Indi an Journal of Experimental Biology Vol. 39, December 2001, pp. 1235-1242

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

Jayat i Chakrabarti , Surjyo Jyoti Biswas & Anisur Rahman Khuda-Bukhsh*

Department of Zoology, University of Kalyani, Kalyani, Nadia 741 235, 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 ex-posures at an interval of 20 days . The mice were then assessed at different intervals, aga inst suitable controls, using pa-rameters like chromosome aberrat ions (CA), mitotic index (M I), sperm head anomaly (SHA) and micronucleated erythro-cytes (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 soni-cated series. AMD had genotoxic effects of its own and also had additive effects on sonication induced genotoxicity . Soni-cated mice fed with Arnica 30 showed appreciably reduced genotox icity as against alcohol 30 and distilled water fed con-trols, thereby showing ameliorating effect which may have human applicat ion.

The effect of sonication on mammali an genetic sys-tem 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 test4-9. 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 loca-tion/site of abnormalities in internal anatomy of hu-man 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 12·13 • 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 coun-tries and developed European L:Ountries 14 after their clinical efficacy was supported by many well-conducted research publications including two major

*Correspondent author: E-mail : arkb@klyuniv .ernet. in Phone : (033) 5828750 Extn. Zoology (313/315)

meta-analysi s 15- 16• Keeping the above in view the ho-meopathic drug, Arnica Montana, commonly used against shock and injury has been examined for its possible protective role against ultrasonication . Inci-dentally, 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 17- 19 •

Materials and Methods Healthy Swiss albino mice (Mus musculus) of both

sexes weighing between 25-30 g, reared and main-tained under the supervision of the Animal Welfare Committee, Department of Zoology, Kalyani Univer-sity, 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 (S2) alone in doses described below and were scanned for the data at the same fixa-tion 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

1236 INDIAN 1 EXP BIOL, DECEMBER 200 1

frequency wave of 23 KHz, and at an energy output percentage of 70 for a total period of 2 min (twice for I min each with an interval of I min in between) . Mice receiving this two min exposure conformed the materials for the sing le exposure study.

Mice were immobilised during sonicati on in a thin wet cloth bag and the lower part of the body sub-merged in water in g lass beaker. The beaker was sur-rounded with ice before start of sonication to avoid any poss ible ri se in temperature of water inside beaker during sonication. 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 with potenti zed homeopathic drug, Ar-nica 30 [HAPCO, Kolkata,(0 .06 ml of liquid Arnica 30 diluted with 20 ml double di still ed water (ddw) to make the stock solut ion of the drug, fro m which l drop, i.e. 0 .06 ml was fed thrice at an interval of 30 min to mice which were sac ri ficed at 2 hr and th rice a day at an interva l of 8 hr to mice sacrificed at 24 and 48 hr)]. Another batch of mice was fed with dilute succussed alcohol (a lcohol 30) (positi ve contro l I) prepared as per homeopathic potenti zation procedure similarl y at corresponding interva ls of time and the third batch of mice were neither fed with the homeo-pathic drug, nor with the diluted alcohol measured as per homeopath ic principle (i.e. a lcohol 30), but was a llowed to take only dd w at corresponding intervals (positive control 2). Another set of unsonicated healthy mice served as negati ve contro l.

Similarl y, in Actinomycin D (AMD) treated series, 5 batches of mice, unsonicated and sonicated , were intramuscularl y inj ec ted with 0 .0005% Actinomycin D @ 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, sonicated and AMD injected mice were either fed with the homeo-pathic drug, Arnica 30, or with alcohol 30 (as contro l of homeopathic drug, Arnica, the "vehicle" of the drug being ethyl alcohol). As AMD itself showed a good amount of clastogenic effect, which could only be partially reduced by the feedi ng of the homeo-pathic drug (Table 1), further experimentation with AMD was not continued.

Multiple exposure series Four sets of mice (each set compnsmg 5 mice)

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 ini-

ti a l exposure actually received 2, 3, 4 and 5 such ex-posures to sonication, respecti vely. In the repeated exposure series, 1 batch of sonicated mice was orally administered with the stock solution of potenti zed homeopathic drug, Arnica 30 in the arne way , but at an interval of 12 hr till sacrificed. Only one control was maintained in this series, that of the 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 observation and scoring of data on various cytogenetical parameters like chromosome aberrations (CA), mitotic index (Ml), mi cronucleated erythrocytes (MNE) and sperm head anomaly (SH A) . MI was not scored in the re-peated exposure series.

Chromosome aberration study-Mice at all fixa-tion intervals were inj ected intraperitoneally with 0 .03 % colchicine solution @ l mill 00 g body weight 1 'h hr prior to sacrifi ce. The conventi onal citrate-fl ame drying-Giemsa technique was fo llowed for the bone marrow chromosome preparation.

Micronuclei testing and mitotic index studies-A part of suspension of bone marrow -::e ll s in 1 % so-dium citrate soluti on was smeared on clean grease free slides. The slides were briefl y fi xed in methanol and subsequently stained with May-G runwald solu-tion followed by Giemsa staining20.

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 sa-line solution. The suspension was filtered th rough 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 21 as per t e routme proce ure .

Results The frequencies of chromosome aberrations in the

sonicated mice, sonicated and unsonicated mice in-jected with AMD, sonicated mice injected with Acti-nomycin D and also fed with the homeopathic drug, Arnica 30, have been presented along with the con-trols in Table 1. The frequencies of CA in the AMD treated unsonicated mice at different fixation interva ls 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 sonicated and AMD treated mice were found to be enhanced,

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 individual s) 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

Unsonicated 0.40 ± 0.25 AMD 23.6 ± 0.75

2 hr Sonicated + AMD 28.00 ± 1.30 Sonicated+ AMD + Arnica-30 21.25 ± 0.51

23.20c 4.40 4.75b

Sonicated + AMD + alcohol 30 26.00 ±0.86

AMD 20.80 ± 0.66

24 hr Sonicated + AMD 23.00 ± 1.41

20.40c 2.20 1.78 Sonicated + AMD + Arnica-30 17.40 ± 0.8 1 Sonicated + AMD + alcohol 30 19.60±0.93

AMD 19.40 ± 1.60

48 hr Sonicated + AMD 21.20 ± 0.97

19.00 1.80 4.27b Sonicated + AMD + Arnica-30 13.40 ± 0.98 Sonicated + AMD + alcohol 30 18.80 ± 0.80

*Chromosome aberrations include stickiness, precocious centromeric separation , erosion, condensed, crumpled, c-mitotic effect, poly-ploidy, aneuploidy etc. SE= Standard Error, bp < 0.001, cp < 0.01 % level of significance at t-test.

showing apparently additive action of AMD on soni-cation-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 in-crease the damage in some cases.

The frequencies of various chromosome aberrations at different fixation intervals encountered in unsoni-cated mice (S 1), unsonicated fed with Arnica 30 (S2), only sonicated mice (S3), in sonicated mice fed with the homeopathic drug Arnica 30 (S4) and sonicated mice fed with alcohol 30 (S5, positive control) have been summarized in Tables 2 and 3 for single expo-sure and Tables 4 and 5 for multiple exposures to sonication. Representative photomicrographs of vari-ous types of chromosome abnormalities (Figs 1-9), micronucleated erythrocytes (Figs I 0 and 11 ) and sperm head anomalies (Figs 12- 14) have been pro-vided.

The chromosome complements in both the experi-mental and control sets of mice were critically studied for possible abnormali ties. In the normal healthy un-sonicated mice (negative control), out of some 500 bone marrow cells examined, normal complements (Fig. I) were obtai ned in all but two cell s, one of which showed an achromatic les ion 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 0.001 % 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 30.00 at 2 hr, 24.20 at 24 hr and 20.00 at 48 hr which were all sta-ti stically significant at various levels (Table 1 ). Simi-larly 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 wa~: 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 un-sonicated normal mice. Therefore, there was a posi-ti ve change in these cytogenetical parameters even for the single exposure to ultrasonication. However. no break type or other more serious type of aberrations

1238

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INDIAN J EXP BIOL, DECEMBER 2001

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ml _· __ ____.,.__. --·---- ill - - _rn. -------Fig. 1-14 - 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.]

CHAKRABARTI eta/.: SONICATION EFFECTS IN MICE & THEIR MODULATIONS 1239

(major type) were encountered in this group of soni-cated mice.

In mice receiving multiple exposures, not only the percentages of CA (Table 4) were increased apprecia-bly, 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 per-centages 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 sonica-tion. 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, S2- unsonicated plus Arnica-30 fed Sr

sonicated, S4-sonicated plus Arnica-30 fed and S5-sonicated plus alcohol-30 fed

Fix. Intervals Series Chromosome aberration Mitotic index

% ±SE % of Erotection

% ±SE % of Erotection

s1 vs s 3 s) vs s4 S4 vs Ss s1 vs s) s ) vs s 4 s 4 VS Ss

sl 0.04 ± 0.21 0.69 ± 0.001 s2 0.03±0.18 0.003 ± 0.02 s ) 30.00 ± 0.84

2hr. s4 19.00 ± 0.50 29.96c 11.00c 10.80c

Ss 29.80 ± 0.42 s) 24.20 ±1.80 2.70 ± 0.15

24hr. s4 12.80 ± 0.65 24.16c 11.40c 6.20b 2.26 ± 0.02 2.01c 0.443 0.44b Ss 19.00 ± 1.06 2.70 ± 0.11 s) 20.00 ±0.71 2.06 ±0.16

48hr s4 12.00 ±0.57 19.96c 8.ooc 6.00< 1.78 ± 1.55 1.37c 0.28 0.22 Ss 18.00 ± 0.61 2.00 ± 1.00

*Chromosome aberrations include stickiness, precocious centromeric separation, erosion, condensed, crumpled, c-mitotic effect, poly-ploidy, aneuploidy etc . SE= Standard Error, •p < 0.05, bp < 0.001,

1240 INDIAN J EXP BIOL, DECEMBER 2001

exposures to ultrasonic waves further increased the extent of cytogenetic damage. Earlier workers3· 24-28

did not get elevated frequencies of SCE in cultured lymphocytes of human subjects exposed to ultrasonic sound waves. On the other hand, several workers7• 29-30

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 system8. Chatterjee and his co-workers4·5•7 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, cavi-tation and direct effects31 . 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 radicals32 and induces temporary local shock waves. On the other hand the "to and fro" mo-tion of the cavitation bubbles produces hydrodynamic shearing stress31 · 33 . This results in degradation of

DNA in aqueous solution and even destruction of cells34·35 . Therfore, present observations of the differ-ent forms of cytogenetic damage caused by ultrasoni-cation 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 responsi-ble 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 homeo-pathic drug Arnica 30 was found to modulate favourably the cytogenetic ill-effects of ultrasonica-tion in mice while the administration of alcohol 30 appeared to increase the damaging effect of sonica-tion. 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, S4-sonicated plus Arnica-30 fed and S5-sonicated plus

alcohol-30 fed

Fix. Intervals Series Chromosome aberration %± SE % of protection

s1 0.40 ± 0.25 s2 0.38 ± 0.27 s3 35.90 ± 0.23

30 days s4 20.00 ± 0.86 35.5o· 15.90 1.60 s5 21 .60 ± 0.83 s3 23.00 ± 0.77

60 days s4 19.80 ± 0.68 22.60c 3.20 4.40 s5 26.80 ± 1.77 s3 25.60 ± 0.50

75 days s4 8.40 ± 0.50 25.20c s5 19.80 ± 1.80 s3 29.40 ± 0.50

90 days s4 24.40 ± 0.40 29 .ooc 5.00 18.00 Ss 42.40 ± 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 ?

CHAKRABARTI eta/.: SONICATION EFFECTS IN MICE & THEIR MODULATIONS 1241

Table 5 - Frequency distribution of micronucle i 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 unson icated plus Amica-30 fed 53-sonicated, 54-sonicated plus Arnica-30 fed and 55-sonicated plus alcohol-30 fed

Fix . Interval s Series Micronuclei in PCE and NCE S~enn head anomal~ 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±0.03 0.45 0.18±0.04 s2 0.04±0.05 0.38 0.14±0.02

s) 0.40±0.09 1.14 0.56±0.11 30 days s4 0.26±0.05 0.8 1 0.34b 0.14 0.17" 0.38±0.06 0.38c 0. 18 0.22

s5 0.43±0.04 0.54 0.60±0. 11

s) 0.42±0.10 0.61 0.88±0.10 60 days s4 0.16±0.02 0.69 0.36b 0.263 1.16b 0.42±0.07 0.70c 0.46c J.24c

s5 1.32±0.25 0.34 1.66±0.1 2

s) 0.49±0. 11 0.60 0.98±0.22 75 days s4 0.38±0.05 0.60 0.43b 0. 11 0.95 0.61±0.07 0.80b 0.37c 2.00c

s5 1.33±0.66 0.33 2.60±0.19

s) 0.60±0.09 0.37 1.48±0. 11 90 days s4 0.24±0.04 0.62 0.54c 0.36b J.06c 0.64±0.12 1.30c 0.84b 1.66b

s5 1.30±0. 14 0.83 2.30±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

Actinomycin-D22

Arnica Montana23

Nature

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 on ly be differentiated from alcohol by NMR studies, otherwise no chemical nature other than alcohol can be substan-ti ated .

It could not also be understood why and how the tiny drops of alcohol had accentuated the effect in soni-cated 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-Bukhsh36 has proposed a hypothesis to explain the mechanism of action by attributing the major pathway through regu-lation of expression of certain genes by the homeo-pathic drugs in an unknown manner. The present find-ings of Arnica in reducing genotoxic effects of sonica-

Source/derived from

Specific strain of Streptomyces

Prepared from fresh roots of Arnica montana, (Composi tae) that grows all over the world; tincture contains some alka-lo ids.

Working principle

The phenoxazone ring of AMD slips in between neigh-bouring base pair of DNA, mainly G-C

Not precisely known, but claimed to act through CNS on skin, venous system, muscular system, digestive organs, ser-ous membranes and circula-tion.

tion in mice may have an application in human subjects (patients) as well, where repeated ultrasonic tests are absolutely necessary for diagnostic or therapeutic pur-poses, to minimize the possible ill-effects of ultrasoni-cation. The use of the homeopathic drug can be consid-ered safe because the administration of repeated doses of Arnica 30 alone in normal healthy mice did not re-veal any clastogenic ill-effects by itselft 7- 19 •

Acknowledgement Financial assistance from the University of Kalyani

is acknowledged. The authors are also grateful to Pro-fessors G. K. Manna, Professor Emeritus, Dept. of Zoology, S. P. Sen, Department of Botany, Universi ty of Kalyani and S. N. Chatterjee, Saha Institute of Nu-clear Physics, Ko1kata for encouragement.

1242 INDIAN J EXP BIOL, DECEMBER 2001

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