Arrest of Mitotic Cycle and Induction of Chromosomal Aberrations by Aflatoxin B2 in Root Cells of Vicia faba L.

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1 2000 The Japan Mendel Society Cytologia 65: , 2000 Arrest of Mitotic Cycle and Induction of Chromosomal Aberrations by Aflatoxin B2 in Root Cells of Vicia faba L. Hanaa H. EI-Shazly1,* and Iman A. EI-Sheikh2 of Biological Sciences and Geology, Faculty of Education, Ain Shams University, Roxy, Cairo, Egypt 2 Department of Botany, Faculty of Science (Girls Branch), Al-Azhar University, Madinet Nasr, Cairo, Egypt Accepted December 6, 1999 Summary Image cytometric measurements demonstrated a dose-dependent effect of aflatoxin B2 (AFB2) treatments on the components of mitotic cell cycle in Vicia faba L. root meristematic cells. The most evident effect appears to be the accumulation of cells in the G0/G1 phase at the expense of other phases of the cycle (S phase, G2 phase, M phase). These results indicate that this toxin acts as an inhibitor of cell cycle progression at the G1 transition point. In addition, a dose-dependent increase in a fraction of cells having <2C DNA and >4C DNA results from the AFB2 treatments. The inhibition of mitotic activity induced by AFB2 treatments is associated with a reduction in seedling growth. Cytological examination of dividing cells revealed an abundance of dose-dependent chromosome abnormalities produced by the applied treatments of AFB2. Chromosomal abnormalities associated with stickiness of chromosomes or due to an action on the mitotic apparatus are the dominant abnormalities induced by this toxin. Some of these abnormalities, particularly chromosome lagging and multipolar ana-telophase configurations, may account for the formation of cells with <the 2C DNA value or more >4C DNA value. However, true clastogenic chromosomal aberrations including chromosome breaks and ring chromosomes at metaphase and chromosomal bridges at ana-telophase were scored in substantial proportion of cells. The induction of whole chromosome breaks at metaphase is congruent with the indication by the cytometric measurements that AFB2 acts on the G1 transition point. The capacity of this toxin to induce clastogenic aberrations may be regarded as an indication of its genotoxic potential. This is also indicated by the formation of micronuclei in interphase cells. Key words Aflatoxin B2, Chromosome aberrations, Mitotic cycle, Vicia faba L. During the last few years, flow and image cytometry has been progressively developed as a convenient, rapid and efficient method to measure nuclear DNA content in cell nuclei (Hammatt et al. 1991, Baranyi and Greilhuber 1996). It is possible with this method to perform cell cycle analysis and study its regulation in plant cells (Zhang et al. 1992, Glab et al. 1994). With this technique, it is possible to estimate cell cycle duration, proportion of proliferative cells, fraction of cells in each phase of cell cycle, DNA ploidy level and mitotic activity. This approach is of increasing value due to recent advances in the area of detection procedures associated with software analysis of data. A possible application of image cytometry may be its use for evaluating cytotoxicity of environmental hazards on biological systems. One major source of environmental hazards is the mycotoxins, which are toxic secondary products of certain fungi, associated with agricultural commodities and cause severe toxicity to animals and man and result in great loss of crop productivity (Ames 1989). One major group of mycotoxins is the aflatoxins produced by few species of the genus Aspergillus, particularly A. flavus and A. parasiticus. There have been several reports documenting the effects of aflatoxins on different plant tissues and organs (Crisan 1973, Reiss 1978, McLean and Dutton 1995). These substances also disturb the * Corresponding author.

2 114 Hanaa H. El-Shazly and Iman A. El-Sheikh Cytologia 65 subcellular organization of plant cells (Walker et al. 1984, Fadl-Allah 1987) and affect physiological processes (Tripathi and Misra 1981, Dashek and Llewellyn 1983, Sinha and Kumari 1990, Sinha and Sinha 1995). The effect of aflatoxins on cell division and chromosomes have been the subject of numerous investigations since their cytotoxic effects were reported by Lilley (1965). Most of these studies have been oriented to demonstrate the antimitotic and chromotoxic activities of various types of these toxins and to point out their danger as environmental carcinogens and mutagens (Reiss 1971, Styler and Cutler 1984, Chakraborty and Nadi 1989, Packa 1991, Ruan et al. 1992, Badr et al. 1995). However, none of these studies dealt with the effect of aflatoxins on cell cycle, although both Fadl-Allah and Abdel-Rahim (1991) and El-Shazly (1995) recorded change in the relative proportion of mitotic phases. In the present study, the effect of different doses of aflatoxin B2 on a number of cell cycle parameters in meristematic cells in the root tips of Vicia faba L. is investigated. The association of changes in cell cycle components with the growth of seedlings, derived from treated germinating seeds, has been observed. In addition, the effect of AFB2 on the chromosomes in dividing cells is documented in order to find out a correlation between the action of aflatoxins on chromosomes and their influence on cell cycle components. Material and methods Seeds of Vicia faba L. cv. Giza 2, of equal size and age, previously soaked for 24 h in tap water, were treated with 5, 25, 50, 100 ppm of AFB2 for 4, 12, 24 h; control roots were simultaneously treated with tap water, and germinated in fine clean wet sand. Four days after treatments, roots, of one group of seedlings were detached, washed several times in water and fixed in 3 : 1 (v : v) ethanol/acetic acid overnight and stored in 70% ethanol in the refrigerator until use for cytological preparations. A second group of treated and control seeds were left to grow and the length of emerging radicle and plumule was measured every 3 days for 15 days. For cytological preparations of treated and control meristematic cells, roots were removed from the fixative, washed several times, blotted on filter paper, hydrolyzed in 1N HC1 for 8 min and stained in Feulgen reagent for 2 h. Inividual roots were then washed and their stained meristems squashed in a drop of 45% acetic acid and coverslips were removed by the dry ice methods. Preparations were then washed for 30 min in SO2 water, dehydrated in ethanol and mounted in Euparal. At least six slides were prepared for each treatment and the control. The amount of DNA in the nucleus, DNA ploidy level and the fraction of cells undergoing different plases of cell cycle were calculated from at least 500 cells sampled from at least 3 slides for each treatment. These include; cells with DNA amount less than the 2C value, cells with 2C DNA (Go/G1), cells with 2C-4C DNA (S-phase), cells with 4C DNA (G2-phase), cells with DNA amount more than the 4C value and cells in mitotic phases. These cytometric determinations were made using SAMBA 4000 cell image analysis system provided with Zeiss Axioskop, CCD video camera, image monitor, open software, statistical package and HP printer. In addition, at least 400 dividing cells were examined and the number and types of chromosomal abnormalities in the mitotic phases were recorded. Statistical analysis for cell cycle measurements and of radicle and plumule length measurements as well as for the total number of abnormal mitotic cells was made using one-way analysis of variance (ANOVA) in the statistical analytical systems (SAS) softwate package. Results The effect of the applied treatments of AFB2 on cell cycle phases is documented in Table 1. Inspection of the values given in this table clearly demonstrate a progressive increase of the proportion of cells with the 2C value (G0/G1-phase) as the concentration of the toxin increased and the pe-

3 2000 Arrest of Mitotic Cycle and Induction of Chromosomal Aberrations 115

4 116 Hanaa H. El-Shazly and Iman A. El-Sheikh Cytologia 65 A B C D Fig. 1. The effect of the applied treatments of AFB2 on the fraction of mitotic cycle phases in V fava root meristems; A) Go/G1 phase, B) S-phase, C) G2 phase, D) cells in M-phase. riod of treatment prolonged. This proportion increased from 45.16±0.45 in control roots to }0.27 in cells treated with the highest used concentration (100 ppm) for 4 h. In root cells treated with the same concentration for 12 and 24 h, the proportion of cells in the Go/GI-phase was increased to and ± 0.31 respectively. On the other hand, the fraction of cells in the DNA synthesis period (S-phase), the G2-phase and in M-phase progressively decreased with the increase of the applied dose of the AFB2. The proportion of cells in the S-phase was reduced from } 0.18 in control roots to } 0.10 and 9.36 } 0.14 in root cells treated with 100 ppm for 4 and 12 h respectively, whereas, in root cells treated with the same concentration for 24 h, the proportion of S-phase was reduced to only 2.28 }0.12. The changes in the fraction of cells in the G2- phase and M-phase closely resemble those of the S-phase. The proportion of G2-phase cells was re-

5 2000 Arrest of Mitotic Cycle and Induction of Chromosomal Aberrations 117

6 118 Hanaa H. E1-Shazly and Iman A. El-Sheikh Cytologia 65 Fig. 2. Some types of abnormalities produced in Vicia faba L. root tip cells by different treatments of AFB2. a) stikiness and ring chromosome at metaphase (12 h, 50 ppm), b) anaphase with chromosome breakage (12 h, 25 ppm), c) disturbed anaphase showing breaks and unbalanced separation of chromosomes (12 h, 25 ppm), d) anaphase with lagging chromosome (6 h, 50 ppm), e) telophase bridge and chromosome lagging (6 h, 50 ppm), f) micronucleus at interphase (6 h, 50 ppm). duced from a control value of }0.29 to 8.36 }0.07, 6.95 }0.08 and 3.54 }0.11 in root cells treated with 100 ppm for 4, 12 and 24 h respectively. Likewise the fraction of cells in M-phase was decreased from 9.89 }0.11 to only 3.30 }0.07, 2.92 } 0.17 and 2.28 }0.12 in root cells treated with the same doses of the toxin. Fig. 1 illustrates the dose-dependent effect of the different treatments of the AFB2 on the G0/G1-phase, S-phase, G2-phase and M-phase respectively. In treated root cells a fraction of cells with DNA amount less than the 2C value or more than the 4C value was scored, the proportion of cells with DNA content less than the 2C value generally increased with the increased dose of the toxin, but that with DNA content less than the 4C value was not dose-dependent (Table 1). The progressive reduction of mitotic activity induced by the AFB2 treatments is associated with a reduced growth rate of seedlings. This is indicated by the values of radicle and plumule length of seedlings after 15 days of germination (Table 1). However, the reduction in these values appears to be concentration-dependent. In cells undergoing mitosis different types of chromosomal abnormalities were recorded. The proportion of total abnormalities was extremely high and increased as the dose of the toxin increased. The types and frequency of abnormalities produced by the different treatments of the toxin in the phases of mitosis are also given in Table 2. Some of these

7 2000 Arrest of Mitotic Cycle and Induction of Chromosomal Aberrations 119 abnormalities are illustrated in Fig. 2. Chromosomal stickiness (Fig. 2a) was the dominant abnormality induced in all phases of mitosis, the frequency of this abnormality increased in prophase, decreased in metaphase and showed little variation at ana-telophase as the dose of the toxin was increased. Other common abnormalities produced by AFB2 treatments include non-oriented chromosomes and chromosome breaks at anaphase (Fig. 2b, c), lagging chromosomes at ana-telophase (Fig. 2b, c, d) chromosomal bridges and lagging at telophase (Fig. 2e). The frequency of bridges was increased, whereas, the frequency of lagging chromosomes was decreased as the dose of the toxin was increased. Other important, but less common abnormalities include ring chromosomes at metaphase (Fig. 2a) disturbed polarity and unbalanced separation of chromosomes at ana-telophase (Fig. 2c). Disturbed prophase and ana-telophase configurations and miconuclei (Fig. 2f) were also induced by most of the applied treatments particularly following treatments with high concentrations of the toxin. The proportion of these abnormalities is also given in Table 2. Discussion The applied treatments of AFB2 clearly demonstrated a dose-dependent effect of this toxin on the components of mitotic cell cycle of meristematic cells in V faba roots. The most evident appears to be the accumulation of cells in the G0/G1 phase at the expense of other phases. This is illustrated by the progressive increase in the fraction of cells in this accompanied by reductions in the proportion of other cell cycle phases. These results indicate that AFB2 acts as an inhibitor of cell cycle at the G1 transition point. In this respect the action by this toxin resembles that of olomoucine, which is an inhibitor of cdc2/cdk2 kinases activity, when applied to Petunia mesophyll cells in culture (Glab et al. 1994). An interesting observation is that the dose-dependent effect induced by the AFB2 matches that induced by olomoucine as reported by these authors. In plant cells phosphorylation of these proteins is probably necessary since inhibition of this phoshorylation by some mitotic cycle inhibitors such as okadaic acid results in the arrest of cell cycle in suspension cell culture of tobacco at early mitosis (Zhang et al. 1992). The action induced by AFB2 at the G1 transition point makes it a possible experimental tool to distinguish between different classes of cdc2 genes that control cell cycle progression in plants. Another prominent effect induced by AFB2 was the progressive reduction in mitotic activity in root cells as the dose of the toxin increased. The inhibition of mitotic activity has been regarded as a common effect induced by numerous chemical compounds. Inhibition of mitosis by aflatoxins was associated with a reduction in the amount of DNA (Yu et al. 1988, Schlemper et al. 1991, Choy 1993, Badr et al. 1995). The reduction in the proportion of cells in the S-phase recorded in cells treated with AFB2 is consistent with results of these and other authors. Reports by some of these authors (Schlemper et al. 1991, Choy 1993) of binding of AFB, to DNA can not be confirmed by the results of the present investigation since the proportion of cells in S-phase was reduced by treatments with this toxin. Reduction in mitotic activity, expressed as a decrease in the proportion of cells in mitotic phases, by the treatments of AFB2 may simply be regarded a result of the arrest of mitotic cycle at the G1 phase. The inhibition of mitotic activity in root meristems is accompanied by a reduction in seedling growth expressed as reduction in radicle and plumule length. Growth inhibition, of V faba seedlings, induced by AFB2 is congruent with similar growth inhibitory effects induced by AFB I on soybean roots (Walker et al. 1984) and by crude toxins of A. flavus, and A. parasiticus and ochratoxin-a on V faba plants (El-Shazly 1995). Plant growth can be considered to consist of two components; cell division and cell expansion, it also involves metabolic events in which growth hormones play a major role. Retardation of plant growth may thus be the result of inhibition of auxin synthesis or the delay in mitotic division. Other views (Datta and Biswas 1985, Padmavahti et al. 1992) relate seedling growth retardation to germination injury and the production of chromosomal

8 120 Hanaa H. El-Shazly and Iman A. El-Sheikh Cytologia 65 abnormalities in dividing cells. Both Bhattacharya and Shama Rao (1978) and Mendhulkar (1993) attributed plant growth inhibition by treatments with mutagenic agents to disturbances in natural growth regulators and mitotic chromosomal irregularities as additional factors. Cytological examination of dividing cells revealed an abundance of dose-dependent chromosome irregularities produced by the applied treatments of AFB2. The production of chromosomal abnormalities provide a valuable genetic assay for screening environmental pollutants (Grant 1994). However, the dominant irregularity induced by this toxin was found to be chromosomal stickiness, which is a physiological and transient phenomenon induced in dividing cells by numerous chemical compounds including aflatoxins (Fadl-Allah and Abdel-Rahim 1991, El-Shazly 1995). Stickiness has been attributed to the entanglement of interchromosomal chromatin fibers that leads to subchromatid connection between chromosomes (Klasterska et al. 1976), but was considered by Patil and Bhat (1992) as a type of physical adhesion involving mainly the proteinacious matrix of chromatin. However, stickiness should be considered as a mitotic disruption that is not likely to lead to chromosomal structural damage (Badr et al. 1995). Other abnormalities that have been induced by an action of AFB2 on mitotic division in root meristems of V faba comprise irregular prophases, disturbed metaphases and ana-telophases, unoriented chromosomes at metaphase, lagging chromosomes at ana-telophase and mutipolar anatelophase configurations. The production of these abnormalities indicates that this toxin induced partial inhibition of mitotic apparatus. Compounds that produce this action are regarded as mitotic poisons and include some aflatoxins (Dashek and Llewellyn 1983, Fadl-Allah and Abdel-Rahim 1991, Bose and Sinha 1991, El-Shazly 1995). The mitotic apparatus is composed of proteinacious fibers and thus the induction of these abnormalities may indicate an action by AFB2 on the cellular proteins. An action by AFB1 on cellular proteins was recorded in maize seedlings (Misra and Tripathi 1980, Tripathi and Mista 1981) and in V faba seedlings by the crude toxins of A. flavus and A. parasiticus and ochrtoxin-a (El-Shazly 1995). The induction of lagging chromosomes and multipolar ana-telophase configurations leads to the separation of unequal number of chromosomes in the daughter nuclei. This is manifested by scoring of fractions of cells with DNA amount<the 2C value and cells with DNA amount>the 4C value following treatment with the AFB2. Another consequence of the effect of AFB2 on mitotic apparatus is the formation of polyploid cells that were only recorded in cells treated with the higher concentration of the toxin used. In addition to the mitotic chromosomal abnormalities discussed above all treatments of AFB2 induced clastogenic chromosome aberrations in V faba root meristems represented by chromosome breakage and ring chromosomes at metaphase and chromosome bridges at ana-talophase. The breaks induced by this toxin are mainly of the chromosome type. This observation supports the findings that the toxin acts during the G1 phase of mitotic cycle. The formation of ring chromosomes and chromosomal bridges may be the result of reunion of broken chromosome ends. However, in the present study, the frequency of bridges is too high to be accounted for by chromosome breaks. Some of the bridges scored here may be the result of chromosomal stickiness, which may cause the chromosomes to remain connected through bridges at ana-telophase. Other aflatoxins have been also reported to induce clastogenic action on the chromosomes (Lilley 1965, Reiss 1971, Sinha et al. 1987, Chakraborty and Nadi 1989, Fadl-Allah and Abdel-Rahim, 1991, El-Shazly 1995). The capacity of these compounds to induce chromosomal aberrations has been discussed by some authors. Schlemper et al. (1991) showed that AFB1 binds to DNA to form a binding adduct which activates the aflatoxin to a mutagenic product. Investigations dealing with the mode of action of camptothecin (CPT) revealed that it produces DNA lesions capable of giving rise to chromosome aberrations by acting on the isomerases involved in DNA replication (Eng et al. 1988). The reaction of CPT with type II topoisomerase catalyzes the breakage and rejoining of the two strands of DNA (Hsiang et al. 1989). Although CPT is an inhibitor of DNA replication, DNA synthesis is required for its clastogenic effect (Andersson and Kihlman 1992).

9 2000 Arrest of Mitotic Cycle and Induction of Chromosomal Aberrations 121 The treatments with AFB2 induced despiralization of chromosomes at ana-telophase and the formation of micronuclei in interphase. Despiralization of chromosomes may indicate an action on the chromosome matrix (El-Shazly 1995). On the other hand, micronuclei may originate from a lagging chromosome at ana-telophase or from a chromosome fragment (Badr and Ibrahim 1987). Both types have been recorded by AFB2 in the present investigation. Micronuclei derived from a whole chromosome have higher probability to survive and undergo condensation in synchrony with the main nuclei than micronuclei derived from a chromosome fragment (Gustavino et al. 1987). Micronuclei are true mutagenic aspects, which may lead to a loss of genetic material and have been regarded as an indication of the mutagenecity of their inducers (Ruan et al. 1992). The production of micronuclei by AFB2 and its capacity to induce chromosomal clastogenic aberrations may be regarded as evidence of its genotoxic potential. Acknowledgement We are grateful to Prof. Dr. Abdelfattah Badr, Professor of Genetics and Head of Botany Department, Faculty of Science, Tanta University for advice on the practical work and help in the preparation of the manuscript and Dr. Ali Saleh of the National Cancer Institute, Cairo University for assistance in conducting the image analysis. References Ames, I. A Mycotoxins, economic and health risks. CAST (Council of Agricultural Science and Technology), Task force Rep. No USA. Andersson, H. C. and Kihlman, B. A Induction of chromosomal aberrations by camptothecin in root tip cells of Vicia faba. Mut. Res. 268: Badr, A. and Ibrahim, A. G Effect of herbicide glean on mitosis, chromosomes and nucleic acids in Allium cepa and Vicia faba root meristems. Cytologia 52: A. S., Kheiralla, Z. H. and El-Shazly, H. H Mutagenic potential of aflatoxin produced by Aspergillus parasiticus and its effect on growth and yield of Vicia faba 3rd Con. Toxcol. Dev. Count., Cairo, Egypt, Nov. proceeding, Vol. II pp Baranyi, M. and Greilhuber, J Flow cytometric and feulgen densitometric analysis of genome size variation in Pisum. Theor. Appl. Genet. 92: Bhattacharya, S. and Shama Pao, H. K Effect of exogenous IAA on radiation induced seedling growth in rice. Ind. J. Exp. Biol. 16: Bose, S. and Sinha, S. P Aflatoxin induced structural chromosomal changes and mitotic disruption in mouse bone marrow. Mut. Res. 261: Chakraborty, A. and Nadi, S Genotoxic studies on root meristem of Allium cepa induced by aflatoxin B1, B2 and G1. Nucleus (Calcutta) 32: Choy, W. N A review of the dose response induction of DNA adducts by aflatoxin B, and its implications to quantitive cancer risk assesment. Mut. Res. 296: Crisan, E. V Effects of aflatoxin on germination and growth of lettuce. Appl. Microbiol. 25: Dashek, W. V. and Llewellyn, G. C Mode of action of hepatocarcinogens of aflatoxin in plant systems, A review. Mycopathologia 81: Datta, A. K. and Biswas, A. K Induced mutagenesis in Nigella sativa L. Cytologia 50: El-Shazly, H. A Genotoxicity of some mycotoxins and their effect on growth and yield of Vicia faba. Ph.D. thesis, AM Shams University, Cairo, Egypt. Eng, W. K., Faucette, L., Johnson R. K. and Sternglanz, R Evidence that bdna topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol. Pharmacol. 34: Fadl-Allah, E. M Studies on the effect of some mycotoxins on plant growth and cell ultrastructure. Ph.D. thesis, Minia University, Minia, Egypt. Abdel-Rahim, A. T The effects of three mycotoxins on mitotic division and somatic chromosomes of roots from Allium cepa. Bull. Fac. Sci., Assiut Univ. 20: Glab, N., Labidi, B., Qin, L., Trehin, Ch., Bergounioux, C. and Meijer, L Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to A and G2 to M cell cycle transitions FEBS Lett. 353:

10 122 Hanaa H. El-Shazly and Iman A. El-Sheikh Cytologia 65 Grant, W. F The present status of higher plant bioassays for the detection of environmental mutagens. Mut. Res. 310: Gustavino, B., Vitagliano, E., Sothili, A. and Rizzoni, A A comparison between short-term evolution of micronuclie induced by x-ray and colchicine in root tips of Vicia faba. Mut. Res. 192: Hammatt, N. M., Blackhall, N. W. and Davey, M. R Variation in the DNA content of Glycine species. J. Exp. Bot. 42: Hsiang, Y. H., Lihou, M. G. and Liu, L. F Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as mechanism of cell killing by camptothecin. Cancer Res. 49: Klasterska, I., Natarajan, A. T. and Roml, C An interpretation of the origin of subchromatic aberrations and chromosome stickiness as a category of somatic aberrations. Hereditas 83: Lilley, L. J Induction of chromosome aberrations by aflatoxins. Nature 207: McLean, M. and Dutton, M. F Cellular interactions and metabolism of aflatoxin. Pharmacology and Therapeutics 65: Mendhulkar, V D Effect of chemical mutagens on Desmodium tortuosum. Ph.D. thesis, Karnatak University, Dharwad, India. Misra, R. S. and Tripathi, R. K Effect of aflatoxin B1 on germination, respiration and alpha-amylase in maize. Z. Pflanzenker. Pflanzenschutz 87: Packa, D Cytogenetic changes in plant cells as influenced by mycotoxins. Mycotoxin Res. 7 (Suppl.) part II: Padmavathi, T., Prathibha, D. and Kiranmai, V Induced variability for different biological parameters in soybean. J. Cytol. Genet. 27: Patil, B. C. and Bhat, G. I A comparative study of MH and EMS in the induction of aberrations on lateral root meristern in Clitoria ternata L. Cytologia 57: Reiss, H chromosomen Aberrationen in den Wurzelspitzen von Allium cepa diirch. Aflatoxin B. Experientia 27: Effects of Mycotoxins on Higher Plants, Algae, Fungi and Bacteria. In: Wyllie, T. D. and Morehouse, L. G. (eds.). Mycotoxic Fungi, Mycotoxins and Mycotoxicoses. Marcel Dekker, New York. pp Ruan, C., Lian, Y. and Lium, J Application of micronucleus test in Vicia faba root tips in the rapid detection of mutagenic environmental pollutants. Chinese J. Environ. Sci. 4: Schlemper, B., Harrison, J., Garner, R., Oesh, F and Steinberg, P DNA binding adduct characterization and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal, kupffer and endothelial cells. Arch. Toxicol. 65: Sinha, K. K. and Kumari, P Some physiological abnormalities induced by aflatoxin B1 in mung bean seeds (Vigna radiata variety Pusa Baishakhi). Mycopathologia 110: Sinha, A. K Effect of aflatoxin B, on some biochemical changes in some seeds of wheat varieties. Indian Phytopathology. 48: Sinha, S. P., Bilgrami, K. S. and Prasad, V Aflatoxin induced clastogeny in bone marrow cells of mice. Proc. Ind. Natl. Sci. Acad. B53: Styler, C. H. and Cutler, H. G Effects of moniliformin on mitosis in maize (Zea mays L.). Plant Cell Physiol. 25: Tripathi, R. K. and Misra, R. S Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds. Appl. Environ. Microb. 42: Walker, S. J., Llewellyn, G. C., Lillehoj, E. B. and Dashek, W. V Uptake and subcellular distribution of aflatoxin B, by excised, cultured soybean roots and toxin effects on root elongation. Environ. Exp. Bot. 24: Yu, F. L., Geronemo, I. H., Bender, W. and Permthamsin, J Correlation studies between the binding of aflatoxin B to chromatin components and the inhibition of RNA synthesis. Carcenogenesis 9: Zhang, K., Tsukitani, Y. and John, P. C. L Mitotic arrest in tobacco caused by the phosphoprotein phosphatase inhibitor okadaic acid. Plant Cell Physiol. 33: