mice {crumpled (2), pulveri sed (3), C mitotic effect (4), stickiness and pol yploidy (5), stickiness and ring (6), chromatid break and constriction (7), terminal association (8) and acentric fragment (9) }. micronucleated polychromatic (I 0) and normochromatic (II) erythrocytes, sperm with norma.! ( 12) and abnormal head shape (13-14). [R= Ring, BS =Break, TA= Terminal Association, F= Fragment; Bar= 10 11m.]

5 CHAKRABARTI eta/.: SONICATION EFFECTS IN MICE & THEIR MODULATIONS 1239 (major type) were encountered in this group of sonicated mice. In mice receiving multiple exposures, not only the percentages of CA (Table 4) were increased appreciably, but also the "major type" (Figs 7-9) aberrations appeared at all the four longer intervals, though not necessarily in a strictly cumulative manner. The percentages of MN, however, increased along with time in sonicated mice (Table 5); the same was true for the incidence of SHA, thereby showing a somewhat "time-dependent" and "cumulative" effect of sonication. The frequency of chromosome aberrations, which was at its peak at 24 hr, however, apparently declined appreciably at 48 hr, presumably because part of the aberrations were either restituted or else heavily damaged ones were eliminated after the cell cycle. Interestingly enough, in the majority of cases, wherever Arnica 30 was fed to sonicated mice, there was a favourable alteration in the damaging effect, practically for all the parameters used (Tables 2-5); and the results were statistically significant at various levels (Tables 2-5). Discussion Even a single exposure to ultrasound in adiation could produce quantifiable genotoxic effects in mice as compared to normal unirradiated controls 10. Repeated Table 2-Frequency distribution of chromosome aberrations in 500 bone marrow cells examined ( 100 cells from each of 5 individuals) and mitotic indices at different fixation intervals in single exposure series: S1-unsonicated, S 2 - unsonicated plus Arnica-30 fed Sr sonicated, S4-sonicated plus Arnica-30 fed and S 5 -sonicated plus alcohol-30 fed Fix. Intervals Series Chromosome aberration Mitotic index % of Erotection % of Erotection % ±SE % ±SE s1 vs s 3 s) vs s4 S4 vs Ss s1 vs s) s ) vs s 4 s 4 VS Ss sl 0.04 ± ± s2 0.03± ± 0.02 s ) ± hr. s ± c 11.00c 10.80c Ss ± 0.42 s) ± ± hr. s ± c 11.40c 6.20b 2.26 ± c b Ss ± ± 0.11 s) ± ± hr s ± c 8.ooc 6.00< 1.78 ± c Ss ± ± 1.00 *Chromosome aberrations include stickiness, precocious centromeric separation, erosion, condensed, crumpled, c-mitotic effect, polyploidy, aneuploidy etc. SE= Standard Error, p < 0.05, bp < 0.001, <p < % level of significance at t-test. Table 3-Frequency distribution of micronuclei in normochromatic erythrocytes (NCE) and polychromatic erythrocytes (PCE) ( 1000 erythrocytes in each individual) and sperm with abnormal head shape (1000 sperm from each individual) at different fixation intervals in single exposure series: S1-unsonicated, Sr unsonicated plus Arnica-30 fed S 3 -sonicated, S4-sonicated plus Arnica-30 fed and S 5 -sonicated plus alcohol-30 fed Fix. Intervals Series M_i-'-c--'ro'-n-'-u cl-'-e'-i i_n-'-p--'c=-=e=-a"-'n.:..:.d-'n_c.::..e=- S::..E"-'e"-rm:..:.:...:.h.:..:.e..:c.ad.::...:c.anc..:o:..;_m:.:.:a.:..:.ly, % ± SE P/N % of Erotection % :!: SE % of Erotection s 1 vs S 3 S 3 vs s 4 s4 vs S 5 s 1 vs S 3 S 3 vs s 4 s4 vs s 5 s, s 2 s ) 2hr s4 Ss s ) 24hr s 4 Ss s ) 48hr s 4 Ss 0.001± ± ± ± ± ± ± ± < c SE= Standard Error, p < 0.05, bp < 0.00 I, <p < 0.01 % level of significance at t--test ± ± ± ± ± ± ± ± c 0.98" 0.84b 1.48b 0.78" 0.70c

6 1240 INDIAN J EXP BIOL, DECEMBER 2001 exposures to ultrasonic waves further increased the extent of cytogenetic damage. Earlier workers did not get elevated frequencies of SCE in cultured lymphocytes of human subjects exposed to ultrasonic sound waves. On the other hand, several workers reported positive effects of ultrasonic sound waves in lymphocytes of human beings and in egg lecithin. Positive genotoxic effects of ultrasonic sound waves were also observed in fish genetic system 8. Chatterjee and his co-workers also documented positive changes in enzymes related to lipid peroxidation and strongly held the view that ultrasonic irradiation caused cytotoxicity. Therefore, ample evidence has accumulated now which would suggest that ultrasonic sound waves really cause some genomic damage to the exposed organisms. The biophysical effects of ultrasound in aqueous solutions can be categorized as thermal effects, cavitation and direct effects 31. The mechanism of action of ultrasound is quite complex; in aqueous media the non-thermal effects of ultrasound is mainly due to cavitation. The degradation of the cavitation bubbles produces free radicals 32 and induces temporary local shock waves. On the other hand the "to and fro" motion of the cavitation bubbles produces hydrodynamic shearing stress This results in degradation of DNA in aqueous solution and even destruction of cells Therfore, present observations of the different forms of cytogenetic damage caused by ultrasonication can be explained in the light of the above findings, as also for the mechanical and psychological stress caused due to exposure of sonication. Further, the present findings suggest that AMD, which is also a transcription-blocker, had genotoxic effect of its own. It possibly bound itself to DNA by intercalating between bases and thereby changed the normal milieu of the DNA and interfered with normal proofreading activities that might in turn be responsible for the different aberrations encountered in AMD treated mjce. Further, it tended to increase the damage already produced in sonicated mice. Thus, the use of AMD may not be advisable as a protective measure against sonication. On the other hand, the homeopathic drug Arnica 30 was found to modulate favourably the cytogenetic ill-effects of ultrasonication in mice while the administration of alcohol 30 appeared to increase the damaging effect of sonication. Although Amica is claimed to have profound regulatory action on various systems like blood vascular, CNS, skin etc., on which sonication might have produced some stressful effects, the exact mechanism of action of this drug could not be known. Table 4-Frequency distribution of chromosome aberrations in 500 bone marrow cells examined ( I 00 cells from each of 5 individuals) at different fixation intervals in repeated exposure series: S 1 -unsonicated, Sr unsonicated plus Arnica-30 fed, Sr sonicated, S 4 -sonicated plus Arnica-30 fed and S 5 -sonicated plus alcohol-30 fed Fix. Intervals Series Chromosome aberration %± SE % of protection 30 days s ± 0.25 s ± 0.27 s ± 0.23 s ± 0.86 s ± o days s ± 0.77 s ± 0.68 s ± c days s ± 0.50 s ± 0.50 s ± c s ± days s ± ooc Ss ± 3.29 *Chromosome aberrations include gap, break, centric fusivn, translocation, fragment, pul verisation, ring, terminal association, polyploidy, aneuploidy, precocious centromeric separation, centromeric stretching, stickiness, c-mitotic effect,, etc. SE =Standard Error, a P < 0.05, b?<0.001, c P < % level of significance at t-test.

7 CHAKRABARTI eta/.: SONICATION EFFECTS IN MICE & THEIR MODULATIONS 1241 Table 5 - Frequency distribution of micronuclei in normochromatic erythrocytes (NCE) and polychromatic erythrocytes (PCE) (I 000 erythrocytes in each individual) and sperm with abnormal head shape ( 1000 sperm from each individual) at different fix ation intervals in single exposure series: S1-unsonicated, Sr unsonicated plus Amica-30 fed 53-sonicated, 54-sonicated plus Arnica-30 fed and 55-sonicated plus alcohol-30 fed Fix. Intervals Series Micronuclei in PCE and NCE S~e nn head a nomal~ o/o ±SE P/N % of ~rotection o/o ±SE % of ~rotection s1 vs s3 s) vs s4 s4 vs s5 s1 vs s) s) vs s4 s4 vs s5 s1 0.06± ±0.04 s2 0.04± ±0.02 s) 0.40± ± days s4 0.26± b " 0.38± c s5 0.43± ±0. 11 s) 0.42± ± days s4 0.16± b b 0.42± c 0.46c J.24c s5 1.32± ±0.1 2 s) 0.49± ± days s4 0.38± b ± b 0.37c 2.00c s5 1.33± ±0.19 s) 0.60± ± days s4 0.24± c 0.36b J.06c 0.64± c 0.84b 1.66b s5 1.30± ±0.33 SE = Standard Error, p < 0.05, b P < 0.00 I, c P < 0.00 I% level of significance at t-test. Some relevant data about the drugs: Actinomycin D and Arnica Montana Name Nature Source/derived from Working principle Actinomycin-D 22 Arnica Montana 23 Polypeptide containing antibiotic; inhibits transcription by binding tightly to DNA, preventing it fro m acting as template for RNA synthesis, binding enhanced by the presence of guani ne residue Potenti zed form in alcohol vehicle can only be differentiated from alcohol by NMR studies, otherwise no chemical nature other than alcohol can be substanti ated. Specific strain of Streptomyces Prepared from fresh roots of Arnica montana, (Compositae) that grows all over the world; tincture contains some alkaloids. The phenoxazone ring of AMD slips in between neighbouring base pair of DNA, mainly G-C Not precisely known, but claimed to act through CNS on skin, venous system, muscular system, digestive organs, serous membranes and circulation. It could not also be understood why and how the tiny drops of alcohol had accentuated the effect in sonicated mice, but it was at least a pointer that intake of alcohol alongside sonication should be discouraged in human subjects as well. It's difficult to perceive at the present state of knowledge how this ultra-low doses of the homeopathic drug could bring such spectacular modulating effect in sonicated mice since the precise mechanism of action of the homeopathic drugs has not yet been completely understood. However, from different scientific evidences, Khuda-Bukhsh 36 has proposed a hypothesis to explain the mechanism of action by attributing the major pathway through regulation of expression of certain genes by the homeopathic drugs in an unknown manner. The present findings of Arnica in reducing genotoxic effects of sonica- tion in mice may have an application in human subjects (patients) as well, where repeated ultrasonic tests are absolutely necessary for diagnostic or therapeutic purposes, to minimize the possible ill-effects of ultrasonication. The use of the homeopathic drug can be considered safe because the administration of repeated doses of Arnica 30 alone in normal healthy mice did not reveal any clastogenic ill-effects by itselft 7-19 Acknowledgement Financial assistance from the University of Kalyani is acknowledged. The authors are also grateful to Professors G. K. Manna, Professor Emeritus, Dept. of Zoology, S. P. Sen, Department of Botany, University of Kalyani and S. N. Chatterjee, Saha Institute of Nuclear Physics, Ko1kata for encouragement.

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