Indi an Jou rnal of Experimenta l Biology Vol. 40, November 2002, pp. 1285- 1294

Assessment of EMS-induced genotoxicity in the Indian climbing perch, Anabas testudineus : Cytogenetical vis-a-vis protein endpoints

B. Guha & A R Khuda-Bukhsh*

Department of Zoology, University of Kalyani, Kalyani 74 1 235, India

Received 18 March 2002; revised 20 JUll e 2002

Genotox ic effects of EMS have been assessed in fish, A. testlldilleus, using widely accepted cytogenetic protocols like chromosome aberrations, nuclear anomalies in red blood cells and abnormal sperm head morphology. In addition, gel elec­trophoretic protein profiles and total protein contents in nine se lected ti ssues were anal ysed for eva luating their utility as po­tential indicators of genotoxicit y. EMS not on ly caused chromosomal aberrat ions in somat ic cell s. nuclear anomali es in red blood cell s, and increased incidence of sperm with abnormal head morphology, but also altered significantl y both protein profiles and tota l protein contents in all ti ssues tested vis-a-vis suitable controls, indicating relevance of protein data in genotoxicity assessment.

Fish are generally considered a relatively difficult cytogenetical material because of the occurrence of a large number of small chromosomes in karyotypes of most of them. onetheless, a few species of fish have been successfully tried as models of genotoxicity studies in India l

-3 and other countries4

-7

. They were used as an in vivo aquatic assay system for testing mutagenic potentials of various toxic chemicals, mostly having relevance to aquatic pollution. To our knowledge the Indian climbing perch , Anabas leslu­din eus, which turned out to have excellent potentials as an in vivo model for such genotoxicity study , has not been tested earlier. Some of the advantages of A. lesludineus over other Indian teleosts are: i) a very suitable karyotype of 2n=46 reasonabl y big rod-like chromosomes, ii) a fairly hi gh rate of mitotic divi sion in its kidney and gi ll cell s, iii) easi ly available and an extremely hardy fish capable of withstanding labora­tory conditions as well as intra-muscular injections of test chemicals, and iv) survivability in toxic/turbid water for relatively longer period of time, even out­side water (for having accessory respiratory organ). Therefore, thi s fish drew our attention as a possible candidate for genotoxicity testing.

The present investigation has been undertaken to test the hypothesis if a known mutagen, ethy l methane sulphonate (EMS) can also cause similar chromoso­mal damage as reported in Notobran chius rochowi5

*Correspondent author : Phone: 033-5828768 E-mail: arkb@kl yuni v.ernet.in

(Cyprinodontidae) and nuclear damage (micronuclei) in peripheral blood as reported in Umbra pygmaea6

(Umbridae) in a phylogenetically distant species like Anabas lesludineus (Anabantidae). The other objec­tive of the study is to determine if EMS can cause sperm head damage, another potential indicator of genotoxicity widely used in mammalian models, but has seldom been used as a parameter of genotoxicity study in fishS-II. Further, in the present study, the nu­cleo-cytoplasmic ratios were determined to assess if there could be any positive correlation between the ratios and the extent of EMS induced toxicity . Lastly, to test if EMS, which is known to intercalate between DNA bases, can also alter protein parameters as a consequence of direct or indirect effect, some other unconventional endpoints, namely, SDS-PAGE gel electrophoretic profiles and variation in protein syn­thesis, if any , in different tissues including certain vital organs of thi s fish were also critically analyzed alongside cytogenetical parameters vis-a-vis suitable controls.

Materials and Methods Live specimens of A. lestudineus weighing be­

tween 20-25 g were collected from a local fresh water pond and were acclimated for 5-6 days in a concrete vat containing fresh tap water (stored from deep tube well ) and fed with artificial diet (rice bran + wheat + oil cake). Separate vats were maintained under the same conditions in which 40 to 42 specimens were kept for the experiments. Six live spec imens each were injected with optimally low doses that produced

1286 INDIAN J EXP BIOL. NOVEMBER 2002

fairly quantifiable changes (obtained through range­finding trials) : (I ) 0.1 % EMS , (2) 0.2% EMS and (3) double distilled water @ I ml 1100 g of body weight for each of the five fixation intervals (viz., 6, 24, 48, 72 and 96 hr; since the peak damaging effect induced by EMS seemed to be obtained at 72 hr and there was a declining trend till 96 hr, fixation interval beyond 96 hr was not considered) . Injected specimens were re­moved into smaller vats containing tap water and were fed with artificial diet till sacrificed. Specimens injected with double di st illed water (bei ng the "vehi ­cle" for EMS) served as control s.

For chromosome anal ysis, experimental and con­trol fish specimens were injected intramuscularly with 0.05 % colchicine @ I mill 00 g bw about 3 hr prior to sacrifice and thei r somatic chromosomes were pre­pared as per the citrate- fl ame drying technique l 2

. For micronucleus testing, slides with blood smears were stained in May-Grunwald as per the procedure of Schmid 13 . The frequency of anomalous nuclei was recorded as per the method of Carrasco et al.14 and nuclear and cytoplasmic volumes were measured ac­cording to the method suggested by Samuels IS to calculate nucleo-cytoplasmic ratio (CytiNu ratio=Ycy/Yn where Ycy,=volume of cytoplasm and Yn=volume of nucleus) in normal erythrocytes l 6

. Sperm smears were prepared on slides and stained with Giemsa8 for the study of sperm head abnormality.

For biochemical study nine different tissues, namely, dorsal muscle (OM), ventral muscle (YM), heart (H), eye (E), brai n (Br), gi ll (G) , liver (Li), spleen (S) and kidney (K) were removed after 72 hr of treatment from each of the treated and control speci­mens separately and homogen ized in 0.1 % NaCI. Af­ter centrifugation at 3000 g for 15 min, the super­natants were collected. Aliquots containing known amounts of protein for each ti ssue (5 to 10 J.lg) were diluted in lOX Laemmli's buffer and loaded in sepa­rate wells. The Sodium Oodecyl Sulphate-Polyacryl­amide gel electrophoresis 17 using 7.5 % separating gel in Tris-glycine-SOS buffer was run for about I hr (current fl ow @ 3mA/Iane at constant 200 V). Gels were stained with 0.5 % Coomessie brilli ant blue and destained with acetic acid-methanol-water (l0:40:50) for visualizing different su b-fractions of prot~in

bands. Standard protein samples of known molecu lar weights were also run on the same gel and a standard curve was prepared from tht: Rm values of the stan­dard proteins against their log of molecul ar weight. The molecular weights of unknown proteins were cal-

culated from thi s standard curve. For estimating the quantity of protein the technique of Lowrey el al. IX

using folin-ciocalteu reagent was followed. A stan­dard curve was constructed from different known con­centrations of Bovine Serum Albumin (BSA) against their 00 values. The amount of unknown proLin (mg/gm ti ssue) was calculated in the routine manner against thi s standard curve. For the analys is of data , Student' s t-test was conducted between data of treated and control fishes and to test the level of signi ficance, the Fisher and Yates statistica l tables 1'1 were used. The similarity index was calcul ated on the bas is of molecular weight of indi vidual band arranged into size-classes of fi ve by adopting the fo llowing r or­mUl a2o.

SI (s imilarity Index)=n/0.5 x (N I + N2)

where n = frequency of identical protein frac tions of both indi vidu als to be compared and NI and N2 repre­sented the number of all frac tions of the electrophore­grams of both the indi viduals under test. The data of protein bands of 6 individuals were initi ally compared and the common bands generally appearing in all the individuals in control were taken as standard for the control series, ignoring the little inter-indi vidual variations. Similarly, the band patterns of each of 6 treated individuals were separately scored and mean values of 6 treated individuals sharing common bands were taken as standard for treated fis h ignoring the minor differences observed among individual treated fish. As bands differing within molecul ar weight of 5kD were clubbed in the same molecular weight class, standard deviations between individuals were not shown for their limited use.

Results Chromosome aberration (Fig. I)-Normal meta­

phase complements (a) as well as the chromosomal abnormalities were carefully examined for any possi ­ble clastogenic change due to the treatments of two doses of EMS (0.1 and 0.2%). Representati ve photo­micrographs of various types of chromosomal aberra­tions like, chromatid break and fragment (b), pul veri­zation (c), ring (d), terminal association (e) and C­mitotic effec t (D etc. have been provided mainl y from EMS-treated fi sh. However, a few aberrations of mi­nor nature were also recorded in fish injected with distilled water (dw). The summarized data on the fre­quencies and types of aberrati on~ observed in vari ow; treated and control fishes have been provided in Table I. The data reveal that the frequenci es of abelTations in

GUHA & KHUDA-BUKHSH : EMS INDUCED GENOTOXICITY IN A. TESTUD INEUS 1287

the EMS-treated fi shes, particul arly in those receiving the higher dose of EMS, were significantly higher (P < 0.00 I) than in the dw-treated controls at all fixa­tion intervals. In both the treated series the aberration frequenc ies appeared to increase (P<O.OO I ) till 72 hr, after which the aberration frequencies gradually de­clined.

Micronucleus study-Micronucleated erythrocytes (MN) (Fig. Ih ,i) and erythrocytes with abnormal nu­clei (AN) U,k,l) were observed in much greater num­ber in fi sh treated with EMS for both the doses as compared to dw treated controls. However, a few MN were also encountered in the dw treated fi sh at the longer interval s. The frequencies of MN and AN ob­served in various treated and control fishes have been summarized in Table I. A critical analys is of the data reveals that in EMS-treated fish the formation of mi­cronuclei and the abnormal nuclei was strikingly higher than in the dw treated fi shes (see Table I) and the differences were stati stically significant at all the fixation intervals (P < 0.05, < 0.0 I and < 0.00 I). The frequency of micronuclei formation was hi gher at 72 hr in both the EMS treated series but the incidence of abnormal nuclei was higher at 96 hr (in case of 0.1 % EMS) and at 48 hr (in case of 0.2% EMS). So no

definite correlation between the micronuclei forma­tion and the formation of abnormal nuclei could be substantiated. Further, the Cyt/N u ratio appeared to increase to some extent in the fish showing greater number of micronuclei at all the fix ation intervals, but no such apparent relationship cou ld be indicated with regard to the frequency of AN and Cyt/Nu. Further, the Cyt/Nu ratios were apparent ly increased in the EMS-treated as compared to dw treated fish , particu­larly in those treated with the higher dose of EMS and the differences were stati stically significant at 24 and 48 hr (P < 0.05 ).

Sperm head abnormality - Sperm with normal (Fig. I m) and abnormal (n) head shapes were encoun­tered in both control and treated fi sh and the frequen­cies of sperm with abnormal head shapes in different series have been summarized in Table 1. The data revea led that the freq uency of sperm with abnormal head was hi gher in the treated series than in the con­trols at all the fixation intervals. The hi gher dose gen­erally produced more abnormality in sperm heads ex­cept at 6 hr, and the differences were stati sticall y sig­ni ficant at various levels (P < 0.05, 0.0 I and 0.00 I) at some interva ls (see Table I).

Table I - Showi ng frequency di stributio n of chromosome aberrat io ns, mieronueleated erythrocytes (MNE), anomalous nuc lei (A ), nucleo-eytoplasmie rat io (CytiN u rati o) and sperm head abnormality (SHA) o f A. l esllldillells treated wi th 0.1 '1c EMS (T 1), 0.2% EMS (T2) separate ly and their respecti ve double di stilled water cont rols (C) at different fixation intervals. [Values are mean±SE from 6 indi-

vidual s in each serieslfixa tion inte rvals]

Fixation Series Chromosome Mi cronucleus Anomalous Cy t/Nu ratio Sperm Head time intervals aberration nuc lei Ano maly

(hI') (%) (%) (%) (%)

C I AO±0.28 O.OO±O.OO 0.98±0. 12 10 .S8± 1.21 O.87±0.11 6 TI 6.88±0.34c 0.06±0.02 " 6.8S±0.S4 ,. II A3±0.92 4.33±0.32 <

T 2 8.30±0.32 < 0.08±0.03 a 7.93± 1.1 2 c 12.08± 1.32 4.28±0.S3 <

C 2.2 1±0.S8 0.04±0.01 1.02±O.2 1 10.26±0.72 O. 8 1±0 .1 3 24 TI 6.10±OAI c 0.IS±0.02 b 7.38±0.S8 c 12.68±0.92 4.78±0.38 c

T2 8.37±O.60 c 0. 18±0.02 c IOA I±O.72 c I 3.84±0.99 " 7.76±0.73 c

C 2.86±0.82 0 .02±0.OI 1.09±0.23 11 .32±0.6S 0.S I±0.2 1 48 TI 8.S2±0.73 c 0. 13±0.02 b 7.67± 1.1 8 c 13.62±0.83 S. 13±0.42 c

'1'2 9.77±O.67 c 0.17±0.02 c 9A3±OAS c 15. 1 S± 1. 52" 6.2S±0.S2 c

C 1.6S±0.23 0 .02±0.0 1 1.23±0.17 10.83±0.27 0 .S2±0.09 72 TI S.72±0.30 c 0. 17±0.03 b S.02±0.9 1 b 10.84±0.60 4.21±0.68 b

T2 12.91±0.67 c 0.21±0.02 c S. 18±0.79 c 10.92±OA7 S.2S±O.9S I>

C 0.92±0. IS 0 .02±O.O I 0.S6±0.12 11.78±0.82 0.7 1±0.IO 96 T I 7.14±0.32 c 0. 12±0.02 b 9 .I S±0.S2c 12.06±O.36 4.S8±0.94b

T2 I 0.OS±OA2 c 0.14±O.01 c 7A7±0.S2 c 13.37±0.6 1 S.73±0.73 c

Chromosome aberrations (CA) include gap, break, centri c fu sio n, translocati on, fragment , pulverisation , ring, terminal assoc iat ion , poly-ploidy, aneuploidy , stickiness, C-mitotic e ffec t, precocious centromeric separation, constric tion, e tc.

Cells scored per individual = 500 for CA, 20000 for MNE. AN etc . 6000 for SHA. P values : "<0.05 ; b<O.O I ; c<O.OO I.

1288

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INDIAN J EXP BIOL. NOV EMBER 2002

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r ig. I - Photomicrographs of normal (a) :l no abe rrated (b-I) Il'letaphuse complc:ments of A. :es{udil/ ('II.\" ~ !II(l Il ~:ll ld 11rcak and fragl11t'n; (b). pulverizat ion (c). ring (d), term inal a ~ soc i a t i o n (c) and C-mi lotic e llen (fL Normal erythrocy tes (g). micro ll llcicatcd erythrocy te, (lI. i,

alld anomalous nuclei U-I), Sperm Will! nornlal (m) and al)l1orl11ai h.· :,, ' s h ap('~ (11) , Gel e! c.:c troryherograms of nine di l'fl;rcnt ti ssue~ 01 (1 , .

till ed water treated control (0). 0.1 1); EM ~ tn'"kif (p) anl' 0.2% EMS tre;I 'cd (q) fish. ICI3 = Chrom:u ic1 Break. F = Fragment. R = R i l ' ~.

TA = Term inal Association. MN ~ Micronuci ated eryth rocytes, AN = Anomalous Nuclei, SHA = Sperm Head Anomal y, DM = DOt sa: Mw,c\e, VM = Velllral Muscle, H = Hear: , E = Eyc, Br = Brain, G = Gill , Li = Li ver, S = Spleen and K = Kidney. Bar = ]0 /lml .

GU HA & KH UDA- BUKH SH : EMS IND UCED GENOTOX ICITY I A. TESTUDINEUS 1289

Qualitative analysis oj protein-Data on gel elec­trophoretic protein profil es of different ti ssues of fi sh injected with di stilled water and two doses of EMS separately (Table 2, Fi g. l ,o-q) reveal that the number of bands was decreased in all the ti ssues in the EMS­treated fish as compared to dw-treated controls except fo r li ver, spleen and kidney ti ssues in which the num­ber of bands was equal (li ver and kidney for 0.2% EMS and spleen for 0. 1 % EMS). The number of bands affec ted/a ltered by EMS was diffe rent (Tables 4 and 5) in respect of di ffe rent ti ssues examined. As fo r exampl e, the number of bands in muscles, heart , eye, brain and gill ti ssues was found to be drasticall y reduced as compared to contro ls while in other ti ssues the red ucti on was less striking or even eq ual. In a bid

to make thi s di ffe renti al response in the diffe rent tis­sues of treated and control fi shes more obvious, a similarity index between band characteri stics of EMS treated versus dw-treated control fi sh has been calcu­lated on the bas is of the molecul ar weight classes of respecti ve bands and presented in Table 3.

Quantitative analysis oj protein-As compared to the dw-treated series the total protein contents in the EMS-treated seri es (for both the doses) were less in all the ti ssues (Table 2). Further, in most of the ti ssues the inhibitory effect of EMS on protein sy nthesis could be noted generally more in the hi gher dose, ex­cept in OM , YM, H and E where the amounts of pro­tein were more or less same as in the lower dose.

Table 2- Showing number of ba nds, ra nges of relati ve mobility (Rm) and molecuia r wc ight (Mo l. W I. ) of SDS-PAGE total protcin profilcs and amount of towl protci n (mg/g m) in diffcrcnt tissucs of A. lesllIdill ells trcatcd with 0.1 % EMS (T I), 0.2% EMS (T2) scpa-

ratel y and thcir rcspcctivc doublc distill cd watcr controls (C) at 72 hr fi xation intcrval

Scrics Tissucs OM YM H E Br G Li S K

C 25 25 19 24 20 22 24 16 19

o. of bands TI IX 20 IS 21 IS 20 24 13 19

T2 24 20 15 IS 15 IX 22 16 15

C 0.030- 0.030- 0.023- 0.023- 0.023- 0.023- 0.023- 0.023- o.on 0.975 0.9XO 0.96'+ 0.974 0.970 0.977 0.9X7 0.9X I D.97'+

T I 0.027- 0.092- 0.02 1- 0. 11 3- 0.025- 0.025- 0.02 1- 0. 11 7- 0.03'+ -

Rangc of Rm 0.9'+0 0.965 0.979 0.965 0.950 0.979 0.979 0.965 0.966 0.0 11- 0.02S- 0.01 4- O.02X- 0.OS5- 0.02S- 0.02 1- 0.0 14- D.OI'+ -

T2 0952 0.979 0.966 0.9S2 0.965 0.979 0.972 0.979 0.96 1

C 30.0- 29.6- 30.S- 30. 1- 30.3- 29.S- 29. 1- 29.5- 30. 1-306.S 306.S 3 11 .S 3 11 .8 3 11.R 3 11.S 3 11 .8 31 1.X 3 11 .8

Rangc of Mol. TI 32.7- 30.X- 29.7- 30.S- 3 1.S- 29.7- 29.7- 30.X- 30.6-

WI. (kDa) 309.0 263 .1 3 13.3 2'+9.7 3 10.6 310.6 3 13.3 247.5 30.+.0 3 1.7- 29.7- 30.6- 29.4- 30.X267 29.7- 30.2- 29.7- 31.0-

T2 32 1.6 307 .9 3 IX.X 307.9 .8 307.9 3 13.3 3 IX.X 3 i lU

C 22.2 1± 23.87± 2'+ .59± 19.39± IX.9 1± 23.05± 49.S5± '+7.85± 39.2l±

Amount or 2.47 3.62 2.70 2.62 2.52 2.4 1 .+ .0'+ 3.'+2 2.05

protcin TI 1.+ .9 1± 19.3'+± IS.74± 16.23± 16.3S± IS.32± 39.0S± 3~L49± 26.32±

(Illg/g lll ) 2.0'+ 2.59 2.32 2.23 3.2 1 3. 10 5. 15 3.24 3.02

T2 16.S8± 19.36± 23.59± 16.93± 13.35± 15. 19± 34.57± 27. 19± 2 1.-+6± 2.22 2.37 3.02 I.I S 1.09 2.56 3.43 2.92 1.35

DM=Dorsa l Muscle, YM=Yentral Musc lc, H=Hean . E=Eye, Br=Brain, G=Gill , Li =Live r, S=Spleen, K=Kidney

Table 3-Showing similarity indiccs based on molec ular wcights or SDS-PAGE total protein profil cs ill dif-fercnt ti ssucs or AllaiJas lesllIdillws trcated with 0. 1 % EMS (1'1 ), 0. 2% EMS (1'2) scparatcly and thcir rcspec-

ti vt; doub lc di stilled water con trol s (C) at 72 hr fixati on interva l

Series Ti ssucs OM YM H E Br G Li S K

C vs TI 0.-+2 0.62 O.X I 0.62 0.63 0.76 0.7 1 'l.-Li 1./1/

C vs T2 0.-+9 0.62 D.53 0.67 0.51 0.65 0.6 1 0.50 0 . .+7

DM=Dorsal Muscle, YM=Ycn tral Musc lc. H=Hean , E=Eye. Br=Brain. G=G ill. Li=Li ver, S=Splec n. K=Kidncy.

- - ---- _ .. - ---

1290 INDIAN J EXP BIOL, NOVEMB ER 2002

Table 4-Dala on band profil es of Dorsal Muscle (OM ), Vcnlral Muscle (VM), Heart (H), Eye (E) and Brain (Br) in A. lesludinells treated with 0.1 % EMS, 0.2% EMS and double distilled waler (dw) arranged into molecul ar weight classes fo r band comparison at a glance

Molecul ar DM VM Weight 0. 1% 0.2% dw 0. 1%

EMS EMS EMS 0.2%

dw EMS

26-30 I I I 3 1-35 2 3 4 2 5 36-4 0 2 2 2 1 1 2 4 1-45 I 3 2 2 3 46-5 0 2 2 1 I 5 I - 55 2 2 2 56-6 0 2 6 1-6 5 2 2 66-70 2 I 2 7 I - 7 5 2 76-8 0 2 I 8 1-8 5 2 86 - 9 0 9 1- 95

96- 100 10 I - I 05 106- 1 10 I I I - I 15 11 6- 120 12 1- 12 5 126- 130 13 1-1 35 136- 14 0 14 1- 145 146- 15 0 15 1- I 55 156 -1 60 16 1- 165 166- 170 17 I -I 7 5 176- 18 0 18 1 - 18 5 186- 19 0 19 1- 195 196-20 0 20 1-205 206-2 10 2 I 1- 2 I 5 2 16-22 0 22 1-225 226-230 23 1-235 236-240 24 1-245 246-25 0 25 1-255 256-260 26 1-265 266-270 27 1- 27 5 276-280 28 1-28 .'1 286-290

Contel

GUHA & KHUDA-BUKHSH : EMS INDUCED GENOTOXICITY IN A. TESTUDINEUS

Molecular Weight

291-295 296-300 301 -305 306 -310 31 1- 315 316-320 321 -325 326 - 330

DM

0.1 % 0.2% EMS EMS

dw YM

0.1 % 0.2% EMS EMS

Table 4 - (Colltd)

dw

H

0.1 % 0.2% EMS EMS

dw

E

0.1 % 0.2% EMS EMS

dw

Br 0.1 % 0.2% EMS EMS

Table 5-Data on band profiles of Gill (G), Liver (Li) Spleen (S) and Kidney (K) in A. testudineus treated with 0.1 % EMS, 0.2% EMS and double dist illed water (dw) arranged into molecular weight classes for band comparison at a glance

Molecular G Li S K Weight

0.1 % 0.2% dw 0.1% 0.2% EMS EMS EMS EMS

dw 0.1 % 0.2% dw

0.1% 0.2% dw EMS EMS EMS EMS

26 - 30 I I 1 I 1 2 1 1 1 3 1-35 2 2 5 3 3 3 2 3 3 1 3 3 36-40 2 2 2 1 2 3 2 3 4 1-45 2 1 2 3 2 2 46-50 2 2 2 1 5 I-55 2 2 I 2 1 2 56-60 2 2 61-65 2 2 66-70 2 2 71- 7 5 2 76-80 81-85 86-90 9 1-95 2

96- 100 101 -105 106-110 1 I 1 - 1 15 116-120 121 - 125 12 1'>-130 131-135 136 - 14 0 141-145 146-150 151-155 156-160 16 1-1 65 166-170 171-175 17 6 - 180 181-185 186 - 190 191- 195 196-200 20 1-205 206-210

1291

dw

Contd

1292 IND IAN J EXP BIOL, NOV EMB ER 2002

Table 5-(Col/ /{/)

Molecular Weight

2 I 1-2 15

2 10-220

22 1-225

22 6-230

23 1- 2 3 5

236-240

24 1-245

246-250

25 1-255

256-260

20 1- 205

266-2 70

27 1-275

270-280

28 1-2R5

280-290

29 1-295

29 6 -30 0

30 1- 305

300-3 10

3 I 1-3 15

3 16-320

32 1-325

320 -33 0

Discussion

G

0 .1 % 0 .2% EMS EMS

Li

dw 0.1 % 0.2% EMS EMS

In the present study, EMS was found to induce vari ous types of chromosome aberrations in kid­ney/g i II ce ll s and nuclear anomal ies i ncl udi ng micro­nucle i formation in the peripheral erythrocy tes of AI/abas leSllldil/ells. Further, it enhanced th' fre­quency of sperm with abnormal heads as compared to contro l. However, change or variati on of nucleo­cy toplasmic ratios noted in EMS-treated fi sh as com­pared to controls cou ld not be convincingly correlated with the ex tent of genotoxicity although there was indica tion of such a relationship to ex ist in some cases. On the \-vho le, it might be more plausible to suggest th at Cyt/Nu ratios could have some relevance to the state of hydration of the cell s. EMS also clearl y induced certain changes in gel-electrophoreti c protein profi les and altered synthes is of protein in nine differ­ent ti ssues examined, as compared to controls. T here­fore, all these fi ndings together would amply corrobo­rate the genotox ic and mutagenic potent ials of EMS in thi s fi sh also and wou ld tes ti fy II. l eSllldil/ ells to be a good model for such tes ts .

S K

dw 0.1 % 0.2% EMS EMS dw

0. 1% 0.2 % EMS EMS

dw

EMS is an alky lating agent which has been ex ten­sively used in genetic research for its ability to alter a base that is already incorporated into a DNA molecule and thereby is of prime importance in the induction of artificial mutations 11 . In fac t, thi s mono-functional agent has been reported to cause maj or chromosomal aberrati ons including chromosomal breakage in vari­ous mammali an models, by bindin o to DN A reo ions o c

ri ch in G-C base pairs, causing those regions to be-come unstable or by di srupting the main chain of DNA, perhaps in regions where protein is bound11

.

Heddle el 01.23 recommended that micronucleus test­ing alone can serve as a potent parameter to des ignate clastogenic efficiency of a chemical because micro­nuclei are believed to be derived fro m chromosomal fragments or chromosomes that are not incorporated into daughter nuclei at the time of ce ll division. How­ever, most recent workers wou ld prefer a more fools­proof method invo lving multiple parameters. In fact, ex tensive studi es invo lving multi ple assays have al­ready been conducted to determine clas tooen ie o '

_ mutag~ni c, teratogenic and carcinogen ic potentials 01"

GU HA & KH UDA- BUK HSH : EMS INDUCED GENOTOX ICIT Y I A. TESTUDINEUS 1293

EMS in va rious mammals24. On the other hand, data

on fish are still meagre. Those avail able are either inadequate in terms of parameters used or deri ved onl y from a limited number of species like Umbra pygl11aea, NOfobranchills rochowis.6 or OreochrOlllis II lOssal1lbicusIO

.II

•

In the present study, a new dimension has been added by including the hitherto fore unknown effects of EMS on protein profiles or total protein contents in diffe rent ti ssues of thi s fi sh, which also seemed to have added va lue in determining genotox ic changes . As genotoxicity may result in DNA damage or may result fro m DN A damage, it becomes imperati ve th at there could be some impairment of gene acti vity as a consequence, either hav ing direct or indirect effects on proteins. In the present study, the concomitant changes observed in the protein profiles of the ti ssues will amply speak fo r thi s. EMS seems to have di ffe r­enti al effects on protein profi les of di fferent ti ssues. However, in some cases where the number of bands was the same in the treated and control seri es (see Tables 4 and 5), the other band characteri stics were somewhat di ffe rent in each ti ssue, e.g., nature and intensity of bands, could di ffe r quite apprec iabl y. This could mean that some di fference in the type of pro­teins could still be there even if a particul ar band was in the same molecul ar weight cl ass. But where the band di stinctl y differed in its molecul ar weight class, the change could be unmistakabl y substantiated. In­deed, some differences as a result of EMS treatment in the indi vidual band profiles became obvious when the profiles of EMS treated fish were compiled vis-a­vis contro ls (Tab les 4 and 5). Thus the response in altered band characterist ics in di ffe rent ti ssues as a resul t of EMS treatment would be mean ingful , whi ch would probabl y refl ect differenti al metaboli c degrada­tion/denaturation or loss of function in protein species of certai n ti ssues. Therefore, possible impact of any test chemi cal on protein profil es of any ti ssue of a fish can also be helpfu l in understanding its impact on genotox icity, as has also recentl y been shown in a different fish, Oreochrolllis II lOssambiclIs 11

• The pos i­ti ve result s obtained in the present in vesti gati on on protein parameters ta ll y qui te well wi th the other ac­cepted protocols of genotox icity assessment , li ke chromosome aberrat ions (CA), nuclear anomal ies (A ) including the occurrence of micronuclei (M ), and sperm head morphology. However, further stud ­ies on protein endpoints in other fish models may be necessary to prov ide more suggest ive insight to the contention that protein parameters coul d fo rm va lid

and potenti al indicators of genotox icity as well for other species of fi sh. Therefore, at least some chemi­cals/agents that are known to di srupt DNA structure, should also be tes ted for their possible effects, direct or indirect, on the proteins/protein-synthesizing ma­chinery in vari ous tissues of some other fishes, along with the routine cytogeneti cal parameters, where ever poss ible, to test the validity of such contention. This would help meaningful ex trapolation fo r most other fishes which would not permit direct chromosomal assay on their numerous and tiny chromosomes, but may need genotox ic assessment in view of rapid pol­lution of many water bodies where they inhabit.

Acknowledgement The authors are grateful to ICA R, New Delhi , for

fin ancial support.

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