J. Biosci., Vol. 10, Number 1, March 1986, pp. 145-151. ... to cells with and without nucleic acids (Lillehoj and Ciegler, 1968), lysogenic and mutagenic properties ...
J. Biosci., Vol. 10, Number 1, March 1986, pp. 145-151. © Printed in India.
Mechanism of action of aflatoxin B1 R. P. TIWARI, T. C. BHALLA, S. S. SAINI, G. S I N G H a n d D . V . VADEHRA Department of Microbiology, Panjab University, Chandigarh 160 0 1 4 , I n d i a MS received 12 December 1984; revised 31 July 1985 Abstract. The inhibitory effects of aflatoxin B1 were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0·05 % w/v) or Tween-80 (0·05 % v/v) addition accentuated the aflatoxin B1 growth inhibition in Salmonella typhi and Escherichia coli at different pH values. The inhibition of lipase production was only 5–20 % in Pseudomonas fluorescence ca. 25–48% in Staphylococcus aureus and Bacillus cereus at different aflatoxin B1 concentrations (4–16 µg/ml).However, inhibition of α-amylase induction was complete in Bacillus megaterium whereas the inhibition was partial in Pseudomonas fluorescence (27–40%) at 32 µg aflatoxin B1 concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B1 concentration. Keywords. Aflatoxin B1; α-amylase; cell content leakage; enzyme induction/inhibition; lipase procaryotes.
Introduction Since 1961, when aflatoxicosis claimed many deaths in turkey poults, pheasants, ducklings and chicks (Asplin and Carnaghan, 1961; Blount, 1961; Carnaghan and Allcroft, 1962; Sargeant et al., 1961; Spensley, 1963), both procaryotes and eucaryotes have been extensively used for detection and quantification of aflatoxins (Arai et al., 1967; Butler and Barnes, 1963; Childs and Legator, 1966; Gablike et al., 1965; Lillehoj and Ciegler, 1968; Vosdingh and Nefe, 1974). Various aflatoxin B1 effects included aberrant cells in Bacillus megaterium (Beuchat and Leochowich, 1971), decrease in cell number, protein concentration, RNA and DNA contents (Gabliks et al., 1965), binding to cells with and without nucleic acids (Lillehoj and Ciegler, 1968), lysogenic and mutagenic properties (Legator, 1966), inhibition of respiration (Nezval and Bosenberg, 1970), inhibition of enzyme biosynthesis (Anand, 1971; Lillehoj and Ciegler, 1970; Black and Altschul, 1965) and alteration of enzyme levels and activity in tissues (Brown and Abrams, 1965). The exact mode of aflatoxin Β1 action still remains unknown. In the present study the action of aflatoxin on gram positive and gram negative bacteria has been delineated. Abbreviations used: EDTA, Ethylene diamine tetra chloroacetic acid; SRBC, sheep red blood cell .
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Materials and methods Aspergillus flavus NRRL 2999, a potent producer of aflatoxin (Hesseltine et al., 1966) procured from Peoria, USA, was used for aflatoxin production using rice as substrate (Shotwell et al., 1966). Aflatoxin B1 was purified and quantitated following the method of Nabney and Nesbitt (1965). Effect on procaryotic cells The test organisms included Salmonella typhi (5 strains), S. paratyphi A and Β (5 strains each), S. typhimurium, S. enteritidis, S. anatum, S. gallinarum (2 strains), S. poona, S. choleraesuis, Bacillus megaterium (5 strains), B. subtilis (5 strains), S. aureus (10 strains), Escherichia coli (8 strains). These were obtained from Departmental Culture Collection and maintained on nutrient agar slants at 4°C, subcultured every month. Effect on growth An appropriate amount of aflatoxin Β1 (20 µg/ml) was added in presterilized trypticase soy broth (5 ml/tube). The tubes were incubated with 0·1 ml of diluted (1:100) 24 h culture of test organism and incubated at 37°C for 24 h. Scoring from – to + + + + + indicates the range, from no growth to maximum growth. In another experiment, growth was monitored by measuring absorbance increase at 540 nm at 4 h intervals during incubation. The aflatoxin Β1 (20µg/ml) growth inhibition was also studied in the presence of Tween-80 (0·05 % v/v) or ethylene diamine tetra chloroacetic acid (EDTA) (0·05 % w/v) at different pH values (pH 6·0–8·0) in E. coli and S. typhi The per cent inhibition was calculated as given below:
Effect on enzyme biosynthesis Strains of P. fluorescence and B. megaterium having inducible α-amylase were induced following the method of Averner and Klein (1963). Samples were removed at 1 h intervals for 4h, centrifuged (10,000 g for 15 min) and the supernatant assayed for amylase activity (Bernfeld, 1955). S. aureus, Β. cereus and P. fluorescence were used to study the effect of aflatoxin Β1 (4–16 µg/ml) on lipase production. A heavy suspension of these organisms (1·0 absorbance at 540 nm) was made in nutrient broth and incubated for 24 h at 37°C. The cells were centrifuged and supernatant assayed for lipase activity (Vadehra and Harmon, 1969). Effect on permeability Inulin uptake and leakage of cellular contents were determined in 1 % sheep red blood cell (SRBC) suspension in normal saline (0·85 % NaCl). The cells were incubated with aflatoxin B1 (20–80 µg/ml) and inulin (8 µg/ml) at 37°C for 2 h. The cells were centrifuged at the end of incubation and supernatant assayed for residual inulin
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(Corcoran and Page, 1939) and haemoglobin leakage at 555 nm. The absorbance of supernatant was also measured at 430 nm (for components other than haemoglobin). Results Aflatoxin B1 (20 µg/ml) inhibited growth in 25 strains out of 48 strains (gram positive and gram negative) used in this study. The inhibited strains included S. aureus (10 strains) and 5 strains each of B. megaterium, B. cereus and B. subtilis (table 1). However, a slight decrease in growth rate in the presence of aflatoxin B1 (20 µg/ml) was observed in trypticase soy broth (pH 7·3) when the test organisms were E. coli and S. typhi (figure 1). The growth decreased further on addition of Tween-80 or EDTA (0·05 %) at various pH values. An overall decrease in growth rate was observed at all pH values (pH 6·0–8·0) in the presence of aflatoxin B1 and EDTA or Tween-80 indicating synergistic inhibitory effects of surfactants with aflatoxin B1 on S. typhi and E. coli. Table 1. Effect of aflatoxin B 1 (20 µg/ml) on growth.
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Figure 1. Aflatoxin B 1 effect on growth of E. coli in the presence of (A) EDTA (0·05 %) at ) pH 7·0( ) and pH 8·0 ( ) or (B) Tween-80 (0·05 %) at pH 6·0 ( ) pH 7·0 ( ) pH 6·0 ( and pH 8·0 ( ) and in the absence at pH 7·3( ) in trypticase soy broth at 37°C. The values for S. typhi growth inhibition have been represented by solid bars ( ).
The aflatoxin B1 effects were also studied on enzyme activity/biosynthesis in both kinds of organisms. The extent of enzyme inhibition (production/induction/activity) was much higher in gram positive organisms than in gram negative organisms (figure 2). The lipase production was approximately 50 % as compared to control in S. aureus and B. cereus at 16 µg/ml aflatoxin B1 concentration whereas, P. fluorescence could show 80% activity at this concentration. The extent of amylase inhibition varied with toxin concentration (8–32 µg/ml) and the time of incubation. The inhibition of enzyme induction ranged from 45·5–100 % in B. megaterium and 29·2–72·7 % in P. fluorescence at different aflatoxin Β1 concentrations (8–32 µg/ml). At all concentrations of toxin, the inhibition was always higher in B. megaterium than in P. fluorescence (figure 3). A continual decrease in inulin uptake (33–100 %) and an increase in leakage of cell constituents were observed with increased amount of toxin (20–80 µg/ml) in suspending medium (table 2). The centrifuged supernatant of toxin incubated cell suspension showed increase in absorbance at 555 nm (for haemoglobin) and 430 nm (other than haemoglobin). Discussion DNA binding and inhibition of nucleic acid synthesis is the most common mechanism suggested for aflatoxin B1 action (Clifford and Rees, 1967; Stark, 1980; Wragg et al., 1967). In the present study gram positive organisms were comparatively more susceptible to aflatoxin B1-mediated growth inhibition than gram negative organisms (table 1). The above observation is in confirmation with the survey of Burmeister and Hesseltine (1966). This indicates the involvement of cell wall and or cell membrane in the action of aflatoxin B1. The observation leading to formation of aberrant cells in B. megaterium (Beuchat and Lechowich, 1971) is also in agreement with the above
Action of aflatoxinB 1
Figure 2. Effect of aflatoxin B1 (4–16 µg/ml) on lipase production in S. aureus ( ( ) and P.fluorescence( ) at 37°C.
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),B cereus
Figure 3. Effect of aflatoxin B1 (8–32µg/ml) on α-amylaseinduction, ( ), ( ), ( ), and ( ) represent α-amylase activity at 8,16,24 and 32 µg/ml aflatoxin B1 in B. megaterium at 1–4 h of incubation. Values for P. fluorescence have been shown in solid bars (▀).
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Tiwari et al. Table 2. Effect of aflatoxin B 1 (20–80 µg/ml) on intracellular contents' leakage and inulin uptake in SRBC (1 %) .
hypothesis. This would indicate either differences in binding or the accessibility of aflatoxin B1 to the effector site. Earlier work done in this laboratory found protoplasts more susceptible to aflatoxin B 1 than the parent organism, despite the fact, that the former bound less aflatoxin Β 1 (Tiwari et al., 1984). Hence, the cell wall appears to be a barrier to the action of aflatoxin B 1 which inference is also supported by accentuated inhibitory effects of aflatoxin B 1 in the presence of Tween-80 or EDTA in this study. EDTA, a chelator is known to affect permeability resulting in the release of cell wall polysaccharides with little or no loss of other cell components (Leive, 1968). The inhibition of enzyme biosynthesis/activity by aflatoxin B1 is also in agreement with earlier studies (Anand, 1971; Lillehoj and Ciegler, 1970; Black and Altschul, 1965). However, the extent of inhibition was found to be related to gram character in this study. The inhibition was more in gram positive organisms. This information along with the conclusions of low oxygen uptake (Nerval and Bosenberg, 1970) and inhibition of electron transport flow by aflatoxin B1 (Doherty and Campbell, 1972) indicate the cell membrane to be the target for aflatoxin B1 action. The decreased inulin uptake and an increased leakage of cell constituents observed in SRBC with increase in aflatoxin B1 concentration further confirm the cell membrane as a target site. This finding is in agreement with an earlier report in procaryotes (Tiwari et al., 1985). The decreased amino acid uptake (Clifford and Rees, 1967) and the changes in nucleous and disappearance of nucleoli (Gabliks et al., 1965) reported earlier, can now be explained considering cell membrane as the primary target for aflatoxin action (Lillehoj and Ciegler, 1968). Therefore, the loss of cell membrane functions would be reflected in terms of the parameter used in the study viz. decrease in protein, nucleic acid content, decrease in the cell number (Gabliks et al., 1965; Wragg et al., 1967) and decrease in enzymes biosynthesis (Anand, 1971; Lillehoj and Ciegler, 1970; Black and Altschul, 1965). Hence, it is concluded that aflatoxin B1 inhibitory effects in procaryotes are related to their gram character and the differences in susceptibility are because of an easy access of aflatoxin B1 to the target site. The cell membrane appears to be the primary target for aflatoxin B1 action. References Anand, S. R. (1971) Indian J. Exp, Biol., 10, 177. Arai, Τ., Ito, Τ. and Koyama, Υ. (1967) J. Bacteriol., 93, 59.
Action of aflatoxin B1 Asplin, F. D. and Carnagham, R. B. A. (1961) Vet. Res., 73, 1215. Averner, Μ. and Klein, H. P. (1963) Biochim. Biophys. Acta, 77, 510. Bernfeld, P. (1955) Methods Enzymol., 1, 149. Beuchat, L. R. and Lechowich, R. V. (1971) Appl Microbiol., 21, 119. Black, H. S. and Altschul, A. M. (1965) Biochem. Biophys. Res. Commun., 19, 661. Blount, W. P. (1961) Turkeys, 9, 52. Brown, J. Μ. Μ. and Abrams, L. (1965) J. Vet. Res., 32, 119. Burmeister, H. R. and Hesseltine, C. W. (1966) Appl. Microbiol., 14, 403. Butler, W. H. and Barnes, J. Μ. (1963) Br. J. Cancer, 17, 699. Carnaghan, R. Β. A. and Allcroft, R. (1962) Vet. Res., 74, 925. Childs, V. A. and Legator, Μ. S. (1966) Life Sci., 5, 1053. Clifford, J. I. and Rees, K. R. (1967) Biochem. J., 103, 467. Corcoran, A. C. and Page, I. H. (1939) J. Biol Chem., 121, 601. Doherty, W. P. and Campbell, Τ. C. (1972) Res. Commun. Pathol Pharmacol., 3, 601. Gabliks, Κ. S., Schaeffer, J. W., Friedman, L. and Wogan, G. (1965) J. Bacteriol., 90, 720. Hesseltine, C. W.,Shotwell, O. L., Ellis, J. J. and Stubblefield, R. D. (1966) Bacteriol Rev., 30, 795. Legator, M. S. (1966) Bacteriol. Rev., 30, 471. Leive, L. (1968) J. Biol. Chem., 243, 2373. Lillehoj, E. B. and Ciegler, A. (1968) J. Gen. Microbiol., 54, 185. Lillehoj, E. B. and Ciegler, A. (1970) Can. J. Microbiol., 16, 1059. Nabney, J. and Nesbitt, B. F. (1965) Analyst, 90, 155. Nezval, J. and Bosenberg, H. (1970) Arch. Hyg., 154, 143. Sargeant, K., Sherioan, Α., Kelly, J. Ο. and Carnaghan, R. B. A. (1961) Nature (London), 122, 1096. Shotwell, G. L., Hesseltine, C. W., Stubblefield, R. D. and Sorenson, W. G. (1966) Appl Microbiol., 14, 425. Spensley, P. C. (1963) Endeavour, 22, 75. Stark, A. A. (1980) Ann. Rev. Microbiol., 34, 235. Tiwari, R. P., Dham, C. K.. Gupta, L. K., Saini, S. S., Bhalla, T. C. and Vadehra, D. V. (1984) J. Gen. Appl Microbiol., 30, 419. Tiwari, R. P., Dham, C. K., Bhalla, T. C, Saini, S. S. and Vadehra, D. V. (1985) Appl Environ. Microbiol., 49, 904. Vadehra, D. V. and Harmon, L. G. (1969) J. Appl Bacteriol., 32, 147. Vosdingh, R. A. and Nefe, M. J. C. (1974) Toxicology, 2, 107. Wragg, I. B., Rose, V. C. and Legator, M. S. (1967) Proc. Soc. Exp. Biol. Med., 125, 1052.
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