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Induction of sister-chromatid exchange in human blood lymphocytes by aqueous extract of palmyrah (Borassus flabellifer) flour
Mutation Research, 224 (1989) 241-245
241
Elsevier MUTGEN 01476
Induction of sister-chromatid exchange in human blood lymphocytes by aqueous extract of palmyrah (Borassus flabellifer) flour Daoroong
Kangwanpong
1, D . M a r a t a n a
2 a n d P. T e m c h a r o e n
2
I Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok 10400 (Thailand) and "~Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand)
(Received 2 January 1989) (Revision received28 April 1989) (Accepted I May 1989)
Keywords: Palmyrah flour; Sister-chromatidexchanges; Human lymphocytes
Summary pPalmyrah palm (Borassus flabellifer) is widely consumed by people in certain tropical countries. The incidence of human malignant lymphomas, mutagenicity and toxicity in rats and bacteria encouraged us to study the potency of palmyrah crude aqueous extracts in inducing sister-chromatid exchanges (SCEs) in human blood lymphocytes in vitro. The extracts induced SCEs in a dose-related manner in both females and males. These effects apparently showed no consistency between batches. This result may be due to the intrinsic variation of different donors in their response to the induction of SCEs by palmyrah extracts. SCE frequency was proportional to chromosome length and SCEs at the centromeric region showed no difficulty in being scored. Concerning methods of short-term cytogenetic testing for detecting mutagenic and carcinogenic chemicals, we found that the SCE test was not more sensitive than the classic chromosome-breakage test.
Flour from the young shoots of the palmyrah palm (Borassus flabellifer), which is widely grown in Asia, is extensively eaten in the northern part of Sri Lanka. Immunological disturbances and toxic effects produced by this flour have been reported (Arseculeratne and Panabokke, 1983; Arseculeratne et al., 1971, 1981; Greig et al., 1980; Panabokke and Arseculeratne, 1976). It also produces clastogenic effects on human blood lymphocytes (Kangwanpong et al., 1981) and acts as a mutagen when tested on S. typhimuriurn and E. coli
Correspondence: Dr. D. Kangwanpong, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok 10400 (Thailand).
(Andersen and Poulsen, 1985). In the present report, the ability of palmyrah crude extracts to induce sister-chromatid exchanges (SCEs), a sensitive means of monitoring D N A damage, is described. Materials and methods Preparation of palmyrah flour extracts
Palmyrah flour (courtesy of Prof. Dr. S.N. Arseculeratne, Sri Lanka) from finely powdered fresh young shoots (3 g / 7 0 ml) was extracted by the m e t h o d previously described elsewhere (Kangwanpong et al., 1981). Five different batches of flour were similarly extracted and tested fresh.
0165-1218/89/$03.50 © 1989 ElsevierScience Publishers B.V. (Biomedical Division)
242 TABLE 1 DISTRIBUTION OF SCEs/METAPHASE INDUCED C O N C E N T R A T I O N S IN F E M A L E S A N D M A L E S
BY 5 B A T C H E S O F
PALMYRAH
E X T R A C T S AT V A R I O U S
Dose ( m g / m l medium)
SCEs/metaphase Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Females control 2 4 8 10 12 MC
6.9+ 3.2 8.8+ ~.4 9.4+ 2.7 10.2_+ 3.6 10.8+ 3.8 12.8_+ 6.0 39.3 _+20.8
7.3+ 2.9 9.9+ 3.6 10.3_+ 3.6 10.7_+ 4.8 11.0-+ 3.6 11.9_+ 5.1 38.0 + 17.3
6.6+ 2.7 8.4+ 3.1 9.1_+ 3.6 10.1+ 3.9 10.9-+ 3.6 12.2_+ 3.5 38.7 -+ 13.5
6.0+ 2.1 6.4+ 2.7 * 7.4_+ 2.5 7.4_+ 2.6 7.6_+ 2.3 8.0_+ 2.6 36.3 ___14.8
5.4+ 1.9 6.6+ 2.3 7.8_+ 2.6 8.2_+ 2.7 8.5_+ 2.6 9.1_+ 3.0 32.5 -+ 13.4
Males control 2 4 8 10 12 MC
6.5-+ 2.9 7.5-+ 3.0 8.7_+ 4.9 8.8-+ 3.8 8.9_+ 3.5 9.3_+ 3.8 31.1 -+ 14.2
5.8-+ 2.6 7.8+ 2.9 8.2± 2.9 8.7-+ 3.3 9.0_+ 3.0 9.9_+ 3.6 31.3 -+ 16.1
4.7-+ 1.6 7.4-+ 2.8 8.2-+ 2.8 8.4-+ 2.4 9.2_+ 2.7 11.0_+ 3.9 34.4+ 14.0
5.3-+ 2.3 6.2_+ 2.1 6.8_+ 2.4 7.4_+ 2.3 7.7_+ 3.1 8.3_+ 3.4 30.1 Jr 14.9
5.3-+ 2.7 6.1_+ 2.9 6.8 + 2.9 7.5_+ 3.1 8.1_+ 3.1 8.9_+ 3.0 32.5 -+ 13.8
MC, mitomycin C, used as a positive control at a final concentration of 1 ~ g / m l . * N o significant difference compared to control ( p < 0.05).
Peripheral blood lymphocytes from one male and one female were used to test each batch of flour. The donors differ from batch to batch.
,2] /
/
!0,
In vitro S C E test
Heparinized venous blood obtained from healthy adults (5 males and 5 females) was cultured as described by Kangwanpong et al. (1981) in the presence of bromodeoxyuridine (BrdU, Sigma, final concentration 1.6 × 10 -5 M). In test samples, after 66 h of cultivation, the lymphocytes were exposed for the next 24 h to final concentrations of 2, 4, 8, 10 and 12 mg of the palmyrah per ml culture medium. Lymphocytes cultured in medium with BrdU served as a negative control. Mitomycin C (1 /~g/ml) was used as a positive control. The cultures were harvested and the chromosomes were prepared using the conventional air-dried technique. Staining was modified from Perry and Wolff (1974). The slides were stained with Hoechst 33258 solution (2.5 m g / 5 0 ml distilled water), exposed to strong sunlight - at noon in high summer - for 18-20 min. The slides were then counterstained with 10% Giemsa solution for 20 rain.
~
8.
~ ~ 6, ®., O"
/ " PE. no. 1
,, ~ 4
-*-PE. no. 2 -,~ PE. no. 3 G - P E . no. 4
2.
% PE. no. 5
o CONe. ( m g / m l )
Fig. 1. D o s e - r e s p o n s e curves of SCE frequencies in blood lymphocytes from male donors after administration of various concentrations of palmyrah extract. PE, palmyrah extract in vitro.
243 14,
S C E analysis Fifty second-division metaphases were observed and SCEs were examined and counted in each culture. SCEs at the centromeric regions were
12,
scored separately. 10,
re.
Results
"
W (.3
"~ PE. no. 1 +
4,
PE. no. 2 PE. no. 3
,8-PE. no. 4 *PE.
no. 5
CONC. ( m g / m l )
Fig. 2. Dose-response curves of SCE frequencies in blood lymphocytes from female donors after administration of various concentrations of palmyrah extract. PE, palmyrah extract in vitro.
The distributions of SCEs per metapb.ase induced by palmyrah extracts at various conc e n t r a t i o n s a r e p r e s e n t e d i n T a b l e 1. T h e S C E frequencies elevated from the baseline values (untreated group) showed a dose-related pattern, when the concentration of extract increased (Figs. 1 and 2). T h e s e h i g h e r S C E f r e q u e n c i e s w e r e s t a t i s t i c a l l y s i g n i f i c a n t ( p < 0 . 0 0 0 5 - 0 . 0 5 ) , e x c e p t f o r t h e extracts of batches 4 and 5 at the concentration of 2 mg/ml culture medium on female and male samples respectively. The SCE frequencies induced by the highest concentration (12 mg/ml culture m e d i u m ) w e r e a b o u t 1.7 t i m e s h i g h e r t h a n t h a t o f the untreated group. SCEs occurred non-randomly in various groups of chromosomes and were found to have the h i g h e s t f r e q u e n c i e s i n g r o u p C, b u t S C E s / c h r o mosome were highest in group A. When SCEs
TABLE 2 DISTRIBUTION OF SCEs/METAPHASE (AS IN TABLE 1) COUNTED SEPARATELY AT THE CENTROMERE Dose (mg/ml medium)
SCEs/metaphase (pq SCEs + cSCEs) Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
6.02 + 0.84 7.80 5- 0.98 8.02 5-1.34 9.00 5-1.24 8.82 4- 2.00 11.44 5-1.36 35.70 5- 3.64
6.78 + 0.52 8.86 + 1.02 9.74 _ 0.54 10.18 4- 0.56 10.38 4- 0.60 11.24 ± 0.62 35.84 5- 2.16
6.46 + 0.12 7.98 5- 0.42 8.38 4- 0.72 9.48 5- 0.66 9.96 5- 0.92 11.38 5- 0.82 36.86 4-1.88
5.52 5- 0.52 6.04 5- 0.36 6.96 5- 0.46 6.96 4- 0.48 7.18 _ 0.38 7.62 5- 0.34 35.32 4-1.0
4.80 5- 0.60 6.16 5- 0.44 7.38 5- 0.40 7.80 5- 0.38 8.08 5- 0.44 8.76 5- 0.36 31.88 4- 0.66
5.60 + 0.92 6.68 5- 0.82 7.70 5- 0.96 8.38 5- 0.46 8.06 4- 0.82 8.62 5- 0.66 30.58 5- 2.52
5.62 ± 0.18 7.08 5- 0.76 7.74 5- 0.44 8.22 5- 0.52 8.46 4- 0.56 9.40 5- 0.46 29.60 5-1.54
4.42 4- 0.26 6.82 4- 0.54 7.58 5- 0.58 7.72 4- 0.64 8.54 4- 0.62 10.20 5- 0.82 32.52 + 1.90
4.74 5- 0.56 5.92 5- 0.30 6.50 5- 0.26 7.04 5- 0.34 7.08 5- 0.62 7.76 5- 0.56 29.12 5-1.0
5.16 4- 0.18 5.60 4- 0.52 6.36 5- 0.48 7.18 5- 0.32 7.60 5- 0.50 8.5 5- 0.38 31.18 5-1.28
Females
control 2 4 8 10 12 MC
Males control 2 4 8 10 12 MC
MC, mitomycin C, used as a positive control at a final concentration of 1 ~g/ml; pq SCEs, SCE frequencies at short and long arms; cSCEs, SCE frequencies at centromere.
244 650 r= 1.00
600,
T 0
t 55oa
I 500-
s c E 45o=
f r •
4oo.
q U e 350. n c
i ~oo.
e s 250 •
200 200
I
I
I
I
I
I
I
250
300
350
4.00
450
500
550
SCE
frequencies
excluded
I
cSCE
Fig. 3. Correlation between total SCE frequencies and SCE frequencies excluding SCEs at the centromeric region (cSCE), determined in second-divisionmetaphases from lymphocytestreated with various concentrations of palmyrah extracts.
were counted separately at the centromeric region, the distributions of SCEs still showed the same phenomenon as with chromosomes as a whole (Table 2). As shown in Fig. 3, there was a significant positive correlation between total SCE frequencies and SCE frequencies counted only on both arms of the chromosomes (y = 11.75 + 1.05x, r = 1.00). N o significant difference was seen between lymphocytes from males and females, although SCE frequencies were observed to be a little higher in males. Extracts of batches 1, 2 and 3 showed equal effects in inducing SCEs, but were significantly different from extracts of batches 4 and 5. Batches 1 and 2 of palmyrah flour were received in May, 1986 and the other batches, 3, 4 and 5, in May, 1987. There is apparently a batch-to-batch variation in causing SCE in cultured human lymphocytes. Discussion
In the present study, an evaluation of SCEs, which is of great value for investigating the effects
of chemical and physical agents on genetic material, was done in vitro. Crude aqueous extracts of palmyrah flour at various concentrations produced primary D N A damage on human lymphocytes in a dose-related manner. Our experiments showed the genotoxic effect of palmyrah extracts without metabolic activation. With the same in vitro test system, this agent was proved to be a clastogen by the production of clear doublestrand breaks (Kangwanpong et al., 1981). The crude extract of palmyrah flour seems to be a direct-acting agent. It contains active material which is of much lower activity than mitomycin C, probably on account of the crude nature of the extracts and the low concentration of the mutagen. A previous study on the mutagenic potential of palmyrah flour using the Ames test showed no influence of metabolizing enzymes in activating the palmyrah extract. Andersen and Poulsen (1985) compared assays on S. typhimurium with and without $9 fraction for metabolizing purpose. They also concluded that this test agent, palmyrah flour, was a weak mutagen. The effect of different batches of palmyrah
245
flour in inducing SCEs was rather difficult to interpret. A statistically significant difference was found between batches 1, 2, 3 and batches 4, 5 in all concentrations of the extract. Each batch of palmyrah flour tested for clastogenicity in previous studies (Kangwanpong et al., 1981) was free from aflatoxins, nitrosamines and pesticides. The possibility that SCE induction was due to contaminating substances could therefore be ruled out. It seems to be an intrinsic factor of the individual's lymphocytes in their response to chemicals that plays a role. It could not be' concluded that an inherent mutagenic activity varies in potency between different batches. Some investigators have suggested that chemical agents induce large numbers of SCEs by the B r d U - d y e method at concentrations below those causing significant numbers of chromosome breaks (Latt, 1974). However, our results indicated that the SCE test was not more sensitive than the classic chromosome-breakage test. Palmyrah extracts at the lowest concentration (2 m g / m l ) induced low SCE frequencies which were not statistically significant compared with the negative control; SCE frequencies at the highest concentration (12 m g / m l ) were 1.7 times higher than the control ~. Using the chromosome-breakage test, all batches of the extracts at the lowest concentration induced a significant increase in aberrations and at the highest concentration, chromosome aberrations were found to be 7.5 times higher than control (Kangwanpong et al., 1981). However, it is easier and faster to score SCEs while scoring chromosome aberrations requires a higher level of skill. Moreover, the SCE test has a more clear-cut end point, i.e., it can be quantitated more accurately. Normally, twisting of chromosomes occurs both at the centromeric region and on both arms. If the twist occurs at the centromeric region, a difficulty arises in analyzing whether it is really an SCE or a distortion. Thus, centromeric SCEs can contribute to errors in the total SCE frequencies. In a good chromosome preparation, such an error ought not to occur if the observers are careful. To avoid a false interpretation, centromeric SCEs were counted separately in our experiments. Nevertheless, the results of our experiment remained the same whether centromeric SCEs were counted separately or not.
The frequency of SCEs seems to be proportional to D N A content and consequently to chromosome length. Our study showed the highest SCE frequencies per chromosome to be in group A chromosomes. When the total chromosome length in each group was considered, we found the highest number of exchanges in group C chromosomes in all subjects. S C E s / m e t a p h a s e in females were always higher than in males, although the difference was not significant.
Acknowledgement We are grateful to Prof. Dr. S.N. Arseculeratne from Sri Lanka for his valuable criticism and for supplying the palmyrah flour.
References Andersen, P.H., and E. Poulsen (1985) Mutagenicity of flour from the palmyrah palm (Borassusflabellifer) in Salmonella typhimurium and Escherichia coli, Cancer Lett., 26, 113-119. Arseculeratne, S.N., and R.G. Panabokke (1983) Studies on the toxicology of the palmyrah palm (Borassus flabellifer). Part III. Development of malignant lymphomas in rats after prolonged feeding of palmyrah flour, J. Natl. Sci. Coun. Sri Lanka, 11, 11-23. Arseculeratne, S.N., R.G. Panabokke, G.E. Tennekoon and C.H.S.R. Bandunatha (1971) Toxic effects of Borassus flabellifer (palmyrah palm) in rats, Br. J. Exp. Path., 52, 524-537. Arseculeratne, S.N., S. Sirisinha, C. Charupatana and D. Kangwanpong (1981) Immunological alterations in rats fed with flour from the palmyrah palm (Borassus flabellifer), Proc. Soc. Exp. Biol. Med., 168, 356-360. Greig, J.B., S.J.E. Kay and R.J. Bennetts (1980) A toxin from the palmyrah palm, Borassus flabellifer: partial purification and effects in rats, Food Cosmet. Toxicol., 18, 483-488. Kangwanpong, D., S.N. Arseculeratne and S. Sirisinha (1981) Clastogenic effect of aqueous extracts of palmyrah (Borassus flabellifer) flour on human blood lymphocytes, Mutation Res., 89, 63-68. Latt, S.A. (1974) Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3162-3166. Panabokke, R.G., and S.N. Arseculeratne (1976) Veno-occlusive lesions in the liver of rats after prolonged feeding with paimyrah (Borassus flabellifer) flour, Br. J. Exp. Path., 57, 189-199. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251,156-158.
241
Elsevier MUTGEN 01476
Induction of sister-chromatid exchange in human blood lymphocytes by aqueous extract of palmyrah (Borassus flabellifer) flour Daoroong
Kangwanpong
1, D . M a r a t a n a
2 a n d P. T e m c h a r o e n
2
I Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok 10400 (Thailand) and "~Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand)
(Received 2 January 1989) (Revision received28 April 1989) (Accepted I May 1989)
Keywords: Palmyrah flour; Sister-chromatidexchanges; Human lymphocytes
Summary pPalmyrah palm (Borassus flabellifer) is widely consumed by people in certain tropical countries. The incidence of human malignant lymphomas, mutagenicity and toxicity in rats and bacteria encouraged us to study the potency of palmyrah crude aqueous extracts in inducing sister-chromatid exchanges (SCEs) in human blood lymphocytes in vitro. The extracts induced SCEs in a dose-related manner in both females and males. These effects apparently showed no consistency between batches. This result may be due to the intrinsic variation of different donors in their response to the induction of SCEs by palmyrah extracts. SCE frequency was proportional to chromosome length and SCEs at the centromeric region showed no difficulty in being scored. Concerning methods of short-term cytogenetic testing for detecting mutagenic and carcinogenic chemicals, we found that the SCE test was not more sensitive than the classic chromosome-breakage test.
Flour from the young shoots of the palmyrah palm (Borassus flabellifer), which is widely grown in Asia, is extensively eaten in the northern part of Sri Lanka. Immunological disturbances and toxic effects produced by this flour have been reported (Arseculeratne and Panabokke, 1983; Arseculeratne et al., 1971, 1981; Greig et al., 1980; Panabokke and Arseculeratne, 1976). It also produces clastogenic effects on human blood lymphocytes (Kangwanpong et al., 1981) and acts as a mutagen when tested on S. typhimuriurn and E. coli
Correspondence: Dr. D. Kangwanpong, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok 10400 (Thailand).
(Andersen and Poulsen, 1985). In the present report, the ability of palmyrah crude extracts to induce sister-chromatid exchanges (SCEs), a sensitive means of monitoring D N A damage, is described. Materials and methods Preparation of palmyrah flour extracts
Palmyrah flour (courtesy of Prof. Dr. S.N. Arseculeratne, Sri Lanka) from finely powdered fresh young shoots (3 g / 7 0 ml) was extracted by the m e t h o d previously described elsewhere (Kangwanpong et al., 1981). Five different batches of flour were similarly extracted and tested fresh.
0165-1218/89/$03.50 © 1989 ElsevierScience Publishers B.V. (Biomedical Division)
242 TABLE 1 DISTRIBUTION OF SCEs/METAPHASE INDUCED C O N C E N T R A T I O N S IN F E M A L E S A N D M A L E S
BY 5 B A T C H E S O F
PALMYRAH
E X T R A C T S AT V A R I O U S
Dose ( m g / m l medium)
SCEs/metaphase Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Females control 2 4 8 10 12 MC
6.9+ 3.2 8.8+ ~.4 9.4+ 2.7 10.2_+ 3.6 10.8+ 3.8 12.8_+ 6.0 39.3 _+20.8
7.3+ 2.9 9.9+ 3.6 10.3_+ 3.6 10.7_+ 4.8 11.0-+ 3.6 11.9_+ 5.1 38.0 + 17.3
6.6+ 2.7 8.4+ 3.1 9.1_+ 3.6 10.1+ 3.9 10.9-+ 3.6 12.2_+ 3.5 38.7 -+ 13.5
6.0+ 2.1 6.4+ 2.7 * 7.4_+ 2.5 7.4_+ 2.6 7.6_+ 2.3 8.0_+ 2.6 36.3 ___14.8
5.4+ 1.9 6.6+ 2.3 7.8_+ 2.6 8.2_+ 2.7 8.5_+ 2.6 9.1_+ 3.0 32.5 -+ 13.4
Males control 2 4 8 10 12 MC
6.5-+ 2.9 7.5-+ 3.0 8.7_+ 4.9 8.8-+ 3.8 8.9_+ 3.5 9.3_+ 3.8 31.1 -+ 14.2
5.8-+ 2.6 7.8+ 2.9 8.2± 2.9 8.7-+ 3.3 9.0_+ 3.0 9.9_+ 3.6 31.3 -+ 16.1
4.7-+ 1.6 7.4-+ 2.8 8.2-+ 2.8 8.4-+ 2.4 9.2_+ 2.7 11.0_+ 3.9 34.4+ 14.0
5.3-+ 2.3 6.2_+ 2.1 6.8_+ 2.4 7.4_+ 2.3 7.7_+ 3.1 8.3_+ 3.4 30.1 Jr 14.9
5.3-+ 2.7 6.1_+ 2.9 6.8 + 2.9 7.5_+ 3.1 8.1_+ 3.1 8.9_+ 3.0 32.5 -+ 13.8
MC, mitomycin C, used as a positive control at a final concentration of 1 ~ g / m l . * N o significant difference compared to control ( p < 0.05).
Peripheral blood lymphocytes from one male and one female were used to test each batch of flour. The donors differ from batch to batch.
,2] /
/
!0,
In vitro S C E test
Heparinized venous blood obtained from healthy adults (5 males and 5 females) was cultured as described by Kangwanpong et al. (1981) in the presence of bromodeoxyuridine (BrdU, Sigma, final concentration 1.6 × 10 -5 M). In test samples, after 66 h of cultivation, the lymphocytes were exposed for the next 24 h to final concentrations of 2, 4, 8, 10 and 12 mg of the palmyrah per ml culture medium. Lymphocytes cultured in medium with BrdU served as a negative control. Mitomycin C (1 /~g/ml) was used as a positive control. The cultures were harvested and the chromosomes were prepared using the conventional air-dried technique. Staining was modified from Perry and Wolff (1974). The slides were stained with Hoechst 33258 solution (2.5 m g / 5 0 ml distilled water), exposed to strong sunlight - at noon in high summer - for 18-20 min. The slides were then counterstained with 10% Giemsa solution for 20 rain.
~
8.
~ ~ 6, ®., O"
/ " PE. no. 1
,, ~ 4
-*-PE. no. 2 -,~ PE. no. 3 G - P E . no. 4
2.
% PE. no. 5
o CONe. ( m g / m l )
Fig. 1. D o s e - r e s p o n s e curves of SCE frequencies in blood lymphocytes from male donors after administration of various concentrations of palmyrah extract. PE, palmyrah extract in vitro.
243 14,
S C E analysis Fifty second-division metaphases were observed and SCEs were examined and counted in each culture. SCEs at the centromeric regions were
12,
scored separately. 10,
re.
Results
"
W (.3
"~ PE. no. 1 +
4,
PE. no. 2 PE. no. 3
,8-PE. no. 4 *PE.
no. 5
CONC. ( m g / m l )
Fig. 2. Dose-response curves of SCE frequencies in blood lymphocytes from female donors after administration of various concentrations of palmyrah extract. PE, palmyrah extract in vitro.
The distributions of SCEs per metapb.ase induced by palmyrah extracts at various conc e n t r a t i o n s a r e p r e s e n t e d i n T a b l e 1. T h e S C E frequencies elevated from the baseline values (untreated group) showed a dose-related pattern, when the concentration of extract increased (Figs. 1 and 2). T h e s e h i g h e r S C E f r e q u e n c i e s w e r e s t a t i s t i c a l l y s i g n i f i c a n t ( p < 0 . 0 0 0 5 - 0 . 0 5 ) , e x c e p t f o r t h e extracts of batches 4 and 5 at the concentration of 2 mg/ml culture medium on female and male samples respectively. The SCE frequencies induced by the highest concentration (12 mg/ml culture m e d i u m ) w e r e a b o u t 1.7 t i m e s h i g h e r t h a n t h a t o f the untreated group. SCEs occurred non-randomly in various groups of chromosomes and were found to have the h i g h e s t f r e q u e n c i e s i n g r o u p C, b u t S C E s / c h r o mosome were highest in group A. When SCEs
TABLE 2 DISTRIBUTION OF SCEs/METAPHASE (AS IN TABLE 1) COUNTED SEPARATELY AT THE CENTROMERE Dose (mg/ml medium)
SCEs/metaphase (pq SCEs + cSCEs) Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
6.02 + 0.84 7.80 5- 0.98 8.02 5-1.34 9.00 5-1.24 8.82 4- 2.00 11.44 5-1.36 35.70 5- 3.64
6.78 + 0.52 8.86 + 1.02 9.74 _ 0.54 10.18 4- 0.56 10.38 4- 0.60 11.24 ± 0.62 35.84 5- 2.16
6.46 + 0.12 7.98 5- 0.42 8.38 4- 0.72 9.48 5- 0.66 9.96 5- 0.92 11.38 5- 0.82 36.86 4-1.88
5.52 5- 0.52 6.04 5- 0.36 6.96 5- 0.46 6.96 4- 0.48 7.18 _ 0.38 7.62 5- 0.34 35.32 4-1.0
4.80 5- 0.60 6.16 5- 0.44 7.38 5- 0.40 7.80 5- 0.38 8.08 5- 0.44 8.76 5- 0.36 31.88 4- 0.66
5.60 + 0.92 6.68 5- 0.82 7.70 5- 0.96 8.38 5- 0.46 8.06 4- 0.82 8.62 5- 0.66 30.58 5- 2.52
5.62 ± 0.18 7.08 5- 0.76 7.74 5- 0.44 8.22 5- 0.52 8.46 4- 0.56 9.40 5- 0.46 29.60 5-1.54
4.42 4- 0.26 6.82 4- 0.54 7.58 5- 0.58 7.72 4- 0.64 8.54 4- 0.62 10.20 5- 0.82 32.52 + 1.90
4.74 5- 0.56 5.92 5- 0.30 6.50 5- 0.26 7.04 5- 0.34 7.08 5- 0.62 7.76 5- 0.56 29.12 5-1.0
5.16 4- 0.18 5.60 4- 0.52 6.36 5- 0.48 7.18 5- 0.32 7.60 5- 0.50 8.5 5- 0.38 31.18 5-1.28
Females
control 2 4 8 10 12 MC
Males control 2 4 8 10 12 MC
MC, mitomycin C, used as a positive control at a final concentration of 1 ~g/ml; pq SCEs, SCE frequencies at short and long arms; cSCEs, SCE frequencies at centromere.
244 650 r= 1.00
600,
T 0
t 55oa
I 500-
s c E 45o=
f r •
4oo.
q U e 350. n c
i ~oo.
e s 250 •
200 200
I
I
I
I
I
I
I
250
300
350
4.00
450
500
550
SCE
frequencies
excluded
I
cSCE
Fig. 3. Correlation between total SCE frequencies and SCE frequencies excluding SCEs at the centromeric region (cSCE), determined in second-divisionmetaphases from lymphocytestreated with various concentrations of palmyrah extracts.
were counted separately at the centromeric region, the distributions of SCEs still showed the same phenomenon as with chromosomes as a whole (Table 2). As shown in Fig. 3, there was a significant positive correlation between total SCE frequencies and SCE frequencies counted only on both arms of the chromosomes (y = 11.75 + 1.05x, r = 1.00). N o significant difference was seen between lymphocytes from males and females, although SCE frequencies were observed to be a little higher in males. Extracts of batches 1, 2 and 3 showed equal effects in inducing SCEs, but were significantly different from extracts of batches 4 and 5. Batches 1 and 2 of palmyrah flour were received in May, 1986 and the other batches, 3, 4 and 5, in May, 1987. There is apparently a batch-to-batch variation in causing SCE in cultured human lymphocytes. Discussion
In the present study, an evaluation of SCEs, which is of great value for investigating the effects
of chemical and physical agents on genetic material, was done in vitro. Crude aqueous extracts of palmyrah flour at various concentrations produced primary D N A damage on human lymphocytes in a dose-related manner. Our experiments showed the genotoxic effect of palmyrah extracts without metabolic activation. With the same in vitro test system, this agent was proved to be a clastogen by the production of clear doublestrand breaks (Kangwanpong et al., 1981). The crude extract of palmyrah flour seems to be a direct-acting agent. It contains active material which is of much lower activity than mitomycin C, probably on account of the crude nature of the extracts and the low concentration of the mutagen. A previous study on the mutagenic potential of palmyrah flour using the Ames test showed no influence of metabolizing enzymes in activating the palmyrah extract. Andersen and Poulsen (1985) compared assays on S. typhimurium with and without $9 fraction for metabolizing purpose. They also concluded that this test agent, palmyrah flour, was a weak mutagen. The effect of different batches of palmyrah
245
flour in inducing SCEs was rather difficult to interpret. A statistically significant difference was found between batches 1, 2, 3 and batches 4, 5 in all concentrations of the extract. Each batch of palmyrah flour tested for clastogenicity in previous studies (Kangwanpong et al., 1981) was free from aflatoxins, nitrosamines and pesticides. The possibility that SCE induction was due to contaminating substances could therefore be ruled out. It seems to be an intrinsic factor of the individual's lymphocytes in their response to chemicals that plays a role. It could not be' concluded that an inherent mutagenic activity varies in potency between different batches. Some investigators have suggested that chemical agents induce large numbers of SCEs by the B r d U - d y e method at concentrations below those causing significant numbers of chromosome breaks (Latt, 1974). However, our results indicated that the SCE test was not more sensitive than the classic chromosome-breakage test. Palmyrah extracts at the lowest concentration (2 m g / m l ) induced low SCE frequencies which were not statistically significant compared with the negative control; SCE frequencies at the highest concentration (12 m g / m l ) were 1.7 times higher than the control ~. Using the chromosome-breakage test, all batches of the extracts at the lowest concentration induced a significant increase in aberrations and at the highest concentration, chromosome aberrations were found to be 7.5 times higher than control (Kangwanpong et al., 1981). However, it is easier and faster to score SCEs while scoring chromosome aberrations requires a higher level of skill. Moreover, the SCE test has a more clear-cut end point, i.e., it can be quantitated more accurately. Normally, twisting of chromosomes occurs both at the centromeric region and on both arms. If the twist occurs at the centromeric region, a difficulty arises in analyzing whether it is really an SCE or a distortion. Thus, centromeric SCEs can contribute to errors in the total SCE frequencies. In a good chromosome preparation, such an error ought not to occur if the observers are careful. To avoid a false interpretation, centromeric SCEs were counted separately in our experiments. Nevertheless, the results of our experiment remained the same whether centromeric SCEs were counted separately or not.
The frequency of SCEs seems to be proportional to D N A content and consequently to chromosome length. Our study showed the highest SCE frequencies per chromosome to be in group A chromosomes. When the total chromosome length in each group was considered, we found the highest number of exchanges in group C chromosomes in all subjects. S C E s / m e t a p h a s e in females were always higher than in males, although the difference was not significant.
Acknowledgement We are grateful to Prof. Dr. S.N. Arseculeratne from Sri Lanka for his valuable criticism and for supplying the palmyrah flour.
References Andersen, P.H., and E. Poulsen (1985) Mutagenicity of flour from the palmyrah palm (Borassusflabellifer) in Salmonella typhimurium and Escherichia coli, Cancer Lett., 26, 113-119. Arseculeratne, S.N., and R.G. Panabokke (1983) Studies on the toxicology of the palmyrah palm (Borassus flabellifer). Part III. Development of malignant lymphomas in rats after prolonged feeding of palmyrah flour, J. Natl. Sci. Coun. Sri Lanka, 11, 11-23. Arseculeratne, S.N., R.G. Panabokke, G.E. Tennekoon and C.H.S.R. Bandunatha (1971) Toxic effects of Borassus flabellifer (palmyrah palm) in rats, Br. J. Exp. Path., 52, 524-537. Arseculeratne, S.N., S. Sirisinha, C. Charupatana and D. Kangwanpong (1981) Immunological alterations in rats fed with flour from the palmyrah palm (Borassus flabellifer), Proc. Soc. Exp. Biol. Med., 168, 356-360. Greig, J.B., S.J.E. Kay and R.J. Bennetts (1980) A toxin from the palmyrah palm, Borassus flabellifer: partial purification and effects in rats, Food Cosmet. Toxicol., 18, 483-488. Kangwanpong, D., S.N. Arseculeratne and S. Sirisinha (1981) Clastogenic effect of aqueous extracts of palmyrah (Borassus flabellifer) flour on human blood lymphocytes, Mutation Res., 89, 63-68. Latt, S.A. (1974) Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3162-3166. Panabokke, R.G., and S.N. Arseculeratne (1976) Veno-occlusive lesions in the liver of rats after prolonged feeding with paimyrah (Borassus flabellifer) flour, Br. J. Exp. Path., 57, 189-199. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251,156-158.