Indi~ Journal of Experimental Biology Vol. 37, February 1999, pp. 161-165

Spindle damaging effect of salbutamol sulphate on bone marrow cells of mice

T V Sujatha & M J Hegde·

Department of Post Graduate Studies and Research in Biosciences, Mangalore University, Mangalagangotri,574 199 India

Received 5 March 1998; Revised 26 November 1998

In vivo assessment and identification of aneuploidy are important phases of genotoxicity evaluation. Considerable effort has been devoted to assess the utility of the existing bioassays and to develope simpler techniques for identifying environmental aneugens. Salbutamol sulphate - an antiasthmatic drug was tested for its spindle damaging effects in bone marrow cells of mice using an in vivo technique, for the evaluation of mitotic index, C-mitotic effects, anaphase reduction and hyperdiploidy. Doses of 0.12, 0.24, 1.2, 2.4, mglkg body weight were dissolved in bidistilled water and administered intraperitoneally to the mice. Colchicine was taken as positive control for its known aneuploidy-inducing effects. The drug showed positive C-mitotic effects accompanied with increases of mitotic index and decreased frequencies of anaphase in higher doses. Significant levels of hypodiploidy also noted at higher doses. The preliminary results indicated that Salbutamol is capable of inducing C-mitotic effects in mouse bone marrow cells, which is suggestive of possible induction of aneuploidy.

Aneuploidy is the most common class of chromosomal abnormality in humans, occuring in at least 0.3% of new borns and approximately 50% of spontaneous abortions I. Chromosome structural aberrations, gene mutations and the occurrence of aneuploidy are all involved in cancer development, fetal wastage and inherited disorders. To prevent human exposure to potential aneuploidy inducers, it is important to identify such agents in the environment through testing in mammalian systems 2.3 .

Much research has been undertaken to establish well­validated mammalian aneuploidy assays. Our study offered an alternative comprehensive system to detect the aneuploidy-inducing potential of chemicals. Through C­mitotic (CM) effects assay, we are able to pre-screen chemicals which may affect somatic cell division and chromosome distribution4

. The occurenance of hyperdi­ploidy/hypodiploidy in a cell is the end result of either the gain or loss of a chromosome in the complement.

Salbutamol sulphate [tert. butylamino-l-(4-hydroxy-3-hydroxymethyl phenyl) ethanol] is one of the most widely used chemotherapeutic agents to bring about a remission and cure of asthmetic attacks and is included in the list of essential drugs by WHO. It is a direct acting sympa­thomimetic agent with predominantly beta-adrenergic activity and has a selective action on beta 2 receptors . It is readily absorbed from the gastro-intestinal tract and is subject to first-pass metabolism in the liver and possibly in the gut wall ; the main metabolite is an inactive suiphate conjugate. It is rapidly excreted in the urine as metabolites

·Correspondent author Fax : 742367 E-mail : root@mnglr.ernet.in

and unchanged drug. Salbutamol does not appear to be metabolised in the lung, therefore its ultimate metabolism and excretion following inhalation depends up on the delivery method used, which determines the proportion of inhaled salbutamol relative to the proportion inadvertently swallowed. It has been suggested that the majority of an inhaled doses is swallowed and absorbed from the guf. The inhibitory effects of beta-receptor-agonists on the release of proinflamatory mediators by macrophages has repeatedly been shown. For example, the super oxide anion production as well as the L TB4-release by alveolar macro­phages, is reduced by salbutamol and isoproterenoI6

•7

•

Development of benign mesovarian leiomyomas in rats has been reported by some authors after long term treatment with salbutamol. But in mutagenicity studies with microorganisms, salbutamol is found to be negative in actions . The studie~ on the mutagenicity of this drug in somatic cells of mice indicated that it was able to induce micronuclei in bone marrow cells of mice9

• Moreover, salbutamol is massively .used and abused and human exposure is frequent. Accordingly, even small sign of genotoxicity requiring rather high concentration may have some as yet undetermined relevance for human risk.

Aneuploidy induction analysis is a part of the genetic study which constitutes an important aspect of toxico­logical evaluation of drugs. Whether or not salbutrnol could be shown to display genotoxic effects should be of importance when assessing the risk to humans of salbutamol intake. In view of this spindle damaging effects of Salbutamol sulphate is undertaken by employing mice bone marrow metaphase analysis for its possible role in aneuploidy induction.

162 INDIAN 1 EXP BIOL, FEBRUARY 1999

Materials and Methods Animals-Swiss albino mice (Mus musculus) were

procured from CFTRl, Mysore and bred in our animal care facility. They were housed in polypropylene cages bedded with paddy husk in constant light cycle and were pmvided with commercial diet (MIS Lipton India Ltd.) and water ad libitum. Adult animals of 8-10 weeks old weighing 25-30 g of either sex were used. Each experimental group consisted of five animals.

Chemical treatment- Salbutamol sulphate obtained from Cipla, Limited Bombay, India (purity 99.6 1 %) in the pure form was used. Different doses 0.12, 0.24, 1.2 and 2.4 mg/kg body weight were dissolved in double distilled water and were administered (ip) to the experimental animals. The distilled water administered animals were kept as negative controls . Colchicine (2 mg/kg body weight) treated animals as positive control. They were treated identically with same dose volume of 10 mllkg body weight. Five animals of either sex per treatment group were considered for the experiment. The animals were sacrificed at different sample intervals after single exposure. In the experiment, bone marrow cell sampling was conducted at 6, 12, 24 and 48 hr time intervals.

CM efJects-Treated mice were killed 6, 12, 24 and 48 h after exposure and bone marrow preparations were made according to the method of Tijo and WhanglO using 0.56% KCI as hypotonic solution. Flame dried slides were coded and stained with buffered Giemsa (PH 6.8). To evaluate CM effects three criteria were considered in parallel according to the description by Miller and Adler4

.

(i) Mitotic Index: Mitoses were scored for 2000 bone marrow ce lls per animal to determine MI.

(ii) C-mitoses: 100 metaphases were scored per animal to determine the percentage of C-mitoses. Cells in meta phases were classified arbitrarily into five groups according to their chromatid spreading and contraction.

Class A- All chromosomes with long and parallel chromatids

Class B- Most chromosomes with parallel chromatids Class C- Most chromatids slightly contracted and

spread Class D- Most chromatids strongly contracted and

spread Class E- As in D, also more than five chromosomes

with totally spread chromatids.

A total of 100 metaphases were scored per animal and the number of ce lls in Classes A-E are determined. An induction of C-mitotic effect can be deduced from a significant increase of the frequencies of cells in classes C­E in treated animals compared to the concurrent controls.

(iii) Anaphase reduction: anaphases among 100 mitotic ce lis were counted per animal. The variations of MI and the frequency of C-mitosis were expressed as a factor of mean of the treated group devided by the mean of the concurrent control. The anaphase reduction was calculated by I-Ana­phase in trea te anaphase in controlx 100.

Hyperdiploidy-The number of diploid, hyperdiploid and hypo-diploid cells were recorded. The frequency of the hyper-hypo diploidy in bone marrow preparations of each animal was determined by the percentage of cells in a total of 100 metaphasesfor the treated and the controls.

Statistical analysis- Statistical tests were performed by using SPSS (Statistical Package for Social Science) Version 5. Student ' t' test was used to determine the significant difference between mean va lue of individual treatment groups and their concurrent controls for CM effects analysis and hyperdiploidy.

Results

CM efJects-The results of the CM effects test with positive control chemical (COL) and the test compound (Salbutamol) are given in Table 1. COL showed significant CM effects for all three criteria after 6, 12, 24 and 48 hr of treatment. Increase of MI and the frequencies of C-mitosis as well as decreased frequencies of anaphase were observed in all test groups.

Salbutamol showed similar CM effects to COL in bone marrow cells after 12 and 48 hr of treatment. However the effect was not as pronounced as with CO L. At 1.2 and 2.4 mg/kg dose groups there was statistically significant increase in MI and C-mitotic effect (P < 0.01 ) and it also showed statistically significant decrease (P < 0.05; P< 0.01) in anaphase frequencies . The anaphase reduction was maximum (75%) for 2.4 mg/kg at 24 hr.

Hyperdiploidy was scored among 100 mitotic meta phases per animal at different intervals for the controls and treated series (Table 2). There was no statistically significant increase in the hyperdiploidy, instead the hypoploidy were found to be statistically significant (P <0.05), and in the colchicine treated senes the hyperdiploidy was highly significant (P <0.01) .

Discussion

The result obtained in the present studies demonstrated· conclusively that the drug salbutamol induces large increase in mitotic index (MI) in bone marrow cells of mice. The increase in MI could arise from interference with spindle apparatus or alternatively, interference with protein synthesis, upon which the continuation of mitosis depends II . The increase in MI might be due to the stimulatory action of the drug on divisional cycle. In the present experiment the increase in MI reaches almost to that of colchicine level clearly indicates the antimitotic action of this drug. This observation can be compared to that of econozole (an antimycotic agent) , bromhexine (a mucolytic agent) and pilocarpine nitrate which are effective in inducing significantly higher frequencies of MI in bone marrow cells of mice4

.12

.1J Vijayalxmil4 reported a dose

dependent increase in MI in bone marrow cells of mice after treatment with salbutamol (doses 0.24, 1.2, 2.4 mg/kg body wt. ). The enhancement of MI in salbutamol treated animals may be due to the stimulatory action of the drug on mitosis. Mesovarian leiomyomas hilS been reported by

SUJATHA & HEGDE: SPINDLE DAMAGING EFFECT OF SALBUTAMOL SULPHATE 163

Poynter et at. 8 after long term treatment with salbutamol. Bishun et at. 15 of the opinion that the increase in mitotic index might give a clue to the fast growth of malignant cells, eventhough the mechanism of cancer growth is not known.

C-mitotic effects ate considered to be due to spindle impairment and chemicals bringing about these changes have been shown to induce aneuploidy l6.17 In the present investigation salbutamol significantly induces. C-rnitoses at different doses. The C-mitotic effect seems to be coupled to the occurrence of abnormal chromosome numbers and there may be a threshold for this effeds. The number of studies suggested that C-mitoses may be produced by an unspecific physico-chemical mechanism which is related to the relative number of molecules that have entered to the hydrophobic compartments of the cells i.e., membranes or hydrophobic pockets of proteins lQ. The chemicals can be expected to effect chromosome condensation along with their effect on the mitotic apparatus as evidenced by the significant induction of C-metaphases by salbutamol in a study of chromosomal aberration analysis in bone marrow cells of mice by Sujatha and Hegde

Q· 20.

Salbutamol shows decrease in anaphase frequencies and this may be due to the influence of the drug on cells entering mitosis and are unable to assemble spindle

microtubules subsequently blocking at metaphase. The decrease in anaphase frequency is dose dependent. Due to the increasing concentration of the drug the anaphase cel(s will become less frequent and this might be the appearance of a block in metaphases, there by inhibiting mitotic progressionl9.Similar conditions were also observed in mouse bone marrow cells treated with pilocarpine, brornhexine, trichloroethylene (TCE)9.20.2 1 .

In vivo treatment was consistently assoc iated with hypodiploidy induction. Generally only an increase in the frequency of hyperdiploidy is regarded as a reliable indicator of aneuploidy because an unknown proportion of hypodiploid cells is often influenced by the technica l procedure of making air-dried slides22

. But in the present investigation there is an indication that hypodiploidy is more than hyperdiploidy at the 24 hr point, it is possible that at 24 hr salbutamol may interfere with anaphase lagging resulting in a loss of chromosome.

Aneuploidy could occur by several different mechanisms. However, the end res.ult is either the gain or loss of a chromosome in the complement. A non­disjunction is usually would result in a simultaneous loss and gain of a chromosome in the progeny of a cell. However, chromosome loss could also occur by mechanisms other than non-disjunction. Thus the

Table I-CM effects in mouse bone marrow cells after treatment with Salbutamol and controls

Chemical dose

(mglkg)

Water control

COL 2

Salbutamol

0.12

0.24

1.2

2A

Interval (hr)

6 12 24 48

6 12 24 48

6 12 24 48 6 12 24 48

6 12 24 48

6 12 24 48

Mitotic Index factor (% ±SE) f

7.2±0.3 1.00 7.3±OA 1.00 7.0±0.S 1.00 72±0.4 1.00

9A± 1.2 1.30" 10.8±1.4 IA8"" IU± 1.2 1.60" · I 08± 1.0 1.50""

72±0.2 1.00 7.3±OA 1.01 7A±0.2 0.94 7.S±OA 1.04 7.8±0.2 1.08 7.9±OA 1.08 7.8±0.3 1.11 74±0.6 1.02

82±0.2 1.13 88±0.S 1.20· 6A±0.6 0.9 1 8.9±0.8 1.23"

8.6±0.8 1.19 8.9±0.S 1.22" 70±0.9 0.88 9.8±0.7 1.36"

f- Mean of the treat ed group divided by mean of the control P value "< O.OS: * *<0.0 I: """< 0.00 I (Student'S ' I' test)

C-mitosis factor Anaphase Reduction frequency f frequency (%) (% ±SE) (% '±SE)

1.8±0.3 1.00 1.2±0.8 0 2.0±0.S 1.00 1.6±0.6 0 2.S±0.6 1.00 1.6±0.6 0 2.4±0.6 1.00 I.S±O.S 0

42.S±0.8 23 .6"" 0.2±0.9 83"" 49 .0±0.S 24 .S""" O.I±O.S 93"· 38.8±0.8 IS .S"" 0.2±0.S 87"" 42 .0±0.8 17.S""" 03±0.6 80""·

3.5±OA 1.94 1.2±0.2 0 6.3±0.2 3. IS 1.3±0.2 18 8.8±0.3 3.S2 1.2±0.2 2S 8.0±0.4 3.33 1.1 ±0.3 26 4A±OA 2.44 1.1 ±OA 8 S.8±0.6 2.90 0.9±0.S 43 9.8±0.S 3.92 1 3±0.6 18 10.7±0.6 4.45 0.9±OA 40

8A±0.S 4.60 08±0.2 33 12.8±0.8 6AO"· 0 .8±OA SO· 9.8±0.6 3.92 1.0±06 33 180±0.S 7.S0" 0.7±OA S3"

IS .S±0.6 4.42·" 08±OA 33 19.5±08 7.S0"· 0.S±0.6 68"" 32.8±0.9 13 .12"" 0.4±0.S 7S" 24A±OA 10.16"" 0.S±0.8 66"

164 INDIAN J EXP BIOL, FEBRUARY 1999

Table 2-Frequency of dipliod and aneuploid cells after exposure to Salbutamol

Treatment Dose

mg/kg

Time Diploidy Aneuploidy (h) No. of cells Analyzed Diploid Cells(%) Hypodiploidy Hyperdiploidy

Water

Salbutamol 0.12

0.24

1.2

2.4

COL 2

24 100 48 100

24 100 48 IDa

24 100 48 100

24 lOa 48 IDa

24 100 48 IDa

24 100 48 100

P value *< 0.05: ** <0.0 1; *** < 0.00 1 (Student's 'I' test)

99 99

99 97 2

94 3.5 2.5 92 5.5 2.5

90.5 7* 2.5 89.5 8* 2.5

82 6 12** 80 7 13**

------------------------------------------------------

freq uency o f chromosome loss would nonnally be higher than chromosome ga in. The frequency of chromosome loss is used as an index for the potential of an environmental agent to induce aneuploidy in mammalian cells23

• A limitat ion . of thi s conservative approach for reporting aneuploidy is that it overlooks the possible induction of aneuploidy by means other than non disjunction. For example, anaphase lagg ing can result in the production of hypoploid ce ll s and may be operational in the case of sa lbutamol. Marchetti et al.2

\ reported that Diazepam (DZ) (an anti epi leptic drug) one of the 10 chemicals chosen in the CEC programme, induced hypodioloidy in oocytes of mice and it also induced mitotic arrest and C-mitoses in mouse bone marrow cells4 Gassner and Adler5 reported that Acrylamide an industria l chemical shown to exhibit a grea t disparity between hyper-and hypodiploidy induction in bone marrow metaphases, the latter being much more freque nt in a ll experimental groups and indicated that an a lterna tive mechanisms of aneuploidy induction rather than produc ing random chromosome loss than non disjunction. Therefore it remains to be detenn in ed whether the induction of hyperd iploidy is due to the mechanisms other than non-disjunction.

The present spindle disturb ing effects of Salbutrnol mi rrored a cytotoxic effect as detennined by the reduced survival of the ce lls i.e., reduction in the MI at 24 hr. Thus, it cannot be excluded that the two effects are related and that some disturbed ce lls are bound for death without risk of aneuploid progeny. However, Onfell '8 has shown that red uced survival and spind le disturbances call occur independently and that cells surviving simi lar challenge can indeed form aneuploid daughter cells . Recently, new tools have emerged in mo lecular cytogenetics, particularly related to multico lour fluorescence in situ hybridization (FISH) for analysis of male gametes26

.2

." with such assays it

should be possible to study risk of aneuploidy from compounds such as salbutamol in vivo.

Acknowledgement One of us (Sujatha T.V) is grateful to UGC, New Delhi

and Mangalore University for awarding Junior Research Fellowship.

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SUJATHA & HEGDE: SPINDLE DAMAGING EFFECf OF SALBUTAMOL SULPHATE 165

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