Chemico-Biological Interactions 113 (1998) 15 – 25
Induction of a SOS repair system in lysogenic
bacteria by zearalenone and its prevention by
vitamin E
L. Ghédira-Chékir a, K. Maaroufi a, A. Zakhama b, F. Ellouz a,
S. Dhouib a, E.E. Creppy c,*, H. Bacha a
a
Laboratoire de Biochimie et de Toxicologie Moléculaire, Faculté de Médecine Dentaire,
Rue A6icenne 5019, Monastir Tunisie, France
b
Laboratoire d’Anatomie Pathologique, Faculté de Médecine, Rue A6icenne 5019,
Monastir Tunisie, France
c
Laboratoire de Toxicologie et d’Hygiène Appliquée, Uni6ersité de Bordeaux II, 146 rue Léo-Saignat,
33076 Bordeaux Cedex, France
Received 13 August 1997; received in revised form 15 January 1998; accepted 20 January 1998
Abstract
Zearalenone (Zen) is an oestrogenic mycotoxin produced by several Fusarium species in
cereals. It induces modifications of haematological parameters in rats with cytotoxicity and
inhibition of macromolecular synthesis (nucleic acids and protein). Zen and its metabolites
have oestrogenic and anabolic activities and interact with human oestrogen receptors. Zen
and its metabolites showed a positive DNA damaging effect in recombination tests with
Bacillus subtilis. It induces sister chromatid exchange and chromosomal aberration in CHO
cells. Zen was found to be capable of inducing DNA-adduct formation in mouse liver. The
genotoxicity of Zen was questionable until the last decade when increasing data tended to
show this toxin to be genotoxic in vivo. However the mechanism of its genotoxicity and
mutagenicity has not been completely clarified. The present investigations were designed to
show whether Zen induces an SOS-DNA repair response in lysogenic bacteria which have an
integrated l-bacteriophage in their genome. Zen was found to be genotoxic in the bacterial
* Corresponding author.
0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-2797(98)00013-1
16
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
systems from a concentration of 1.50 mM and it was also bactericidal (IC50 = 1.45 mM). In
addition vitamin E (6.0–12.0 mM) added 1 h prior to the toxin proved to prevent both the
genotoxic and bactericidal effects of Zen. This vitamin could be active both as an antioxidant
and as a radical scavenger. The specificity of this prevention is probably due to the similarity
of structure between vitamin E and Zen. © 1998 Elsevier Science Ireland Ltd. All rights
reserved.
Keywords: Zearalenone; SOS repair system induction; Lysogenic bacteria; Vitamin E
1. Introduction
Zearalenone (Zen) (Fig. 1) is a non-steroidal oestrogenic mycotoxin produced as
a secondary metabolite by several species of Fusarium which are contaminants of
cereals all over the world [1 – 3]. The concentrations of this toxin can reach 289 mg/g
in human food [4,5]. Zen is able to bind to oestrogen receptors in mammary glands,
brain and liver [6 – 10]. Zearalenone has been implicated in numerous mycotoxicoses
in animals, especially in pigs [11], in which oestrogenic response was produced,
impairing the normal reproductive function, [11,12].
The LD50 24 h i.p. in the rat is 10 mg/kg [12]. Injected i.p. to the rat, Zen
produced modifications of haematological parameters [13]. Various studies have
shown that zearalenone was metabolised by two main pathways: reduction to a and
b isomers of zearalenol (Zel) and conjugation with glucuronic acid [14–16]. Studies
in various species, (rodent, pig and monkey) have shown that zearalenone and its
above quoted metabolites, have oestrogenic and anabolic activities [4,5,11,17–22].
Furthermore Miksicek (1994), has shown that zearalenone interacts with human
oestrogen receptors by competition with 17 b-estradiol, [23].
Fig. 1. Structure of zearalenone.
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
17
A limited number of studies of genotoxicity have been conducted with zearalenone. Zen and its metabolites showed a positive DNA damaging effect in
recombination tests with Bacillus subtilis [24]. It induced sister chromatid exchange
and chromosomal aberration in CHO cells, [25,26] and DNA-adducts, [27]. Interaction between the DNA and xenobiotics are considered to be the critical step in the
initiation of mutagenesis and carcinogenesis, [28,29]. Furthermore a good correlation was found between the number of DNA adducts formed and the frequency of
mutations and tumour incidence during chronic carcinogen exposure in rodents
[30].
To fully assess the genotoxic potential of zearalenone and its implication in
mutagenesis and carcinogenesis, we tested the SOS repair gene response in lysogenic
bacteria (E. coli which have an integrated l-bacteriophage in their genome) after
incubation with zearalenone. This response was measured by the bacterial lysis
induced by the release of l-bacteriophage as a consequence of DNA-damage
caused by Zen.
Since Li et al. (1991) have shown that vitamin E (a-tocopherol) in diet was
efficient in preventing lipid-peroxidation, [31] and since vitamin E, a zearalenonestructurally related compound was found to prevent the inhibition of DNA and
protein synthesis induced by Zen and its global cytotoxicity (unpublished data) and
to avoid DNA-adduct formation [32], we also investigated its preventive effect on
SOS repair system induced by Zen in lysogenic bacteria.
2. Materials and methods
2.1. Chemicals
Zearalenone and vitamin E, obtained from Sigma (St. Louis, MO) were dissolved
in ethanol in high stock concentrations (10 and 25 mg/ml, respectively), in order to
add a minimal volume to the assays. Concentrations of zearalenone used for
bacteriostatic test and IC50 determination varied from 0 to 2.50 mM. For lytic cycle
induction, zearalenone concentrations varied from 0.1 to 2.0 mM. Vitamin E
concentrations used were in the range of 4-fold that of the zearalenone concentration. Other reagents and solvents of analytical grade were provided by Merck,
Prolabo, Sigma and Difco, France.
2.2. Bacterial strain
Lysogenic bacterial strain used, E. coli C600, Thr, Leu, Thi, Lac Y, Ton A, Sup
E (Huynh et al., 1985), [33] was a gift from Dr H. Karoui (Institut Pasteur, Tunis).
2.3. Culture media
Luria broth medium, tryptone agar and soft agar were prepared according to
Boyer and Roulland-Dussoix (1969) [34].
18
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
2.4. Selection of the lysogenic bacteria and lysing process
The selection of the lysogenic bacteria was performed as previously described by
Huynh et al. (1985), [33]. Since l-bacteriophage contains the factor CI857, temperature-sensitive repressor, the simplest way to express the lytic cycle of the phage is to
induce the lysis of E. coli by a temperature shift from 32 to 41°C, thus inactivating
the repressor and releasing the l-bacteriophage [35].
Briefly, the selected lysogenic bacteria were incubated in liquid L.B. medium in the
presence or the absence of Zen (0.1–2.0mM) and/or vitamin E (at least 4-fold the
concentration of Zen, 6 or 12 mM) or both, for at least 20 h at 32°C. At determined
times (0 – 20 h), 200ml of each lysogenic bacterial culture were taken and loaded on
a wild type of E. coli in tryptone agar plate after required dilution, followed by 24
h incubation. After this additional incubation period, the lysis plaques were
numbered, their number corrected using the dilution coefficient and expressed as
mean 9 S.E.M. The values were analysed using the Wilcoxon rank sum test, [36].
3. Results
3.1. Bacteriostatic test and induction of the lytic cycle of the l-bacteriophage on E.
coli after incubation with Zen and/or 6itamin E
The Zen 50% inhibitory concentration (IC50) of the growth of E. coli C 600 strain,
as determined in the presence of increasing concentrations (0–2.50 mM) was 1.45
mM.
The Zen-induced lysogenic activity was first determined using 0.1–2.0 mM of the
toxin. It appeared that the induction of the lytic cycle of the l-bacteriophage in
lysogenic bacteria, occurred only at Zen concentrations of about 1.35 mM. Thus a
concentration of 1.50 mM of Zen was chosen for further investigations on the
induced lysogenic process. Indeed this concentration offered a good compromise
between the bactericidal activity and the induction of the lytic cycle of the
l-bacteriophage.
3.2. Induction of the SOS repair system by zearalenone
The data shown in Fig. 2 confirmed that zearalenone at the concentration of 1.50
mM inhibited bacterial growth as compared to the control. The bacterial multiplication was clearly slower with 1.50 mM of Zen as compared to the control and
reached a maximum at 6 h then the bacterial density decreases towards a minimum
at 20 h.
Zearalenone (1.50 mM) induced the lytic cycle of the l-bacteriophage at 32°C, the
lysis of lysogenic bacteria occurred approximately after 6 h of incubation and
reached a maximum after 12 h of incubation with the toxin (Table 1). As a control
test, a culture of lysogenic bacteria at 32°C in the absence of zearalenone, showed
no lysis after 20 h of incubation.
No lysis
No lysis
0
0
No lysis
0
No lysis
No lysis
0
0
No lysis
3
Incubation times (h)
No lysis
No lysis
3
2
No lysis
5
No lysis
No lysis
3
2
No lysis
6
No lysis
No lysis
10
30
No lysis
8
No lysis
No lysis
7009 12
499 2
No lysis
9
No lysis
No lysis
20509 70
6992
No lysis
10
No lysis
No lysis
24339 493
1909 14
No lysis
11
No lysis
No lyses
25509636
400938
No lysis
12
No lysis
No lysis
26009566
700952
No lysis
13
No lysis
No lysis
25509636
24009 566
15
No lysis
No lysis
26009566
24509636
20
Vitamin E was either added simultaneously or preincubated for 1 h before the addition of zearalenone.
The number of the lysis plaques was evaluated in the following conditions: selected lysogenic bacteria were incubated in liquid L.B. medium for at least 20 h at 32°C. At determined times (0 – 20 h), 200 ml
of each lysogenic bacterial culture was taken and loaded on a wild type of E. coli in tryptone agar plate after required dilution, followed by 24 h incubation. After this incubation period, the plaques of lysed
bacteria were numbered and their number corrected by the dilution coefficient.
The values presented as the mean 9S.E.M. were analysed using the Wilcoxon rank sum test.
Control (without Zen)
Control with Vitamin E (6 or 12 mM)
Zen (1.5 mM)
Zen (1.5 mM)+Vitamin E (6mM)
Vitamin E(6 mM) preincubated 1h + Zen (1.5
mM)
Plaques of lysis (×106/ml)
Table 1
l-Bacteriophage-induced lysis of E. coli (C600) in the presence or the absence of zearalenone (1.5 mM) and/or vitamin E (6 or 12 mM)
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
19
20
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
Fig. 2. Lysogenic bacteria (E. coli C600) growth in the presence of zearalenone (1.5 mM) ( —
)
" ) without zearalenone. The number of bacteria was evaluated in the
compared to the control ( —
following conditions: lysogenic bacteria (E. coli C600) were incubated in liquid L.B. medium for at least
20 h at 32°C. At determined times (0–20 h), 200 ml of the culture medium was taken and loaded on a
tryptone agar plate after required dilution (four different dilutions were performed), followed by 24 h
incubation. After this additional incubation period, the bacteria were numbered, their number corrected
by the dilution coefficients and expressed as the mean 9S.E.M. The values were analysed using the
Wilcoxon rank sum test.
The exponential phase of the multiplication of lysogenic bacteria in the presence
of zearalenone, ended after 5 h of incubation, whereas the stationary phase was
very brief and lasted only 1 h approximately. In the control test without zearalenone, the exponential phase was prolonged up to approximately 10 h.
The lysogenic bacteria were followed during 20 h of incubation and remained for
an additional 10 h period, at the stationary phase in the control. The rate of
bacterial growth in the presence of zearalenone decreased by approximately 57% as
compared to the control, as shown by the equation of the slope: r= y2 − y1/t2 − t1
in which r is the rate of multiplication, y2 is the number of bacteria at determined
time (t2) and y1 is the number of bacteria at determined time (t1) (Fig. 2).
The beginning of the bacterial lysis was perfectly correlated with the sudden
bactericidal activity and with the shortness of the stationary phase (Table 1).
3.3. Effect of 6itamin E added simultaneously to cultured bacteria with zearalenone
The addition of vitamin E (6 mM) simultaneously with zearalenone (1.50 mM)
induced the following changes as compared to incubation with Zen alone: the
bacterial growth was 25% higher, the maximal bacterial density was reached after
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
21
10 h of incubation instead of 6 h as compared to the control lacking the toxin.
However the bacterial density decreased after 11 h to reach the same minimal level
after 20 h. In these conditions, the Zen-induced lysis mediated by l-bacteriophage
release was obtained to the same extent with however a shift of 4 h as compared to
bacteria incubated with Zen alone (Table 1). There was no noticeable increase in
bacterial production with vitamin E alone as compared to the normal control.
The culture medium with vitamin E, prolonged the exponential phase of multiplication of lysogenic bacteria by approximately 4 h. The vitamin E did not modify
the length of the stationary phase which remained very brief, (about 1 h), (Fig. 3).
Fig. 3. Lysogenic bacteria (E. coli C600) growth in the presence of Zen (1.5 mM) and vitamin E (6 or
12 mM). Vitamin E was either added simultaneously (6 mM, —
), preincubated (6 mM, —
) or (12
mM, —
x ) for 1 h before the addition of zearalenone and compared to the control with vitamin E (6 mM)
" ). The number of bacteria was evaluated in the following conditions: lysogenic bacteria (E.
alone ( —
coli C600) were incubated in liquid L.B. medium for at least 20 h at 32°C. At determined times (0 – 20
h), 200 ml of the culture medium were taken and loaded on tryptone agar plate after required dilution
(four different dilutions were performed), followed by 24 h incubation. After this additional incubation
period, the bacteria were numbered, their number corrected by the dilution coefficients and expressed as
the mean9 S.E.M. The values were analysed using the Wilcoxon rank sum test.
22
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
The introduction of vitamin E in the culture medium of lysogenic bacteria (6
mM) at the same time than Zen (1.5 mM), did not prevent the induction of lysis
mediated by l-bacteriophage release (Fig. 3). In this case, the beginning of the
bacterial lysis was similarly correlated with the bactericidal effects and explained
the shortness of the stationary phase.
It is noticeable that the induction of the bacterial lysis in these conditions
occurred after approximately 11 h of incubation with the toxin and vitamin E. This
time shift of approximately 4 h was exactly the one observed for the bactericidal
activity and could be attributed to vitamin E (Fig. 3 and Table 1).
The control cultures of lysogenic bacteria at 32°C in the absence of zearalenone
and in the presence of vitamin E did not show any lysis after 20 h of incubation
(Table 1).
3.4. Effect of 6itamin E preincubated 1 h prior to the addition of zearalenone
A preincubation of 1 h of vitamin E (6 mM) prior to addition of Zen (1.5 mM),
totally prevented the Zen-induced release of l-bacteriophage (Fig. 3). However, the
growth of lysogenic bacteria was still 25% lower than the control. Again incubation
of lysogenic bacteria at 32°C with vitamin E but without Zen did not induce any
lysis.
In the presence of Zen (1.5 mM), bacteria previously incubated 1 h with vitamin
E (6 mM) showed an exponential phase of multiplication close to the control
without bactericidal activity within the incubation period of 20 h. However the
maximal bacterial density was lower by 18% as compared to the control with or
without vitamin E. When vitamin E (12 mM) was added instead of 6 mM, the
bacterial multiplication rate was totally restored (Fig. 3).
When vitamin E (6 mM) was added 1 h before the addition of Zen (1.5 mM), it
totally prevented the induction of the lysis by l-bacteriophage which was observed
with Zen alone or with Zen and vitamin E added simultaneously (Table 1).
4. Discussion
The E. coli SOS-DNA repair system could be induced by mutagenic substances,
UV irradiation or temperature shift from 32 to 41°C. This last induction process is
based on the inactivation of CI857 temperature-sensitive gene repressor by the shift
of temperature. DNA lesions leading to DNA repair can also induce the inactivation of this factor without any temperature shift. In this case the l-bacteriophage
incorporated in the genome of E. coli C600 is released and thus induces the lysis of
the host.
Our assumption was that zearalenone already found to induce DNA-adducts,
[27] could induce DNA-damage in E. coli, and then induce SOS-DNA repair and
lysis of lysogenic bacteria. This was confirmed by our results. Zearalenone at 1.5
mM and without any temperature shift induced an almost complete lysis of
lysogenic bacteria after 11 – 12 h of incubation (Table 1). This concentration of 1.5
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
23
mM of zearalenone is in the same range as the IC50 found (1.45 mM). This
concentration is by far higher than the IC50 value (20 mM) found for Zen in Vero cells
(unpublished results). This discrepancy could be due to differences between bacteria
and mammalian cells in the uptake and/or metabolism of zearalenone.
The abrupt arrest of the lysogenic bacteria proliferation (shown in Fig. 2) was
perfectly correlated with the lysis of these bacteria and could be explained by the
combination of bactericidal effect and bacterial lysis. In the control however, the
bacterial growth reached the maximum at 12–13 h and the bacterial density remained
to the same level until 20 h of incubation. The same was found after addition of
vitamin E alone (6 – 12 mM).
The simultaneous addition of vitamin E (6 mM) with Zen (1.5 mM), did not prevent
either the bactericidal or bacterial lysis induced by zearalenone, since bacterial
multiplication remained low and bacterial lysis still occurred (Table 1, Fig. 3). Vitamin
E in this case seemed solely to delay both the bactericidal effect and the induction of
bacterial lysis for approximately 4 h. This delay of the effects of Zen, could be the
result of a competition between Zen and vitamin E for possible binding to membranes
and uptake which could likely be due to the structural analogy between the toxin and
vitamin E.
When vitamin E (6 – 12 mM) was preincubated 1 h before addition of Zen, it
restored normal bacterial growth and totally prevented bacterial lysis, indicating that
the preincubation period of 1 h favoured the fixation and uptake of vitamin E.
Vitamin E could be acting as an antioxidant of oxygen reactive species, at the
membrane level or inside the bacteria. It could thus prevent the lipid peroxidation and
preserve the integrity of membranes, and DNA. This is in agreement with previous
data [31] on the prevention of genotoxic effects in general by vitamin E and that of Zen
in particular, [32]. Vitamin E could also act as a radical scavenger in preventing the
formation of free radicals possibly produced by Zen and/or its metabolites.
Altogether the present data confirmed that zearalenone is genotoxic, and a likely
mutagenic since it induced SOS-DNA repair system. They also showed a specific
prevention by vitamin E especially when present in the medium before the addition of
the toxin.
Acknowledgements
This research was supported by Ministère Tunisien de l’Enseignement Supérieur,
Secrétariat d’Etat de la Recherche Scientifique (PNM P95/SP12), Ministère Français
des Affaires Etrangères (Action intégrée, CMCU Tunisie/France) and Accords
INSERM-DGRST 1996 – 1997. The authors thank Dr H. Karoui (Institut Pasteur,
Tunis) for his help by the gift of lysogenic E. coli C600.
References
[1] G.A. Bennet, O.L. Shotwel, Zearalenone in cereals grains, J. Am. Oil Chem. Soc. 56 (1979)
812–819.
24
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
[2] J.C. Sutton, W. Balik, H.S. Funnett, Relation of weather variables to incidence of zearalenone in
corn in Southern Ontario, Can. J. Lant. Sci. 60 (1980) 149 – 155.
[3] H.L. Trenholm, B.K. Thompson, J.F. Standish, W.L. Seamen, Mycotoxins in feeds and feedstuffs,
in: P.M. Scott, H.L. Tranholm, M.D. Sutton (Eds.), Mycotoxins: A Canadian Perspective, National
Research Council of Canada, Ottawa, Ont. 22848., 1985, pp. 43 – 49.
[4] J.C. Kim, H.J. Kang, D.H. Lee, Y.W. Lee, T. Yoshizawa, Natural occurrence of Fusarium
mycotoxins (trichothecenes and zearalenone) in barley and corn in Korea, Appl. Environ. Microbiol. 59 (1993) 3798–3802.
[5] K.E. Yuwai, K.L. Rao, K. Singh, T. Tanaka, Y. Ueno, Occurrence of nivalenol, deoxynivalenol
and zearalenone in imported cereals in Papua New Guinea, Natural Toxins 2 (1994) 19 – 21.
[6] P. Boyd, J. Wittliff, Mechanism of Fusarium mycotoxin action in mammary gland, J. Toxicol.
Environ. Health 4p (1978) 1–8.
[7] D.T. Kiang, B.J. Kennedy, S.V. Lathre, C.J. Mirocha, Binding characteristics of zearalenone
analogues to estrogens receptors, Cancer Res. 38 (1978) 3611 – 3615.
[8] D.L. Greenman, R.G. Metha, J.L. Wittliff, Nuclear interactions of Fusarium mycotoxins with
estradiol binding sites in the mouse uterus, J. Toxicol. Environ. Health 5 (1979) 593 – 598.
[9] W. Powell-Jones, S. Raeford, G.W. Lucier, Binding properties of zearalenone mycotoxins to hepatic
estrogen receptors, Mol. Pharmacol. 20 (1981) 35 – 42.
[10] M. Kitagawa, F. Tashiro, Y. Ueno, Interaction between zearalenone, an estrogenic mycotoxin and
the estrogen receptor of the rat brain, Proc. Jpn. Assoc. Mycotoxicol. 15 (1982) 28 – 30.
[11] M. Etienne, M. Jemmali, Effects of Zearalenone (F2) on oestrus activity and reproduction in gilt,
J. Anim. Sci. 55 (1982) 1–10.
[12] H. Bacha, L. Chekir, F. Ellouz, R. Hadidane, E.E. Creppy, Effect of zearalenone on fertilisation
and gestation in rats, in: K.A. Scudanore (Ed.), Occurrence and Significance of Mycotoxins,
Proceedings of a UK Workshop, Central Science Laboratory, 1993, pp. 258 – 262.
[13] K. Maaroufi, L. Chekir, E.E. Creppy, F. Ellouz, H. Bacha, Zearalenone induces modifications of
haematological and biochemical parameters in rats, Toxicon 34 (1996) 535 – 540.
[14] K.H. Kiessling, H. Pettersson, Metabolism of zearalenone in rat liver, Acta Pharmacol. Toxicol. 43
(1978) 285–290.
[15] C.J. Mirocha, S.V. Pathre, T.S. Robinson, Comparative metabolism of zearalenone and transmission into bovine milk, Food Cosmet. Toxicol. 19 (1981) 25 – 30.
[16] Y. Ueno, S. Ayakis, N. Sato, T. Ito, Fate and mode of action of zearalenone, Ann. Nutr. Alim. 31
(1977) 935–948.
[17] P.H. Hidy, R.S. Baldwin, R.L. Greasham, C.L. Keith, J.R. Mc Mullen, Zearalenone and some
derivatives: production and biological activities, Adv. Appl. Microbiol. 22 (1977) 59 – 82.
[18] US Food and Drug Administration, Foods and Drugs, US Code Federal Regulations, Title 21, Part
522, 1980, pp. 192 and 239.
[19] T. Kuiper-Goodman, P.M. Scott, H. Watanabe, Risk assessment of the mycotoxin zearalenone,
Reg. Toxicol. Pharmacol. 7 (1987) 253 – 306.
[20] U. Thrane, Fusarium species and their specific profiles of secondary metabolites, in: J. Chelkowski
(Ed.), Fusarium Mycotoxins, Taxonomy and Pathogenicity, Elsevier, Amsterdam, 1989, pp. 199 –
225.
[21] IARC (International Agency for Research on Cancer), Some naturally occurring substances: food
items and constituents, heterocyclic aromatic amines and mycotoxins, in: IARC Monographs on the
Evaluations of the Carcinogenic Risk of Chemicals to Humans, vol. 56, IARC, Lyon, 1993, pp.
397–444.
[22] M. Etienne, J.Y. Dourmad, Effects of zearalenone or glucosinolates in the diet on reproduction in
sows: a review, Livestock Prod. Sci. 40 (1994) 99 – 113.
[23] R.J. Miksicek, Interactions of naturally occurring non steroidal estrogens with expressed recombinant human estrogen receptor, J. Steroid Biochem. Mol. Biol. 49 (1994) 153 – 160.
[24] Y. Ueno, K. Kubota, DNA attacking ability of carcinogenic mycotoxins in recombination-deficient
mutant cells of Bacillus subtilis, Cancer Res. 36 (1976) 445 – 451.
L. Ghédira-Chékir et al. / Chemico-Biological Interactions 113 (1998) 15–25
25
[25] R. Thust, S. Kneist, V. Huhne, Genotoxicity of Fusarium mycotoxins (nivalenol, fusarenon-X,
T2-toxin, and zearalenone) in Chinese hamster V79-E cells in vitro, Arch. Geschwulsforsch. 53
(1983) 9–15.
[26] S.M. Galloway, M.J. Amstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F.
Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson, E. Zeiger,
Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluation
of 108 chemicals, Environ. Mol. Mutagen. 10 (1987) 1 – 175.
[27] A. Pfohl-Leszkowicz, L. Chékir-Ghédira, H. Bacha, Genotoxicity of zearalenone, an oestrogenic
mycotoxin: DNA adduct formation in female mouse tissues, Carcinogenesis 16 (1995) 2315 – 2320.
[28] E.C. Miller, J.A. Miller, Searches for ultimate chemical carcinogens and their reactions with cellular
macromolecules, Cancer 47 (1981) 2327 – 2345.
[29] F.A. Beland, F.F. Kadlubar, Formation and persistence of arylamine DNA adducts in vivo,
Environ. Health Perspect. 62 (1985) 19 – 33.
[30] M.C. Poirier, F.A. Beland, DNA adduct measurements a tumour incidence during carcinogen
exposure in animals models: implications for DNA adduct-based human cancer risk assessment,
Chem. Res. Toxicol. 5 (1992) 749–755.
[31] D. Li, Y.M. Wang, R.G. Nath, S. Mistry, K. Randerath, Modulation by dietary Vitamin E of
I-compounds (putative indigenous DNA modifications) in rat liver and kidney, J. Nutr. 12 (1991)
65–71.
[32] Y. Grosse, L. Chékir-Ghédira, A. Huc, S. Obrecht-Pflumio, G. Dirheimer, H. Bacha, A. PfohlLeszkowicz, Retinol, ascorbic acid and a-tocopherol prevent DNA adduct formation in mice
treated with the mycotoxins ochratoxin A and zearalenone, Cancer Lett. 114 (1997) 225 – 229.
[33] T. Hyunh, R. Young, R. Davis, Constructing and screening cDNA libraries in l gt 10 and l gt 11,
in: D. Glover (Ed.), DNA Cloning: A Practical Approach, IRL Press, Oxford, 1984.
[34] H.W. Boyer, D. Roulland-Dussoix, A complementation analysis of the restriction and modification
of DNA in a Escherichia coli, J. Mol. Biol. 41 (1969) 459 – 472.
[35] G.D. Davis, M.D. Dibner, J.F. Battey, Preparation of l gt 10 and l gt 11 in cDNA cloning vectors,
in: L.G. Davis (Ed.), Basic Methods in Molecular Biology, Elsevier, New York, 1986, pp. 194 – 198.
[36] S.C. Gad, C.S. Neil, Statistics for toxicologists, in: W. Hayes, C.S. Neil, (Eds.), Principles and
Methods of Toxicology, Raven Press, New York, 1982, pp. 273 – 308.
.