aflatoxin b1-induced ultrastructural alterations in maturezea mays embryos

Mycopathologia 128: 181-192, 1994. @ 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Aflatoxin Bl-induced ultrastructural alterations in mature Zea mays embryos

M i c h e l l e M c L e a n Department of Physiology, Faculty of Medicine, University of Natal, Congella, Rep. of South Africa

Received 23 June 1994; accepted in original form 29 August 1994

Abstract

Mature maize (Zea mays L.) embryos were exposed to aflatoxin B1 (AFB1) concentrations ranging from 0.1 to 25 #g/ml for 9 days. With increasing toxin concentration above 2 #g/ml,primary root elongation of germinated embryos was progressively inhibited, to reach a maximum value of 81% at 25 #/ml toxin. An ultrastructural investigation of the subcellular alterations induced following toxin exposure provided evidence of deteriorative changes in several compartments of the plant cell. Alteration in membrane integrity (e.g., the tonoplast, plasmalemma and inner mitochondrial membrane) was a frequent feature of many cells. Apparent fusion of vacuoles, incorporation of cytoplasmic components into vacuoles and intravacuolar membrane whorls might be interpreted as deteriorative alterations. The results are discussed in the light of ultrastructural findings for other plant systems exposed to similar AFBI concentrations, as well as findings for animal systems.

Key words: Aflatoxin B l, Embryo, Mature, Ultrastructure, Zea mays L.

Introduction

Aflatoxin B~, a secondary metabolite produced by Aspergillusparasiticus and A.flavus on growing plants and stored plant products, is a potent hepatocarcinogen [1]. While the experimental evidence for AFB~ involvement in the development of hepatocellular car- cinoma in several laboratory animals is convincing [2], the role of AFB1 in the high incidences of liver cancer in human populations in Africa, Asia and South America [3--4] has long been a contentious issue. Other aetiological agents (e.g., hepatitis B virus) are regarded as complicating factors [5]. Following the discovery of AFB 1 as an animal toxin and carcinogen, the phytotox- icity of this mycotoxin (and other aflatoxins) has been investigated since 1965 [6, 7]. Since then, there have been several publications, documenting the inhibitory responses of AFB1 on many different plant tissues and organs [8-14]. Only a few researchers have, however, attempted to explain the metabolic role of AFBt in the observed inhibitory responses, or its fate in the plant cell [15-17]. Similarly, the number of reports of AFBl-induced subcellular alterations in plant cells is limited [18-2@ The present investigation reports

on the ultrastructural changes induced by AFB~ in the root tips of mature, germinating Zea mays embryos exposed to toxin concentrations which did not impair germination, but influenced root elongation at higher toxin concentrations. The research presents the final results of a comprehensive investigation of the phy- totoxic effects of AFB1 on several plant tissues. To this end, the inhibitory effects of AFB1 on tobacco callus [25] and tobacco plantlet [26] growth and metabolism have already been investigated. Addition- ally, the inhibitory effects of AFB 1 on root and shoot development in matureZea mays embryos [24, 27], and the AFBl-induced ultrastructural alterations in tobacco callus [25], maize callus [ 19, 21 ] and immature Zea mays embryos [23, 24] have been documented.

Materials and methods

Aflatoxin exposure. Mature Zea mays seeds were surface-sterilised in 2% Hibitane [(v/v) (ICI Pharma- ceuticals] for 15 rain and, in order to facilitate embryo excision, caryopses were soaked overnight in sterile distilled water. Following excision, embryos were

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surface-sterilised in 2% Hibitane for 15 rain, soaked for 10 rain in a 0.1 mg/ml each of a penicillin/streptomycin mixture (Highveld Biological), followed by a 5 rain immersion in 1% NaOC1. Embryos were rinsed three times in sterile distilled water and plated aseptically onto a maize embryo germination medium (pH 5.8) [28] into which AFB1 had been incorporated (eight concentrations, ranging from 0.1-25 #g/ml AFB 1) following autoclaving of the medium. Aflatoxin B1 was added to the medium from a stock solution such that concentrations of 0.1, 0.5, I, 2, 5, 10, 20 and 25 /zg/ml were attained. Since DMSO (dimethyl sulphox- ide) was used to dissolve the AFB 1 for the stock solution, the possible effects of DMSO on embryos had to be considered. Controls comprised embryos plated on a medium containing no DMSO, and a medium containing 1% DMSO (as found in 25 #g/ml AFB1). Embryos were allowed to germinate and establish for 9 days on the toxin-containing medium at 25 4- 3 °C, with a 16 h photoperiod r~gime at a photon flux density of 200 #moles/m2/sec. Three replicates of twenty-five embryos were assessed for each treatment.

Transmission electron microscopy. At the termina- tion of the 9 day incubation period, the primary root tips were processed for electron microscopy. The selected material was fixed in Karnovsky's fixative (pH 6.0) at room temperature overnight, washed twice in 0.2 M sodium cacodylate buffer (pH 6.0) and post-fixed in i% aqueous OSO4 for 2 h (4 °C in the dark). Fixed material was then washed twice in distilled water, and dehydrated in an acetone series (25, 50, 75, 90 and 100%), for 30 min at each concentration, with two changes. During dehydration in the 75% solvent, material was block-stained for 1 h (4 °C) with saturated uranyl acetate. Material was then placed in a 50:50 solution of acetone: epoxy resin [29] overnight. The resin was then replaced with fresh, pure resin for 8-12 h (three changes), after which the resin was replaced and allowed to polymerise for 8-12 h at 70 °C.

Sections were cut using a Reichert Ultracut ultra- microtome, stained for 10 min with 2% uranyl acetate, washed twice in sterile distilled water and post-stained for 30 rain with lead citrate [30]. Sections of five root tips (three replicates) from each toxin concentration (including controls) were viewed with either a Zeiss EM10B or a Joel 100C transmission electron microscope at an accelerating voltage range of 60-100 kV.

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Fig. I. Percentage inhibition of primary root elongation in mature, germinating Zea mays embryos exposed to aflatoxin B~ for 9 days. The I50 value was approximately 7.5 /zg/ml. Alphabetical symbols were assigned according to a multiple range test.

R e s u l t s

Following 9 days of incubation in the presence of AFBb mean root (primary) length was unaffected at toxin concentrations below 5 #g/ml. With increasing toxin concentration above, (and including) this concentration, exposure to AFB1 resulted in a progres- sive inhibition of root elongation (Fig. 1). In compari- son with control values, mean root length of embryos exposed to 25 #g/ml was inhibitedby 81%. The I50 value (50% inhibition, relative to controls) was approximately 7.5 #g/ml AFB1.

The cells of root tips from control material (DMSO- free and 1% DMSO) of mature maize embryos were characterised by a dense cytoplasm, resulting at least partly from the large number of polysomes (Figs. 2 - 5). The rough endoplasmic reticulum of cells of control root tips was present as many shortish cisternae and the cells contained numerous mitochondria with many cristae and dense matrices, reflecting the high level of metabolic activity. While most mitochondria appeared normal, occasional organelles exhibited a diffuse area within the matrix (Figs. 2 and 5). This diffuse appearance was slightly more conspicuous in DMSO- treated cells. Small vacuoles and occasional small lipid droplets were also present in these cells. Plastids var- ied from relatively undifferentiated organelles to more differentiated, starch-containing organelles. A clear area, contiguous with some organelles (Fig. 5), was observed in some DMSO-treated cells.

Low dose levels (0.1-2 pg/ml) AFBI had little effect on the ultrastructure of cells of the root tips of mature seedlings (Figs. 6-9). Polysome disposi- tion appeared similar to control root tip cells, as was the distribution of rough endoplasmic reticulum, mitochondria and plastids. Some ultrastructural features observed in cells treated with low AFB1 concentrations might perhaps be interpreted as indicative of potential deteriorative changes, e.g., the appearance and persis- tence of a diffuse central region in mitochondria (Figs. 6, 8 and 9); a clearing of cytoplasm in the immediate vicinity of some plastids and mitochondria (Fig. 9); and apparent fusion of vacuoles (Figs. 6 and 7). Tono- plast membranes were not always clearly visible (Figs. 6 and 7). Membrane whorls within vacuoles were also observed (Fig. 8).

As the toxin concentration increased above 2.0 #g/ml, cell ultrastructure became progressively dete- riorated (Figs. 10-18). At 5 /~g/ml AFBI, a slight cytomatrical dissolution was observed in many cells (Figs. 10 and 11), particularly in the area immediately surrounding organelles. The matrix of some mitochondria exhibited an extensive central clearing (Fig. 11), while others with well developed cristae were visible (Figs. 10 and 11). Plastids were generally observed as small, relatively undifferentiated organelles containing a few or no starch deposits (Figs. 10 and 11). Appar- ent incorporation of lipid droplets into vacuoles was frequently observed (Fig. 10). Other small membrane- bound structures, which may be interpreted as the remains of small lipid droplets, the contents of which appeared shrunken and diffuse, were observed (Fig. 11). In these instances, the delimiting membrane was clearly visible.

In the 10-25 #g/ml AFBl dose range, many of the features observed at 5 #g/ml AFB1 became exacerbated (Figs. 12-19). While some cells still possessed a relatively normal complement of cytoplasmic ribosomal subunits/polysomes, many others exhibited a diminution in this population (Figs. 12-19). Tonoplast membranes could not always be visualised in their entirety and this may reflect localised membrane disruption (Figs. 13 and 16). It would appear that vacuoles had increased in size (Figs. 12 and 13) and many of these organelles contained remnants of cellular components, including membrane whorls, some of which were extensive (Figs. 12 and 15). Fusion of vacuoles appeared to have occurred in several cells (Figs. 12, 13 and 17). Peripheral multivesicular bodies which could have originated from plasmalemmal vesiculation were frequently observed (Figs. 12 and 13).

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Plastid and mitochondrial morphologies were vari- ably affected by the toxin. Many intact, apparently normal mitochondria were observed (Figs. 12-15, 17 and 19). Mitochondria exhibiting a diffuse internal area, as observed at lower AFBI concentrations, were no longer apparent. Instead, a few of these organelles with a swelling of the inner membrane were observed (Figs. 4 and 18). Starch grains within several plastids were shrunken (Figs. 13 and 14). Areas of lipid accumulation and coalescence were observed in some cells (Figs. 17 and 19).

In spite of the deterioration in organellar and cytoplasmic integrity, the nucleus and nucleolus generally remained morphologically intact (Figs. 13 and 19). In some instances, however, chromatin was observed to be abnormally condensed (not shown), while in others, the nuclear matrix appeared relatively homogeneous (Fig. 19).

Discussion

An ultrastructural investigation of tissues exposed to AFB1 has proven to be an informative exercise to explain the inhibitory effects of the toxin on primary root elongation of germinating, mature maize embryos. Early major ultrastructural alterations induced by AFB1 in this study include alterations in vacuole morphology (apparent enlargement, fusion and tonoplast disruption), with many of these organelles appearing autophagic. A marked decrease in the cytoplasmic ribosomal subunit/polysome population was observed. These features generally became more exaggerated in cells exposed to AFB1 concentrations in excess of 5 #g/ml, and probably more likely from a concentration close to the I50 (50% inhibition) value for root elongation of mature embryos (approximately 7.5 pg/ml). A comprehensive discussion of the possible events underlying these AFBl-induced subcellular changes has been presented in previous ultrastructural studies of immature Zea rnays embryos [23] and tobacco callus [25]. These tissues exhibited similar (albeit more exacerbated, in the case of tobacco callus) subcellular deteriorative alterations when exposed to the same AFB~ regime.

Ultrastructurally, several organelles/subcellular components exhibited marked deteriorative changes that may have contributed to the general cellular dis- organisation of Zea mays root tip cells. Of particular importance in this regard, is the apparent disruption in vacuolar morphology (and by implication, func-

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Plate 1. Figs 2 and 3 (DMSO-free controls); Figs. 4 and 5 (1% DMSO) Cells of root tips of germinated control embryos were characterised by a cytoplasm containing a large number of polysomes, mitochondria (m), plastids (p), many of-which contained starch (s) deposits (Figs. 1 and 2). Several small vacuoles (v) and occasional small lipid droplets (I) were additional features of these cells. DMSO (1%) generally had little effect on cell ultrastructure (Figs. 3 and 4). Some mitochondria exhibited a diffuse internal area (*), which was slightly more exaggerated than in DMSO-free controls. Polysomes and ribosomal subunits appeared to he excluded from the cytoplasmic zone immediately contiguous (arrows) with some plastids (Fig. 5). Bar on micrographs = 1 ~m (unless stated otherwise). Fig. 2 × 11900; Fig 3. X 10 700; Fig. 4 X 15 500; Fig. 5 × 18 000.

t ion). Several of these organelles contained cellular

debris in var ious stages of degenerat ion, perhaps indi-

cat ing an abnormal induct ion o f au tophagy which may

have been t r iggered by AFB1 exposure. The frequent ly

observed intravacuolar membrane whorls may possi-

bly be a manifes ta t ion of the final stages of the process

of autophagy. Dis rupt ion of the tonoplas t was observed in many vacuoles , a feature which may be v iewed as

a severe deter iorat ive alteration. The local ised cyto-

plasmic dissolut ion observed in many cells may have

185

Plate 2. Fig. 6 (0.5/~g/ml AFB1 ); Figs. 7 and 8 (1 #g/ml); Fig. 9 (2 ~g/ml) Mitochondria containing a central diffuse region persisted (Figs. 6, 8 and 9). Organelles with well developed cristae were also observed (Figs. 6 and 7). Abnormal vacuolar morphology was apparent in many cells (Figs 6-8). Membrane whorls can be observed within vacuoles illustrated in Fig. 8, while lipid droplets (1) appear to be in the process of being incorporated into vacuoles in Fig. 7. Incipient vacuolar fusion is illustrated in Fig. 7 (arrows), while there may be slight cytoplasmic dissolution in the vicinity of a small vacuole, the tonoplast of which is no longer discernible (arrow) in Fig. 6. A cytoplasmic zone from which polysomes and/or ribosomal subunits were excluded (arrows) was marked in some cells (Fig. 9). Fig. 6 x 21500; Fig 7. × 12 900; Fig. 8 × 14 200; Fig. 9 × 19 500.

arisen as a result of hydrolys is of cel lular const i tuents

by lysosomal enzymes released fo l lowing tonoplast

damage.

Several other membrane systems exhibi ted dete-

r iora t ive alterations (e.g., occasional swel l ing of the

inner mi tochondr ia l membrane) fo l lowing toxin expo-

sure. Peripheral vesicle aggregat ions (some of which

may have been mul t ives icular bodies) were a feature of

many cells and may be indicat ive of a level o f p lasma

membrane (and therefore cellular) deterioration.

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Plate 3. Figs. 10 and iI (5 t~g/ml AFB,); Figs. 12 and 13 (10 gg/ml) The most striking alteration in cellular ultrastructure in cells of root tips exposed to higher AFB] concentrations, as compared with lower toxin doses, was the less compact nature of the cytoplasm, presumably resulting largely from a decrease in the ribosomal subunit/polysome population (Figs. 10-14). There was an apparent increase in frequency and size of the vacuoles, several of which appeared to be in a state of incipient fusion or actually fusing (Figs. 10 and 12). Vacuoles in which the tonoplast could no longer be visualised (arrows) are illustrated in Fig. 13. Mitochondria generally appeared normal, with well-developed cristae. Organelles containing a diffuse area were generally not present (except in Fig. 11 [5 lzg/ml]), perhaps reflecting the disappearance of those organelles that were deranged. Peripheral multivesicular bodies or areas (encircled) were observed, possibly resulting from plasmalemmal vesiculation (Figs. 12 and 13). Starch deposits in plastids in Fig. 13 appear somewhat shrunken. Figs. 10, l 1 and 13 × 16 500; Fig. 12 × 13300.

It has been documented that the pr imary effect

of several host-specif ic toxins is damage to plant

cell membranes , part icularly the p l a sma lemma [31].

Membrane damage, part icularly if it affects several organelles, may be difficult to repair and so wil l con-

siderably exacerbate any deter iorat ive process wi th in

the cell. Addit ional ly, membrane damage may result in

an inabil i ty o f the cell to control the entry and exit o f

molecules , result ing in leakage, a c o m m o n occurrence

in fungal or bacterial tox in- induced plant pathogene-

sis [32]. Afla toxin B1 (or more specifically, the 8,9-

epoxide) in animal cells binds to the macromolecu les

187

Plate 4. Figs. 14 and 15 (20 #g/ml AFB1); Figs. 16-19 (25 ~zg/ml) Many apparently autophagic vacuoles were present in cells treated with 20 and 25 ~g/ml AFBt, some containing extensive membrane whorls (Fig. 15). Starch depletion was apparent in several plastids (Fig. 14). Areas of Iocalised cytoplasmic dissolution were frequently observed (*), particularly in the vicinity of some organelles (Figs. 14 and 19). In some instances, this may have arisen as a result of a loss of tonoplast integrity (Fig. 16 - arrow). In several mitochondria, there was evidence of swelling of the cristae (Figs. 14 and 18). Normal mitochondria were frequently observed (Figs. 14, 15, 17 and 18). In Fig. 14, abnormal withdrawal of the plasmalemma from the cell wall can be seen. At 25 /zg/ml AFB1, some cells exhibited abnormal lipid droplets, many of which might be interpreted as fusing (Fig. 17). The nuclear matrix illustrated in Fig. 19 has a homogeneous appearance. Fig. 14 x 20 300; Fig. 1 5 x 9900;Fig. 16× 18000;Fig. 17 × 16300;Fig. 18 ×54000 ;F ig . 19 × 17400.

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[1, 33]. Cytoplasmic and membrane proteins are thus likely to be targets for both non-covalent and covalent binding of AFB 1.

The generation of excessive free radicals (or the failure of the efficient scavenging of free radicals generated through normal metabolic reactions) may explain several of the observed deteriorative responses in cells. Free radicals can react with proteins and nucleic acids, potentially causing denaturation and/or mutagenesis [34]. The superoxide anion generated as a result of the univalent reduction of oxygen can react with hydrogen peroxide, forming an hydroxyl radical, which in turn can cause peroxidation of membrane lipids. In turn, the products of lipid peroxidation may result in damage to proteins, DNA, RNA and pig- ments [35-36]. In this regard, Amstad et al. (1984) have proposed that in human lymphocytes, AFB1 may also induce DNA lesions by an indirect, membrane- mediated mechanism. According to those authors, as a membrane-active agent, AFB1 may stimulate the arachidonic acid cascade and induce the formation of hydroperoxy-arachidonic acid derivatives. Degrada- tion products of hydroperoxy-arachidonic acid, such as active oxygen, lipid hydroperoxides and small alde- hydes, may induce chromosomal damage by indirect action. Anti-oxidants (e.g., superoxide dismutase) and inhibitors of the arachidonic acid cascade were found to suppress the clastogenic activity of AFB1, thereby confirming the involvement of intermediary free radicals in cellular damage [35].

Superoxides, normally produced in the unstressed, illuminated chloroplast, are generally scavenged, e.g., by glutathione, superoxide dismutase and ascorbates, to protect the photosynthetically-active plant cell [37]. It is possible that following AFB ~ exposure, aside from attack by AFB 1 or its highly reactive epoxide, the normal mechanisms of free radical scavenging become disrupted within the cell (both in the cytoplasm and within organelles). In this regard, it may not be pre- sumptive to assume that the deteriorative disruption of several membranes observed in AFB 1-treated cells may (in part) be explained in terms of free radical damage to the membranes. The early deteriorative alterations in chloroplast morphology (e.g., thylakoid disruption; membrane evagination) observed in AFB 1- sensitive tobacco callus cells [25] may be a manifesta- tion of either an excessive production of free radicals, or failure to effectively scavenge the free radicals normally produced.

The ribosomal subunit/polysome population was markedly affected by AFB1. There was a measured

dose-dependent decrease in the cytoplasmic ribosomal subunit/polysome population [24]. Disturbance of ribosome integrity is likely to result in an inhibition of protein synthesis. Many cellular reactions, catalysed by enzymes, will therefore be inhibited as result of deranged enzyme activity per se (e.g., by toxin binding to protein molecules), or from impaired de novo protein synthesis. As a result, general cell deterioration is likely to occur, largely as a result of failure to maintain normal cellular processes and possibly from failure to repair damage incurred (especially to the membranes). The indications are that the higher the AFB 1 concentration, the more exaggerated will be these effects and the less likely will repair processes be initiated. The recovery of tobacco calli (assessed as an increase in fresh mass accumulation following transfer to a toxin-free medium) previously treated with < 10 #g/ml AFB1 following toxin removal, would suggest that there may have be a temporary suppression of one or more metabolic processes (including inhibition of DNA replication and RNA and protein synthesis), and/or a stasis in the ability of callus cells to initiate repair mechanisms [24-25]. Failure of cells previously treated with AFB1 concentrations in excess of 10 #g/ml to recover, would indicate that lethal damage had occurred within these cells.

Mitochondrial morphological changes in cells of Zea mays root tips in response to toxin treatment could possibly be described as biphasic. Initially, following exposure to low doses of AFB 1, the matrix of several of these organelles appeared quite diffuse and generally devoid of Visible cristae. Normal mitochondria, with well developed cristae were also present. At higher toxin concentrations, however, mitochondria exhibiting this diffuse appearance were only rarely observed, while mitochondria with well-developed cristae pre- dominated. The most plausible explanation for this occurrence would be the apparent elimination of the mitochondria that had sustained injury during early toxin exposure. If this is indeed the case, then the presence of extensive intravacuolar membrane whorls may explain the autolytic fate of those mitochondria that had sustained precocious injury. The occurrence of normal mitochondria even at 25 pg/ml, however, would suggest perhaps that a certain population of these organelles may be resistant to the toxic effects of AFB1. Terao & Ueno (1978) have commented that the higher levels of free radical-scavenging enzymes in mitochondria may protect them to some extent from toxin attack. The inner mitochondrial membrane com- prises only about 25% phospholipid, and as such, the

potential for peroxidation of the membrane lipids may be reduced, particularly in the presence of active free radical scavengers.

This proposal of resistance of mitochondria (or at least a sub-population) is, however, in direct contradiction to the findings of Niranjan et al. (1982). Those authors found that rat hepatic mitochondrial DNA had a three to four-fold greater capacity for AFB1-DNA adduct formation than did nuclear DNA, resulting in pronounced inhibition of mitochondrial RNA and protein synthesis. Furthermore, the DNA lesions within mitochondria persisted for longer, possibly because of a lack of appropriate excision mechanisms [38]. Previ- ously, Niranjan & Avadhani (1980) had demonstrated that rat liver mitochondria contain a cytochrome P- 450 type of mono-oxygenase system that was capable of activating AFB1 to an electrophilic reactive form which could covalently modify mitochondrial DNA, RNA and protein. The results of Ch'ih & Devlin (1984), however, indicate that translocation of AFB1 within the hepatocyte follows the cytoplasm ---+ nucleus --+ mitochondria route, suggesting that mitochondrial damage and inhibitory responses arising from this damage may be detected only following nuclear alterations. By utilising an immunocytochemical localisation technique specific for AFBI, toxin could be localised in several compartments of the cells of Zea mays root tips and stem cells of Nicotiana tabacum plantlets [24, 25, 42]. The label was not associated specifically with any organelle-type, but large numbers of gold particles were found in the nucleus, vacuoles and the cytoplasm of these cells. On the other hand, only a few particles were apparently located within mitochondria and plastids, suggesting that in plant cells, most of these organelles are not primary targets for the toxin. These findings are, however, in contradiction of the findings of McLean et al. (1994a) that the alterations in chloroplast ultrastructure was an early event following AFB1 exposure. It is possible therefore that AFB1 may not be directly involved in effecting disruption of chloroplast integrity. The immunocytochemical localisation of AFB1 intravac- uolarly may have resulted from autophagy of badly affected mitochondria (and plastids). It is also possible that since this study was conducted following a con- tinuous supply of AFB1, removal of the source may result in redistribution of toxin molecules within the cell, possibly to organelles.

The accumulation of lipid bodies and their possible coalescence within the cells of maize root tips exposed to the higher doses of AFB~ would indicate

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a possible interference with lipid metabolism and/or impaired lipid transport within the cell. An additional consideration may be impaired mitochondrial capacity for lipid oxidation, resulting in the accumulation of lipid bodies. Increased numbers of lipid droplets in cells may also be explained in terms of localised membrane disruption, i.e., of the plasmalemma and/or of damaged organelles. It is interesting to note that a pathological feature of suspected cases of aflatoxicosis is the presence of fatty degeneration of the liver [43]. The presence of large quantities of lipid in the liver and hence within the hepatocytes of AFBl-treated animals, has been interpreted by Hamilton (1975) not as increased lipid synthesis, but as impaired intracellular transport. Tung et al. (1972), on the other hand, have expressed the opinion that AFB 1-induced altered lipid metabolism is a primary effect and not a secondary lesion arising from the general effects of AFBl on nucleic acid metabolism.

In the present investigation, nuclear alterations, except perhaps abnormal chromatin clumping or general loss of nucleoplasm, were not obvious features in the cells of Zea mays root tips exposed to concentrations up to 25 #g/ml of toxin. In maize callus [19, 21] and maize embryos treated with higher AFB~ concentrations (up to 1 #g/ml; data not presented), nuclear damage became a regular feature; Crisan (1973b) reported irregularly-shaped nuclei and abnormal nuclei in 100 /~g/ml AFBl-treated Lepidium sativum root cells. These nuclei exhibited a less granular nucleoplasm, an increase in interchromatin granules and the occurrence of ring-shaped nucleoli with macrosegre- gation (separation into granular and fibrillar components). Similar observations have been reported in the aflatoxin-induced changes in animal cells [46, 47]. It was suggested as early as 1965 [6] that, in the plant cell, aflatoxins may act at the nucleic acid level. Several researchers have reported on AFBI -induced inhibitory effects on mitosis and the development of abnormal anaphases [6, 8, 9, 48]. These changes are not unlike those reported in animal cells treated with AFBI [35, 49, 50]. Other mycotoxins have also been reported to interfere with mitosis in plant cells, resulting in abnormal metaphase chromosomes [51-53].

Hanchey (1981), in a review on ultrastructural effects induced by host-specific fungal toxins, has commented that nuclear changes have generally not been observed until the late stages of pathogenesis. It is probable that by the time severe nuclear alterations were visible, a chain of deteriorative events has already proceeded within the cell. It is not known whether

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AFB1 requires metabolic activation to its epoxide to exert its toxic effects in plant systems, although Tri- pathi & Misra (1981) have demonstrated the ability of

AFB~ to bind to DNA of Zea mays seedlings in vitro without metabolic activation. In animal systems, AFB1 (as with many noxious substances) requires metabolic activation to its 8,9-epoxide [33], achieved by a

cytochrome P-450-dependent, microsomally located

mono-oxygenase system. Irvin and Wogan (1984) have reported that in vivo

AFB1-DNA adduct formation in animal cells, rDNA contained four to five-fold more AFB1 residues than did any other nuclear DNA. The results of those authors support the hypothesis that rDNA regions are pref-

erentially accessible to carcinogen-induced alteration, which may result from the diffuse conformation of these transcribing genes. These are similar to the find-

ings of Yu (1983), where AFB1 was found to bind preferentially to physiologically active regions of rat liver nucleolar DNA. Yu (1977) had reported previously that RNA polymerase II activity was inhibited directly, as was impairment of the nucleolar DNA tem- plate in hepatocytes. Danley et al. (19 81 ) have reported a decrease in the RNA content of the nuclear fraction

of excised AFB 1-treated G[ycine max roots, providing additional evidence for an AFB 1-induced inhibition of RNA synthesis.

From the observations of the present investigation,

and studies involving the same AFB1 concentration range [23-25], it would appear that exposure to AFB1

initiates a cascade of metabolic reactions within the plant cell. These events are manifested as the degen- erative alterations observed within these ceils, ranging from plasmalemmal vesiculation, loss of polyri-

bosomes to nuclear abnormalities. Based on the (lack of) current knowledge regarding the mode of action on the plant cell, it is necessary to extrapolate from the literature pertaining to animal systems. While much of the AFB 1-induced cellular damage reported for animal cells is mirrored in plant cells, it is not known whether

the underlying metabolic factors effecting this damage are the same. Membrane damage, for example, may arise as a result of a number of reactions (lipid peroxi-

dation; binding to membrane proteins). For researchers interested in further understanding this aspect of AFB 1 toxicity, several options are available, ranging from assessment of free radical damage, epoxide formation and the involvement of cytochrome P-450s and glutathione S-transferases in the metabolism of AIq31.

Acknowledgements

The authors would like to thank the Electron Micro- scope Unit, Faculty of Medicine, University of Natal, for assistance with sectioning of material, and for the printing of micrographs. Funding for this research was

provided by the University of Natal Research Fund.

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Address for correspondence: Dr M. McLean, University of Natal, Department of Physiology, Faculty of Medicine, EO. Box 17039, Congella, 4031 South Africa Phone: (31) 260 4275; Fax: (31) 260 4455; E-mail: mclean@ med.und.ac-za.

aflatoxin b1-induced ultrastructural alterations in maturezea mays embryos

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