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Field bean protease inhibitor mitigates the sister-chromatid exchanges induced by bromoform and depresses the spontaneous sister-chromatid exchange frequency of human lymphocytes in vitro
Environmental Mutagenesis
ELSEVIER
Mutation Research 360 (1996) 29-35
Field bean protease inhibitor mitigates the sister-chromatid exchanges induced by bromoform and depresses the spontaneous sister-chromatid exchange frequency of human lymphocytes in vitro Ashutosh P. Banerji *, Augustine O. Fernandes Biological Chemistry Dh'ision, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay 400 012, India Received 3 July 1995; revised 6 October 1995; accepted 31 October 1995
Abstract The mutagenicity of a trihalomethane-bromoform (CHBr3)-was assessed by the in vitro sister-chromatid exchange (SCE) assay using human peripheral blood lymphocytes. CHBr 3 was found to induce SCEs significantly in a dose-dependent manner. When the cells were exposed to 600 ng CHBr3/ml of the medium, the SCE/cell mean reached a value as high as 18.78 + 0.17. Beyond this concentration, CHBr 3 proved to be cytotoxic. A protease inhibitor (PI), purified appreciably by affinity chromatography from fieldbean (FB), was able to suppress significantly in a dose-dependent way the high SCE frequencies induced by this specific concentration of CHBr 3 (600 ng/ml). Addition of 600 /xg of FBPI/ml of the medium brought down the CHBr3-induced high SCEs to near (8.80 + 0.15) base line or control value (8.45 +__0.21). A study of the effect of FBPI on the normal low SCE frequencies in these cells indicated that the FBPI has the intrinsic property to suppress in a dose-dependent manner these SCEs in the lymphocytes. This functional property of FBPI, which is related to its protease inhibitory activity and which is destroyed when it is inactivated by autoclaving, makes it an effective antimutagenic/chemopreventive agent. Keywords: Cultured human lymphocyte; Bromoform; Field bean protease inhibitor; Sister-chromatid exchange suppression
1. Introduction Trihalomethanes (THMs) are volatile organohalides formed during chlorination of water as
Abbreviations: THMs, trihalomethanes; CHBr 3, bromoform; PI, protease inhibitor; SCE, sister chromatid exchange; FBPI, field bean protease inhibitor; BBI, Bowman-Birk inhibitor * Corresponding author. Tel.: 91-22-4123803; Fax: 91-224146089.
a result of the interaction of chlorine with humic substances present in drinking water (Rook, 1974). Though chloroform is the principal THM congener, chlorination in the presence of bromide ions also generates chlorinated-brominated THMs such as dichlorobromomethane, dibromochloromethane and bromoform (CHBr3). THMs are of public concern because of the health risk they pose to the population at large (Pieterse, 1988). Epidemiological studies have indicated an association of human cancers with
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A.P. Baneui, A.O. Fernandes / Mutation Research 360 (1996) 29-35
concentrations of THMs in drinking water (Cantor, 1983; KIWA, 1986). While animal studies have shown chloroform and CHBr 3 to be carcinogenic in rodents (Eschenbrenner, 1945; Theiss et al., 1977: Davidsom et al., 1982), testing of mutagenicity by the Ames Salmonella test indicated that CHBr 3, dibromochloromethane, dichlorobromomethane but not chloroform are mutagenic (Simmon et al., 1977; Davidsom et al., 1982). However, all four known THMs, including chloroform, can induce sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (Morimoto and Koizumi, 1983). In 1990, Fujie et al. (1990) presented evidence for the in vivo mutagenicity of THMs. Their intraperitoneal or oral administration in the rat induced significant chromosomal aberrations in the bone marrow cells of the animals. Subsequently, they also examined the ability of a crude catechin extract of green tea to suppress the SCEs induced by the four principal THMs in rat erythrocytic leukemia cells in culture (Fujie et al., 1993). They observed that the tea catechin could bring about a significant dose-dependent suppression of SCEs induced by chloroform or CHBr 3 among the THM congeners. Interestingly, the soybean-derived protease inhibitor (PI), the Bowman-Birk inhibitor (BBI) has also been reported to significantly suppress the high levels of chromosomal aberrations and SCEs present in lymphocytes from Bloom syndrome patients (Kennedy et al., 1984). Since, like green tea catechins PIs are also chemopreventive agents (Wattenberg, 1985) capable of reducing SCEs, it was felt interesting to examine the ability of a PI purified from the Indian legume field bean to suppress SCEs induced by the THM, CHBr 3 in normal human lymphocytes. CHBr 3 was selected as it has been shown to induce, in a dose-dependent manner, the highest increase in SCEs in human lymphocytes in vitro (Morimoto and Koizumi. 1983) besides being a common THM contaminant found in the disinfected water of metropolitan cities (Dowty et al., 1975). Our present findings indicate that field-bean-derived PI can significantly suppress, in a dose-dependent manner, SCEs induced by CHBr 3 in human lymphocytes in vitro. Additionally, we observed that it had the intrinsic ability to repress the spontaneous SCE frequency of human lymphocytes.
2. Materials and methods
2.1. Materials All reagents used were of reagent grade. CHBr 3 was purchased from Fluka (Germany). Stock solution of CHBr 3 was made by dissolving a small aliquot of it in the minimal required volume of dimethyl sulfoxide (Merck, spectroscopic grade). Aliquots of the freshly made stock solution were added to culture medium to give the required final concentrations of CHBr 3. The final concentration of dimethyl sulfoxide in the medium did not exceed 0.1%. Good quality field beans (Dolichos lablab) were purchased from the local market.
2.2. Purifi'cation of the field bean protease inhibitor by affini O' chromatography Field-bean protease inhibitor (FBPI) used in these studies was obtained by further purification of a partially-purified preparation of the same from field bean flour reported previously (Banerji and Fernandes, 1994) by chromatography on a trypsin-Sepharose affinity column. To 100 ml cyanogen bromide (Sigma) activated Sepharose 4B (Pharmacia, Uppsala), 1 g of porcine trypsin type IX (Sigma) was coupled according to the procedure described by March et al. (1974), the unreacted groups being masked by glycine. The trypsin-coupled Sepharose 4B (93% coupling efficiency) was then treated by a modification of the procedure of Axen and Emback (1971) with three cycles of 5 vols each of 0.1 M sodium acetate buffer, pH 4.1 (0.5 M NaC1 and 0.1 M sodium bicarbonate buffer), pH 8.3 (0.5 M NaCI). The affinity gel was then suspended in 0.1 M TrisHC1 buffer, pH 8.0 (0.5 M NaC1) and then packed into a glass column (2.5 × 30.0 cm) and finally equilibrated in the same buffer. About 25 ml of the partially purified FBPI containing 50 mg protein/ml, after adjustment of its pH to 8.0, was loaded on to the column, and 3-ml fractions were collected at a flow rate of 20 m l / h . After washing the column with the equilibrating buffer till washings showed no absorption at 280 nm, nonspecifically adsorbed material was eluted with 0.1 M sodium acetate buffer,
A.P. Banerji, A.O. Fernandes / Mutation Research 360 (1996) 29-35
pH 4.1, containing 0.5 M NaC1. The trypsin inhibitors were finally eluted from the column with 0.2 N HC1 containing 0.5 M NaC1 (Kortt, 1979). The eluting peak of protein at 280 nm was pooled, dialysed against distilled water and concentrated by lyophilization. The trypsin (TIU/mg protein) and chymotrypsin (CIU/mg protein) inhibitory activities of the final product (17 700 × 10 -3 T I U / m g protein and 1015 × 10 -3 C I U / m g protein) determined by the procedures reported previously (Banerji and Fernandes, 1994) indicated a 16-fold purification with respect to the trypsin inhibitor activity of the starting field bean flour extract (1145 × 10 -3 T I U / m g protein). The purity of the final product when assessed by SDS-polyacrylamide gel (15%) electrophoresis (Laemmli, 1970) revealed two components in the molecular mass range of 10-12 kDa. Protein was estimated by the method of Lowry et al. (1951). An aqueous stock solution of the purified FBPI (10 m g / m l ) was made from which aliquots were added to the culture medium to give the required concentration of the inhibitor. The stock was stored at - 70°C.
31
method of Hungerford (1965). SCEs were analysed by staining slides by the modified 'fluorescenceplus-Giemsa technique' (Perry and Wolff, 1974). The slides were stained for 20 min in Hoechst 33258 solution (5 /xg/ml in Sorensen's buffer, pH 6.8). After the slides were washed, dried, and mounted with the buffer under coverslips, they were exposed in a tray to 360 nm light from fluorescent black light tubes at 60°C for 30-45 min. After removal of the coverslips, the slides were stained in 2% Giemsa (Merck) for 15 min made in the same Sorenson's buffer.
2.4. Statistical analysis SCEs were scored from 50 second division cells per culture. Statistical significance between different groups was determined by the Student's t-test.
3. Results
3.1. CHBr3-induced SCEs in lymphocytes in vitro 2.3. Sister-chromatid exchange For these in vitro studies human peripheral blood drawn by heparinized syringe from healthy adults was used. Two replicate cultures were prepared by adding 0.2 ml of heparinized whole blood to 5 ml of RPMI 1640 culture medium containing 20% fetal calf serum, 3% phytohaemagglutinin (PHA-M, Gibco), 5 / x g / m l of 5-bromo-2-deoxyuridine (BrdU) (Sigma), penicillin (100 U / m l ) and streptomycin (100/zg/ml). Each specific concentration of CHBr 3 and that of the FBPI were added prior to incubation and the cells were exposed to CHBr 3 and or the FBPI for the entire culture period. Control plates comprising of cells exposed to 0.1% dimethyl sulfoxide and others containing cells treated with highest concentrations of CHBr 3 (600 ng/ml) plus 600 /xg/ml of heat-inactivated FBPI (autoclaved for 30 min at 121°C at 15 p.s.i.) or with 600 /zg/ml of heat-inactivated FBPI were also included in the experiment. The cultures were incubated for 72 h in complete darkness at 37°C. About 3 h prior to fixation, colchicine ( 1 0 0 / x g / m l ) was added to arrest the cells in metaphase. Air-dried chromosome preparations were made by minor modifications of the
It was interesting to note that exposing cultured lymphocytes to CHBr 3 in the concentration range of 25 to 150 n g / m l of the medium induced SCEs in a dose-dependent manner (Fig. 1). From a SCE/cell mean of 9.10 + 0.17 at 25 ng CHBr3/ml concentration, close to the control base line (8.45 + 0.21)
15.
| 10O
5-
0
Bromof~rm dace (ng/ml culture medium)
Fig. 1. Frequencies of SCEs in human lymphocytes induced by 72 h of bromoform (CHBr 3) treatment in culture. Mean SCE counts were ascertained from 50 second-division cells per culture.
A.P. Banelji, A.O. Fernandes/ Mutation Research 360 (1996) 29 35
32
value, t h e r e was a steady a n d s i g n i f i c a n t i n c r e a s e in the S C E
which
reached
13.70_+ 0.19 S C E / c e l l
a frequency
as h i g h as
m e a n at a 150 n g / m l
con-
c e n t r a t i o n o f C H B r 3. B e y o n d this c o n c e n t r a t i o n , the S C E r e s p o n s e o f the c u l t u r e d cells t e n d e d to plateau. A t c o n c e n t r a t i o n as h i g h as 6 0 0 n g / m l o f the m e d i u m , the l y m p h o c y t e s s h o w e d the h i g h e s t S C E / c e l l m e a n ( 1 8 . 7 8 _+ 0.19) v a l u e (Fig. l). C H B h p r o v e d to b e c y t o t o x i c a b o v e this c o n c e n t r a t i o n . T h i s c o n c e n t r a t i o n o f C H B r 3, i n d u c i n g the h i g h e s t S C E f r e q u e n c y , was t h e r e f o r e s e l e c t e d to study the supp r e s s i v e activity o f F B P I on C H B r 3 - i n d u c e d S C E s in the c u l t u r e d cells.
3.2. The effect o f F B P I on CHBo-induced SCEs q[ human lymphocytes in culture T h e ability o f the F B P I to b r i n g a b o u t e f f e c t i v e s u p p r e s s i o n o f S C E s i n d u c e d in l y m p h o c y t e s is quite e v i d e n t f r o m T a b l e 1. E v e n at a c o n c e n t r a t i o n o f 50 /xg o f F B P I p r o t e i n / m l , the r e d u c t i o n in the S C E / c e l l m e a n was h i g h l y s i g n i f i c a n t ( p < 0 . 0 0 1 ) c o m p a r e d to the S C E f r e q u e n c y o f cells treated w i t h 6 0 0 n g C H B r 3 / m l . A s the t r e a t m e n t dose o f F B P I i n c r e a s e d , the s u p p r e s s i v e e f f e c t o f F B P I o n C H B r 3i n d u c e d S C E s i n c r e a s e d in a d o s e - d e p e n d e n t m a n n e r such that at F B P I c o n c e n t r a t i o n s o f 6 0 0 a n d 8 0 0 / x g / m l the S C E s i n d u c e d b y 6 0 0 n g C H B % / m l w e r e r e p r e s s e d to n e a r n o r m a l a n d b e l o w b a s e l i n e v a l u e s r e s p e c t i v e l y ; the s u p p r e s s i o n at e a c h c o n c e n tration o f the i n h i b i t o r w a s statistically h i g h l y sign±f-
Table 2 Effect of field bean protease inhibitor (FBPI) on baseline (control) sister-chromatid exchange (SCE) frequencies in human lympho cytes Treatment
SCE mean ± S.E.M.
Inhibition (/,
Control FBPI (200 /.tg/lnl) FBP1 (300 /xg/ml) FBPI (400 /*g/ml) FBPI (600 /*g/ml) Autoclaved FBPI (600 /xg/ml) *
8.45 ±0.21 ~' 7.09±0.21 I, 6.(10 +(l.24 ~ 5.(10±0.21 d 4.00_+0.17 ~ 8.53_+0.13 i
16.1 29.0 40.8 52.6 _
a:b or c or d or e: p <0.001 by Student's t-test. a: f: not significant. FBPI was autoclaved at 121°C for 30 rain at 15 p.s.i, to destroy its protease inhibitory activity.
±cant ( p < 0.001). T h i s S C E s u p p r e s s i v e activity o f F B P I was, h o w e v e r , lost w h e n it was i n a c t i v a t e d by a u t o c l a v i n g ( T a b l e 1).
3.3. Effect o f FBPI on SCE o f human lymphocytes in culture T o see the effect o f F B P I itself on the n o r m a l low level S C E f r e q u e n c i e s o f c u l t u r e d n o r m a l l y m p h o cytes, the cells w e r e m a i n t a i n e d in the p r e s e n c e o f graded doses of FBPI ranging from 200-600 #g F B P I p r o t e i n / m l m e d i u m for 72 h. T h e S C E / c e l l m e a n w h e n r e c o r d e d i n d i c a t e d that the F B P I has the i n h e r e n t ability to s u p p r e s s e v e n the b a s e l i n e (8.45 ±
Table 1 Inhibition by field bean protease inhibitor (FBPI) of the high frequencies of sister-chromatid exchanges (SCEs) induced by bromoform (CHBh) in human lymphocytes Treatment
SCE mean ± SEM
Control CHBr 3 (600 ng/ml) CHBr 3 (600 ng/ml) + FBPI (50/xg/ml) CHBr 3 (600 ng/ml) + FBPI (100 # g / m l ) CHBr 3 (600 ng/ml) + FBPI (200/xg/ml) CHBr 3 (600 ng/ml) + FBPI (400 p.g/ml) CHBr 3 (600 ng/ml) + FBPI (600/xg/ml) CHBr 3 (600 ng/ml) + FBPI (800 txg/ml) CBr 3 (600 ng/ml) + * Autoclaved FBPI (600 tzg/ml)
8.45 ± 18.81 + 16.70 ± 15.33 + 12.50 ± 10.16 ± 8.80 ± 7.35 ± 18.88 +
0.21 a 0.18 h 0.30 ~ 0.16 a 0.20 ~ 0.1 I i O. 15 ~ 0.17 h 0.14
a:b: p < 0.001. b:c or d or e or f or g or h: p < 0.001 by Student's t-test. FBPI was autoclaved at 121°C for 30 rain at 15 p.s.i, to destroy its protease inhibitory activity.
Inhibition %
20.4 33.6 60.9 83.5 96.6 110.6
A.P. Banerji, A.O. Fernandes / Mutation Research 360 (1996) 29-35
0.21) SCE value of the cells in a dose-dependent manner without having any effect on their growth or morphology when compared to the controls. However, the heat-inactivated FBPI(600 /zg/ml) had no effect on the spontaneous SCE frequencies (8.53 + 0.13) of the normal lymphocytes (Table 2).
4. Discussion In an earlier study, and an only study of its kind reported so far, Kennedy et al. (1984) showed that protease inhibitors, such as antipain, soybean trypsin inhibitor as well as crude and purified BBI could suppress the high levels of chromosomal aberrations and SCEs seen in the cultured cells of Bloom syndrome patients. Although the reductions were significant the SCE frequencies were not depressed to the levels noted in normal cells. The inherently increased SCEs seen in cells of Bloom syndrome individuals makes them more susceptible to the incidence of spontaneous cancers and the BBI indicated its potentials as an effective antimutagenic agent. Inspired by this report, we decided to investigate whether a PI derived from the Indian legume field bean could also suppress exceedingly high frequencies of SCEs induced in normal human lymphocytes by a strong mutagenic agent. The mutagen we chose was the THM congener CHB% (Simmon et al., 1977; Uehleke et al., 1977), a common contaminant in drinking water (Dowty et al., 1975; NAS-NRC, 1980), considered to be the most active among the THMs in inducing high levels of SCEs in human lymphocytes and in a dose-dependent manner (Morimoto and Koizumi, 1983; Fujie et al., 1993). Moreover, it has been shown to be a carcinogen in rodents (Eschenbrenner, 1945; Theiss et al., 1977). Our experimental findings (Fig. 1) confirm the reported ability of CHBr 3 to induce remarkably and in a dose-dependent manner, SCEs in normal human lymphocytes. However, addition of purified FBPI to cells exposed to a high concentration of the mutagen repressed the generation of high SCE frequencies (Table 1). This blocking effect, which was highly significant, increased steadily with increasing concentration of the FBPI in the culture medium until the SCE/cell mean almost reached the
33
base line value (Table 1) when they were treated to a FBPI concentration of 600 /zg/ml. A further increase in the concentration of FBPI in the medium repressed the CHBr3-induced SCEs even below the baseline value. These findings indicated an oncopreventive feature of FBPI in common and another at variance with the soybean-derived BBI. Like BBI, it suppresses high levels of SCEs generated in cells; but unlike BBI and like green-tea crude catechin extract (Fujie et al., 1993) can extenuate, in a dose-related way, high frequencies of SCEs induced in cells by a mutagen like CHBr 3. BBI, on the other hand, was unable to reduce the spontaneous high SCE frequencies of Bloom syndrome cells below 50%, even when its concentration was increased to above 300 /xg/ml (Kennedy et al., 1984). The present data thus indicated that the intake of PIs through food could prove to be beneficial to the population at large who drink considerable quantities of water containing THMs at relatively high concentrations (Dowty et al., 1975; NAS-NRC, 1980). Considering that the FBPI's antimutagenic activity is effective only at concentrations above 200 /~g/ml (Table 1), it is doubtful whether such levels can be achieved in tissues by adult humans through dietary intake of PI which has been estimated to be about 330 rag/day for the normal Western diet (Kennedy, 1993). The question that we addressed next is whether the SCE-repressing activity is related to the protease inhibitory activity of the FBPI. The findings enumerated below support our contention that this activity is, indeed, associated with the protease inhibitory activity of the FBPI. The loss in SCE-depressing activity of heat-inactivated FBPI seen on its addition to cell cultures treated with very high concentrations of CHBr 3 (600 ng/ml) (Table 1) and the inherent ability of FBPI to reduce significantly dose-wise the base-line frequencies of normal cultured lymphocytes (Table 2) prompted us to conclude that this activity is, indeed, associated with its protease inhibitory activity. Additionally, the inability of autoclaved FBPI to inhibit such spontaneous frequencies of normal untreated lymphocytes (Table 2) supported this conclusion. It is pertinent to mention that FBPI appears to be different from other protease inhibitors such as BBI, antipain and soybean trypsin inhibitor, which have no effect on the spontaneous low-level
34
A.P. Banetji. A.O. Fernandes / Mutation Research 360 (1996) 29-35
SCE frequencies of normal cells (Kennedy et al., 1984). Having established its antimutagenic activity, the underlying mechanism of its SCE-repressing activity needed to be defined. Since this is not yet known, some speculations can be made. It is possible that FBPI inhibits cytochrome P450 activity of the lymphocytes and thereby interferes with the formation of the ultimate mutagen from CHBr 3. A decrease in hepatic cytochrome P450 following intraperitoneal treatment of BBI to mice has been reported (Oreffo et al., 1991). However, since CHBr 3 is a direct-acting mutagen (Fujie et al., 1993), the hypothesis relating to the FBPI interfering with cytochrome P450 activity and indirectly affecting SCEs of cells does not appear to be tenable. The other possible mechanism, and the most plausible one, through which FBPI could lower SCEs is by regulating the level of free radicals in cells which are known to induce SCEs as well as chromosomal aberrations (MacRae and Stich, 1979). There are no reports which indicate that PIs interact with or neutralize free radicals; however, the ability of the PIs to mitigate tumor promoter-mediated generation of the superoxide anion radicals ( 0 2) and H 202 by human polymorphonuclear leukocytes has been noted (Goldstein et al., 1979). These radicals, produced in large amounts by normal cellular processes, are capable of damaging DNA and are implicated in mutagenesis and in the etiopathogenesis of various diseases including cancer (Feig and Loeb, 1994). Our findings that FBPI can suppress quite appreciably, in a dose-dependent manner, low levels of spontaneous SCEs seen in normal cells lends credence to the possibility that the FBPI acts through the suppression of production of active oxygen radicals rather than by neutralizing them. It is also likely that it acts on some cellular targets such as the membrane on which the THM may also act, and blocks the latters ability to generate free radicals through the stimulation of arachidonic acid cascade and oxidative burst (Ide et al., 1983). A direct interaction of FBPI with CHBr 3 resulting in the repression of SCEs, an argument proffered to explain green tea catechin's ability to subdue CHBr3-induced SCE frequencies in lymphocytes (Fujie et al., 1993), could also not be ruled out. These mechanisms, indeed, need to be investigated.
Acknowledgements We thank Mr. D.S.Chavan for his help during this study and Ms. Prema Salian and Mr. Athavale for helping to prepare the manuscript. A.O.F. was supported by a grant from the Council of Scientific and Industrial Research (CSIR), New Delhi.
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ELSEVIER
Mutation Research 360 (1996) 29-35
Field bean protease inhibitor mitigates the sister-chromatid exchanges induced by bromoform and depresses the spontaneous sister-chromatid exchange frequency of human lymphocytes in vitro Ashutosh P. Banerji *, Augustine O. Fernandes Biological Chemistry Dh'ision, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay 400 012, India Received 3 July 1995; revised 6 October 1995; accepted 31 October 1995
Abstract The mutagenicity of a trihalomethane-bromoform (CHBr3)-was assessed by the in vitro sister-chromatid exchange (SCE) assay using human peripheral blood lymphocytes. CHBr 3 was found to induce SCEs significantly in a dose-dependent manner. When the cells were exposed to 600 ng CHBr3/ml of the medium, the SCE/cell mean reached a value as high as 18.78 + 0.17. Beyond this concentration, CHBr 3 proved to be cytotoxic. A protease inhibitor (PI), purified appreciably by affinity chromatography from fieldbean (FB), was able to suppress significantly in a dose-dependent way the high SCE frequencies induced by this specific concentration of CHBr 3 (600 ng/ml). Addition of 600 /xg of FBPI/ml of the medium brought down the CHBr3-induced high SCEs to near (8.80 + 0.15) base line or control value (8.45 +__0.21). A study of the effect of FBPI on the normal low SCE frequencies in these cells indicated that the FBPI has the intrinsic property to suppress in a dose-dependent manner these SCEs in the lymphocytes. This functional property of FBPI, which is related to its protease inhibitory activity and which is destroyed when it is inactivated by autoclaving, makes it an effective antimutagenic/chemopreventive agent. Keywords: Cultured human lymphocyte; Bromoform; Field bean protease inhibitor; Sister-chromatid exchange suppression
1. Introduction Trihalomethanes (THMs) are volatile organohalides formed during chlorination of water as
Abbreviations: THMs, trihalomethanes; CHBr 3, bromoform; PI, protease inhibitor; SCE, sister chromatid exchange; FBPI, field bean protease inhibitor; BBI, Bowman-Birk inhibitor * Corresponding author. Tel.: 91-22-4123803; Fax: 91-224146089.
a result of the interaction of chlorine with humic substances present in drinking water (Rook, 1974). Though chloroform is the principal THM congener, chlorination in the presence of bromide ions also generates chlorinated-brominated THMs such as dichlorobromomethane, dibromochloromethane and bromoform (CHBr3). THMs are of public concern because of the health risk they pose to the population at large (Pieterse, 1988). Epidemiological studies have indicated an association of human cancers with
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A.P. Baneui, A.O. Fernandes / Mutation Research 360 (1996) 29-35
concentrations of THMs in drinking water (Cantor, 1983; KIWA, 1986). While animal studies have shown chloroform and CHBr 3 to be carcinogenic in rodents (Eschenbrenner, 1945; Theiss et al., 1977: Davidsom et al., 1982), testing of mutagenicity by the Ames Salmonella test indicated that CHBr 3, dibromochloromethane, dichlorobromomethane but not chloroform are mutagenic (Simmon et al., 1977; Davidsom et al., 1982). However, all four known THMs, including chloroform, can induce sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (Morimoto and Koizumi, 1983). In 1990, Fujie et al. (1990) presented evidence for the in vivo mutagenicity of THMs. Their intraperitoneal or oral administration in the rat induced significant chromosomal aberrations in the bone marrow cells of the animals. Subsequently, they also examined the ability of a crude catechin extract of green tea to suppress the SCEs induced by the four principal THMs in rat erythrocytic leukemia cells in culture (Fujie et al., 1993). They observed that the tea catechin could bring about a significant dose-dependent suppression of SCEs induced by chloroform or CHBr 3 among the THM congeners. Interestingly, the soybean-derived protease inhibitor (PI), the Bowman-Birk inhibitor (BBI) has also been reported to significantly suppress the high levels of chromosomal aberrations and SCEs present in lymphocytes from Bloom syndrome patients (Kennedy et al., 1984). Since, like green tea catechins PIs are also chemopreventive agents (Wattenberg, 1985) capable of reducing SCEs, it was felt interesting to examine the ability of a PI purified from the Indian legume field bean to suppress SCEs induced by the THM, CHBr 3 in normal human lymphocytes. CHBr 3 was selected as it has been shown to induce, in a dose-dependent manner, the highest increase in SCEs in human lymphocytes in vitro (Morimoto and Koizumi. 1983) besides being a common THM contaminant found in the disinfected water of metropolitan cities (Dowty et al., 1975). Our present findings indicate that field-bean-derived PI can significantly suppress, in a dose-dependent manner, SCEs induced by CHBr 3 in human lymphocytes in vitro. Additionally, we observed that it had the intrinsic ability to repress the spontaneous SCE frequency of human lymphocytes.
2. Materials and methods
2.1. Materials All reagents used were of reagent grade. CHBr 3 was purchased from Fluka (Germany). Stock solution of CHBr 3 was made by dissolving a small aliquot of it in the minimal required volume of dimethyl sulfoxide (Merck, spectroscopic grade). Aliquots of the freshly made stock solution were added to culture medium to give the required final concentrations of CHBr 3. The final concentration of dimethyl sulfoxide in the medium did not exceed 0.1%. Good quality field beans (Dolichos lablab) were purchased from the local market.
2.2. Purifi'cation of the field bean protease inhibitor by affini O' chromatography Field-bean protease inhibitor (FBPI) used in these studies was obtained by further purification of a partially-purified preparation of the same from field bean flour reported previously (Banerji and Fernandes, 1994) by chromatography on a trypsin-Sepharose affinity column. To 100 ml cyanogen bromide (Sigma) activated Sepharose 4B (Pharmacia, Uppsala), 1 g of porcine trypsin type IX (Sigma) was coupled according to the procedure described by March et al. (1974), the unreacted groups being masked by glycine. The trypsin-coupled Sepharose 4B (93% coupling efficiency) was then treated by a modification of the procedure of Axen and Emback (1971) with three cycles of 5 vols each of 0.1 M sodium acetate buffer, pH 4.1 (0.5 M NaC1 and 0.1 M sodium bicarbonate buffer), pH 8.3 (0.5 M NaCI). The affinity gel was then suspended in 0.1 M TrisHC1 buffer, pH 8.0 (0.5 M NaC1) and then packed into a glass column (2.5 × 30.0 cm) and finally equilibrated in the same buffer. About 25 ml of the partially purified FBPI containing 50 mg protein/ml, after adjustment of its pH to 8.0, was loaded on to the column, and 3-ml fractions were collected at a flow rate of 20 m l / h . After washing the column with the equilibrating buffer till washings showed no absorption at 280 nm, nonspecifically adsorbed material was eluted with 0.1 M sodium acetate buffer,
A.P. Banerji, A.O. Fernandes / Mutation Research 360 (1996) 29-35
pH 4.1, containing 0.5 M NaC1. The trypsin inhibitors were finally eluted from the column with 0.2 N HC1 containing 0.5 M NaC1 (Kortt, 1979). The eluting peak of protein at 280 nm was pooled, dialysed against distilled water and concentrated by lyophilization. The trypsin (TIU/mg protein) and chymotrypsin (CIU/mg protein) inhibitory activities of the final product (17 700 × 10 -3 T I U / m g protein and 1015 × 10 -3 C I U / m g protein) determined by the procedures reported previously (Banerji and Fernandes, 1994) indicated a 16-fold purification with respect to the trypsin inhibitor activity of the starting field bean flour extract (1145 × 10 -3 T I U / m g protein). The purity of the final product when assessed by SDS-polyacrylamide gel (15%) electrophoresis (Laemmli, 1970) revealed two components in the molecular mass range of 10-12 kDa. Protein was estimated by the method of Lowry et al. (1951). An aqueous stock solution of the purified FBPI (10 m g / m l ) was made from which aliquots were added to the culture medium to give the required concentration of the inhibitor. The stock was stored at - 70°C.
31
method of Hungerford (1965). SCEs were analysed by staining slides by the modified 'fluorescenceplus-Giemsa technique' (Perry and Wolff, 1974). The slides were stained for 20 min in Hoechst 33258 solution (5 /xg/ml in Sorensen's buffer, pH 6.8). After the slides were washed, dried, and mounted with the buffer under coverslips, they were exposed in a tray to 360 nm light from fluorescent black light tubes at 60°C for 30-45 min. After removal of the coverslips, the slides were stained in 2% Giemsa (Merck) for 15 min made in the same Sorenson's buffer.
2.4. Statistical analysis SCEs were scored from 50 second division cells per culture. Statistical significance between different groups was determined by the Student's t-test.
3. Results
3.1. CHBr3-induced SCEs in lymphocytes in vitro 2.3. Sister-chromatid exchange For these in vitro studies human peripheral blood drawn by heparinized syringe from healthy adults was used. Two replicate cultures were prepared by adding 0.2 ml of heparinized whole blood to 5 ml of RPMI 1640 culture medium containing 20% fetal calf serum, 3% phytohaemagglutinin (PHA-M, Gibco), 5 / x g / m l of 5-bromo-2-deoxyuridine (BrdU) (Sigma), penicillin (100 U / m l ) and streptomycin (100/zg/ml). Each specific concentration of CHBr 3 and that of the FBPI were added prior to incubation and the cells were exposed to CHBr 3 and or the FBPI for the entire culture period. Control plates comprising of cells exposed to 0.1% dimethyl sulfoxide and others containing cells treated with highest concentrations of CHBr 3 (600 ng/ml) plus 600 /xg/ml of heat-inactivated FBPI (autoclaved for 30 min at 121°C at 15 p.s.i.) or with 600 /zg/ml of heat-inactivated FBPI were also included in the experiment. The cultures were incubated for 72 h in complete darkness at 37°C. About 3 h prior to fixation, colchicine ( 1 0 0 / x g / m l ) was added to arrest the cells in metaphase. Air-dried chromosome preparations were made by minor modifications of the
It was interesting to note that exposing cultured lymphocytes to CHBr 3 in the concentration range of 25 to 150 n g / m l of the medium induced SCEs in a dose-dependent manner (Fig. 1). From a SCE/cell mean of 9.10 + 0.17 at 25 ng CHBr3/ml concentration, close to the control base line (8.45 + 0.21)
15.
| 10O
5-
0
Bromof~rm dace (ng/ml culture medium)
Fig. 1. Frequencies of SCEs in human lymphocytes induced by 72 h of bromoform (CHBr 3) treatment in culture. Mean SCE counts were ascertained from 50 second-division cells per culture.
A.P. Banelji, A.O. Fernandes/ Mutation Research 360 (1996) 29 35
32
value, t h e r e was a steady a n d s i g n i f i c a n t i n c r e a s e in the S C E
which
reached
13.70_+ 0.19 S C E / c e l l
a frequency
as h i g h as
m e a n at a 150 n g / m l
con-
c e n t r a t i o n o f C H B r 3. B e y o n d this c o n c e n t r a t i o n , the S C E r e s p o n s e o f the c u l t u r e d cells t e n d e d to plateau. A t c o n c e n t r a t i o n as h i g h as 6 0 0 n g / m l o f the m e d i u m , the l y m p h o c y t e s s h o w e d the h i g h e s t S C E / c e l l m e a n ( 1 8 . 7 8 _+ 0.19) v a l u e (Fig. l). C H B h p r o v e d to b e c y t o t o x i c a b o v e this c o n c e n t r a t i o n . T h i s c o n c e n t r a t i o n o f C H B r 3, i n d u c i n g the h i g h e s t S C E f r e q u e n c y , was t h e r e f o r e s e l e c t e d to study the supp r e s s i v e activity o f F B P I on C H B r 3 - i n d u c e d S C E s in the c u l t u r e d cells.
3.2. The effect o f F B P I on CHBo-induced SCEs q[ human lymphocytes in culture T h e ability o f the F B P I to b r i n g a b o u t e f f e c t i v e s u p p r e s s i o n o f S C E s i n d u c e d in l y m p h o c y t e s is quite e v i d e n t f r o m T a b l e 1. E v e n at a c o n c e n t r a t i o n o f 50 /xg o f F B P I p r o t e i n / m l , the r e d u c t i o n in the S C E / c e l l m e a n was h i g h l y s i g n i f i c a n t ( p < 0 . 0 0 1 ) c o m p a r e d to the S C E f r e q u e n c y o f cells treated w i t h 6 0 0 n g C H B r 3 / m l . A s the t r e a t m e n t dose o f F B P I i n c r e a s e d , the s u p p r e s s i v e e f f e c t o f F B P I o n C H B r 3i n d u c e d S C E s i n c r e a s e d in a d o s e - d e p e n d e n t m a n n e r such that at F B P I c o n c e n t r a t i o n s o f 6 0 0 a n d 8 0 0 / x g / m l the S C E s i n d u c e d b y 6 0 0 n g C H B % / m l w e r e r e p r e s s e d to n e a r n o r m a l a n d b e l o w b a s e l i n e v a l u e s r e s p e c t i v e l y ; the s u p p r e s s i o n at e a c h c o n c e n tration o f the i n h i b i t o r w a s statistically h i g h l y sign±f-
Table 2 Effect of field bean protease inhibitor (FBPI) on baseline (control) sister-chromatid exchange (SCE) frequencies in human lympho cytes Treatment
SCE mean ± S.E.M.
Inhibition (/,
Control FBPI (200 /.tg/lnl) FBP1 (300 /xg/ml) FBPI (400 /*g/ml) FBPI (600 /*g/ml) Autoclaved FBPI (600 /xg/ml) *
8.45 ±0.21 ~' 7.09±0.21 I, 6.(10 +(l.24 ~ 5.(10±0.21 d 4.00_+0.17 ~ 8.53_+0.13 i
16.1 29.0 40.8 52.6 _
a:b or c or d or e: p <0.001 by Student's t-test. a: f: not significant. FBPI was autoclaved at 121°C for 30 rain at 15 p.s.i, to destroy its protease inhibitory activity.
±cant ( p < 0.001). T h i s S C E s u p p r e s s i v e activity o f F B P I was, h o w e v e r , lost w h e n it was i n a c t i v a t e d by a u t o c l a v i n g ( T a b l e 1).
3.3. Effect o f FBPI on SCE o f human lymphocytes in culture T o see the effect o f F B P I itself on the n o r m a l low level S C E f r e q u e n c i e s o f c u l t u r e d n o r m a l l y m p h o cytes, the cells w e r e m a i n t a i n e d in the p r e s e n c e o f graded doses of FBPI ranging from 200-600 #g F B P I p r o t e i n / m l m e d i u m for 72 h. T h e S C E / c e l l m e a n w h e n r e c o r d e d i n d i c a t e d that the F B P I has the i n h e r e n t ability to s u p p r e s s e v e n the b a s e l i n e (8.45 ±
Table 1 Inhibition by field bean protease inhibitor (FBPI) of the high frequencies of sister-chromatid exchanges (SCEs) induced by bromoform (CHBh) in human lymphocytes Treatment
SCE mean ± SEM
Control CHBr 3 (600 ng/ml) CHBr 3 (600 ng/ml) + FBPI (50/xg/ml) CHBr 3 (600 ng/ml) + FBPI (100 # g / m l ) CHBr 3 (600 ng/ml) + FBPI (200/xg/ml) CHBr 3 (600 ng/ml) + FBPI (400 p.g/ml) CHBr 3 (600 ng/ml) + FBPI (600/xg/ml) CHBr 3 (600 ng/ml) + FBPI (800 txg/ml) CBr 3 (600 ng/ml) + * Autoclaved FBPI (600 tzg/ml)
8.45 ± 18.81 + 16.70 ± 15.33 + 12.50 ± 10.16 ± 8.80 ± 7.35 ± 18.88 +
0.21 a 0.18 h 0.30 ~ 0.16 a 0.20 ~ 0.1 I i O. 15 ~ 0.17 h 0.14
a:b: p < 0.001. b:c or d or e or f or g or h: p < 0.001 by Student's t-test. FBPI was autoclaved at 121°C for 30 rain at 15 p.s.i, to destroy its protease inhibitory activity.
Inhibition %
20.4 33.6 60.9 83.5 96.6 110.6
A.P. Banerji, A.O. Fernandes / Mutation Research 360 (1996) 29-35
0.21) SCE value of the cells in a dose-dependent manner without having any effect on their growth or morphology when compared to the controls. However, the heat-inactivated FBPI(600 /zg/ml) had no effect on the spontaneous SCE frequencies (8.53 + 0.13) of the normal lymphocytes (Table 2).
4. Discussion In an earlier study, and an only study of its kind reported so far, Kennedy et al. (1984) showed that protease inhibitors, such as antipain, soybean trypsin inhibitor as well as crude and purified BBI could suppress the high levels of chromosomal aberrations and SCEs seen in the cultured cells of Bloom syndrome patients. Although the reductions were significant the SCE frequencies were not depressed to the levels noted in normal cells. The inherently increased SCEs seen in cells of Bloom syndrome individuals makes them more susceptible to the incidence of spontaneous cancers and the BBI indicated its potentials as an effective antimutagenic agent. Inspired by this report, we decided to investigate whether a PI derived from the Indian legume field bean could also suppress exceedingly high frequencies of SCEs induced in normal human lymphocytes by a strong mutagenic agent. The mutagen we chose was the THM congener CHB% (Simmon et al., 1977; Uehleke et al., 1977), a common contaminant in drinking water (Dowty et al., 1975; NAS-NRC, 1980), considered to be the most active among the THMs in inducing high levels of SCEs in human lymphocytes and in a dose-dependent manner (Morimoto and Koizumi, 1983; Fujie et al., 1993). Moreover, it has been shown to be a carcinogen in rodents (Eschenbrenner, 1945; Theiss et al., 1977). Our experimental findings (Fig. 1) confirm the reported ability of CHBr 3 to induce remarkably and in a dose-dependent manner, SCEs in normal human lymphocytes. However, addition of purified FBPI to cells exposed to a high concentration of the mutagen repressed the generation of high SCE frequencies (Table 1). This blocking effect, which was highly significant, increased steadily with increasing concentration of the FBPI in the culture medium until the SCE/cell mean almost reached the
33
base line value (Table 1) when they were treated to a FBPI concentration of 600 /zg/ml. A further increase in the concentration of FBPI in the medium repressed the CHBr3-induced SCEs even below the baseline value. These findings indicated an oncopreventive feature of FBPI in common and another at variance with the soybean-derived BBI. Like BBI, it suppresses high levels of SCEs generated in cells; but unlike BBI and like green-tea crude catechin extract (Fujie et al., 1993) can extenuate, in a dose-related way, high frequencies of SCEs induced in cells by a mutagen like CHBr 3. BBI, on the other hand, was unable to reduce the spontaneous high SCE frequencies of Bloom syndrome cells below 50%, even when its concentration was increased to above 300 /xg/ml (Kennedy et al., 1984). The present data thus indicated that the intake of PIs through food could prove to be beneficial to the population at large who drink considerable quantities of water containing THMs at relatively high concentrations (Dowty et al., 1975; NAS-NRC, 1980). Considering that the FBPI's antimutagenic activity is effective only at concentrations above 200 /~g/ml (Table 1), it is doubtful whether such levels can be achieved in tissues by adult humans through dietary intake of PI which has been estimated to be about 330 rag/day for the normal Western diet (Kennedy, 1993). The question that we addressed next is whether the SCE-repressing activity is related to the protease inhibitory activity of the FBPI. The findings enumerated below support our contention that this activity is, indeed, associated with the protease inhibitory activity of the FBPI. The loss in SCE-depressing activity of heat-inactivated FBPI seen on its addition to cell cultures treated with very high concentrations of CHBr 3 (600 ng/ml) (Table 1) and the inherent ability of FBPI to reduce significantly dose-wise the base-line frequencies of normal cultured lymphocytes (Table 2) prompted us to conclude that this activity is, indeed, associated with its protease inhibitory activity. Additionally, the inability of autoclaved FBPI to inhibit such spontaneous frequencies of normal untreated lymphocytes (Table 2) supported this conclusion. It is pertinent to mention that FBPI appears to be different from other protease inhibitors such as BBI, antipain and soybean trypsin inhibitor, which have no effect on the spontaneous low-level
34
A.P. Banetji. A.O. Fernandes / Mutation Research 360 (1996) 29-35
SCE frequencies of normal cells (Kennedy et al., 1984). Having established its antimutagenic activity, the underlying mechanism of its SCE-repressing activity needed to be defined. Since this is not yet known, some speculations can be made. It is possible that FBPI inhibits cytochrome P450 activity of the lymphocytes and thereby interferes with the formation of the ultimate mutagen from CHBr 3. A decrease in hepatic cytochrome P450 following intraperitoneal treatment of BBI to mice has been reported (Oreffo et al., 1991). However, since CHBr 3 is a direct-acting mutagen (Fujie et al., 1993), the hypothesis relating to the FBPI interfering with cytochrome P450 activity and indirectly affecting SCEs of cells does not appear to be tenable. The other possible mechanism, and the most plausible one, through which FBPI could lower SCEs is by regulating the level of free radicals in cells which are known to induce SCEs as well as chromosomal aberrations (MacRae and Stich, 1979). There are no reports which indicate that PIs interact with or neutralize free radicals; however, the ability of the PIs to mitigate tumor promoter-mediated generation of the superoxide anion radicals ( 0 2) and H 202 by human polymorphonuclear leukocytes has been noted (Goldstein et al., 1979). These radicals, produced in large amounts by normal cellular processes, are capable of damaging DNA and are implicated in mutagenesis and in the etiopathogenesis of various diseases including cancer (Feig and Loeb, 1994). Our findings that FBPI can suppress quite appreciably, in a dose-dependent manner, low levels of spontaneous SCEs seen in normal cells lends credence to the possibility that the FBPI acts through the suppression of production of active oxygen radicals rather than by neutralizing them. It is also likely that it acts on some cellular targets such as the membrane on which the THM may also act, and blocks the latters ability to generate free radicals through the stimulation of arachidonic acid cascade and oxidative burst (Ide et al., 1983). A direct interaction of FBPI with CHBr 3 resulting in the repression of SCEs, an argument proffered to explain green tea catechin's ability to subdue CHBr3-induced SCE frequencies in lymphocytes (Fujie et al., 1993), could also not be ruled out. These mechanisms, indeed, need to be investigated.
Acknowledgements We thank Mr. D.S.Chavan for his help during this study and Ms. Prema Salian and Mr. Athavale for helping to prepare the manuscript. A.O.F. was supported by a grant from the Council of Scientific and Industrial Research (CSIR), New Delhi.
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