Redox-dependent toxicity of diepoxybutane and mitomycin C in sea urchin embryogenesis
Ludmila G. Korkina,
Irina B. Deeva,
Antonella De Biase1,
Mario Iaccarino1,
Rahime Oral2,
Michel Warnau3 and
Giovanni Pagano1,4
Department of Molecular Biology, Russian State Medical University, 1 Ostrovityanova, Moscow, 117513, Russia,
1 Italian National Cancer Institute, G.Pascale Foundation, via M.Semmola, I-80131 Naples, Italy,
2 Department of Fisheries, Ege University, TK-35000 Bornova, Izmir, Turkey and
3 Marine Biology Laboratory, Free University of Brussels, 50, avenue F.D.Roosevelt, B-1050 Brussels, Belgium
 |
Abstract
|
---|
The effects and mechanisms of action of diepoxybutane (DEB) and mitomycin C (MMC) were investigated on sea urchin embryogenesis, (Sphaerechinus granularis and Paracentrotus lividus). DEB- and MMC-induced toxicity was evaluated by means of selected end-points, including developmental defects, cytogenetic abnormalities and alterations in the redox status [oxygen-dependent toxicity, Mn-superoxide dismutase (MnSOD) and catalase activities and glutathione (GSH) levels]. Both DEB and MMC exhibited developmental toxicity (at concentrations ranging from 3 x 105 to 3 x 104 M and 3 x 106 to 3 x 105 M, respectively) expressed as larval abnormalities, developmental arrest and mortality. The developmental effects of both compounds were significantly affected by oxygen at levels ranging from 5 to 40%. These results confirmed previous evidence for oxygen-dependent MMC toxicity and are the first report of oxygen dependence for DEB toxicity. Both DEB and MMC exerted significant cytogenetic abnormalities, including mitotoxicity and mitotic aberrations, but with different trends between the two chemicals, at the same concentrations as exerted developmental toxicity. The formation of reactive oxygen species was evaluated using: (i) luminol-dependent chemiluminescence (LDCL); (ii) reactions of the main antioxidant systems, such as GSH content and MnSOD and catalase activities. The results point to clear-cut differences in the effects induced by DEB and MMC. Thus, DEB suppressed GSH content within the concentration range 1073 x 105 M. The activity of catalase was stimulated at lower DEB levels (107106 M) and then decreased at higher DEB concentrations (
105 M). Increasing MMC concentrations induced LDCL and MnSOD activity (
106 M) greatly and modulated catalase activity (107 106 M). GSH levels were unaffected by MMC. The results suggest that oxidative stress contributes to the developmental and genotoxic effects of both toxins studied, although through different mechanisms.
Abbreviations: D, dead embryos/larvae; DEB, diepoxybutane; E(Ab+), percentage of embryos having
1 mitotic aberration; FSW, filtered seawater; GSH, reduced glutathione; GSSG, oxidized glutathione; IE, interphase embryos; LDCL, luminol-dependent chemiluminescence; M/A, metaphase:anaphase ratio; MMC, mitomycin C; MnSOD, Mn-superoxide dismutase; MPE, mitoses per embryo; N, normal larvae; P1, malformed larvae; P2, embryos/larvae unable to achieve the pluteus stage; PMA, phorbol 12-myristate 13-acetate; R, retarded larvae; ROS, reactive oxygen species; TMA, total mitotic aberrations.
 |
Introduction
|
---|
In order to maintain cellular functions, antioxidant enzymes such as catalase and Mn-superoxide dismutase (MnSOD) as well as a number of glutathione-metabolizing enzymes are a key part of the inducible pathways for antioxidant defense (1,2). On the other hand, all chemicals which can either metabolically generate reactive oxygen species (ROS) or modify intracellular thiols may cause a depletion of reduced glutathione (GSH) and/or an impairment of redox balance in favour of a prooxidant state (35).
Diepoxybutane (DEB) is one of the key metabolites of a genotoxic carcinogen, 1,3-butadiene (6,7), which is known to exert both cytogenetic and cytotoxic effects in mammalian cells (810). The main genotoxic metabolite in butadiene carcinogenicity was shown to be mediated by DEB, which derives from butadiene bioactivation by cytochrome P450 monoxygenase (7). The reproductive toxicity of 1,3-butadiene is also thought to be mainly caused by DEB (9). Little is known so far about the molecular mechanisms in DEB toxicity. Some studies of DEB toxicity in Fanconi's anaemia cells and human lung epithelial cells have focused on DEB-induced cross-linking effects (1113), as well as DEBadenine adduct formation in calf thymus DNA (14). Other findings have shown that DEB-induced toxicity may be modulated by DEB interaction with GSH (810). It was shown that DEB was enzymatically conjugated to GSH by glutathione S-transferases, thus GSH depletion led to a substantial increase in DEB-induced toxicity to mouse germ cells (9), suggesting that GSHDEB conjugation is the main metabolic pathway in DEB detoxification. Thus, expression of the glutathione S-transferase T1 gene (GSST1) has been related to resistance to DEB toxicity (10,15).
The anticancer antibiotic mitomycin C (MMC), similarly to a variety of quinones, possesses DNA damaging properties due to its well-documented prooxidant action (1618). Its bioactivation is effected by a number of enzymes, including NADPH-cytochrome P450 reductase, mitochondrial NADH-dehydrogenase, phagocyte NADPH-oxidase, DT-diaphorase and xanthine oxidase (1924). Thus, MMC can be reversibly reduced and oxidized through a one-electron reduction reaction, resulting in the formation of semiquinones and superoxide; the latter dismutates giving rise to hydrogen peroxide, ultimately producing hydroxyl radicals (5,17,21).
In the present work we investigated DEB- and MMC-induced developmental and cytogenetic toxicity to sea urchin embryos, as related to oxidative stress [assessed by luminol-dependent chemiluminescence (LDCL), GSH levels and catalase and MnSOD activities]. The use of sea urchin bioassays has been applied to a number of investigations on environmental toxicants, drugs and complex mixtures, providing a multiple set of toxicological end-points, encompassing developmental, reproductive and cytogenetic toxicity (2528). Moreover, we have reported the modulation of redox state in developing sea urchin embryos, both as a function of developmental stage and as a consequence of exposure to antioxidants or xenobiotics (28,29).
In the cases of DEB and MMC, the precise mechanisms of their toxicity are not fully understood and these xenobiotics are mostly referred to as `cross-linkers' (1113), by overlooking the published evidence for the diverse involvement of oxidative stress in their toxicity. Thus, it appeared worthwhile to verify whether, and to what extent, these agents were effective in affecting sea urchin embryogenesis via redox mechanisms.
The results corroborated previous evidence for redoxmediated superoxide-dependent mechanisms in MMC toxicity (1618,23,24) and pointed to a direct involvement of oxygen in DEB toxicity, which results in a number of effects, including GSH depletion and induction of catalase.
 |
Materials and methods
|
---|
Reagents
All reagents, media and enzymes were purchased from Sigma-Aldrich Co. (St Louis, MO and Milwaukee, WI) of the purest grade available.
Sea urchin bioassays
Adult urchins of the species Sphaerechinus granularis and Paracentrotus lividus were collected from Naples Bay by the staff of the Zoological Station. Gametes were obtained and embryos were reared as reported previously (26). The control embryos were reared either in natural filtered seawater (FSW, blank) or in FSW containing 2.5x104 M CdCl2 as a positive control (25). Exposure of embryos to DEB or MMC in FSW was carried out from the zygote stage (10 min after fertilization) up to the pluteus larval stage (72 h after fertilization). For the sperm pretreatment schedule, 50 µl sperm aliquots were suspended in 50 ml of FSW and exposed to the test agents for 120 min. Thereafter, 50 µl of sperm suspension was used to inseminate untreated eggs in 10 ml wells. Cultures were run in 6-well, 10 ml culture plates and incubated at 18 ± 1°C. Each experiment was replicated at least six times. Cultures were analysed microscopically for larval survival and morphology on living plutei, which were first immobilized by adding chromium sulphate (final concentration 104 M). In each culture, 100 plutei were scored for the determination of: (i) normal larvae (N); (ii) retarded larvae (R); (iii) malformed larvae (P1); (iv) embryos/larvae unable to achieve the pluteus stage (P2); (v) dead embryos/larvae (D). The results are expressed as percentages. The end-points of the sperm pretreatment schedules were: (i) fertilization rate (per cent fertilized eggs), demonstrating any spermiotoxic action; (ii) offspring quality, scored as described above for embryo exposure, demonstrating any transmissible damage from exposed sperm cells to embryos.
Oxygen treatment schedule
Gas mixtures containing nitrogen and various concentrations (5, 20 or 40%) of oxygen were bubbled through FSW 1 h prior to addition of the embryos, with or without test chemicals at the following concentrations: (i) 104 M DEB; (ii) 5x106 M MMC. The fertilized eggs were incubated throughout embryogenesis in FSW containing the same oxygen levels at normal pressure and a temperature of 18 ± 1°C. After a 72 h incubation the embryos were examined for developmental defects as described above.
Cytogenetic analysis
Cytogenetic determinations were carried out on 30 cleaving embryos from each of four replicate cultures, fixed in Carnoy's fluid 5 h after fertilization and stained with acetocarmine and rinsed in 20% acetic acid. The parameters analysed were quantitative and morphological abnormalities. Quantitative abnormalities were: (i) mean number of mitoses per embryo (MPE); (ii) per cent interphase embryos (IE); (iii) metaphase:anaphase ratio (M/A) (26). The morphological abnormalities scored were: (i) anaphase bridges; (ii) lagging chromosomes; (iii) acentric fragments; (iv) scattered chromosomes; (v) multipolar spindles. These were both scored individually and reported as total mitotic aberrations (TMA) per embryo. The percentage of embryos having
1 mitotic aberration [E(Ab+)] was also scored.
Reactive oxygen species formation
Spontaneous and phorbol 12-myristate 13-acetate (PMA)-stimulated release of ROS from embryos at the gastrula stage was performed as described previously (29). Briefly, 100 µl of embryo suspension was added to a polysterene cuvette containing 900 µl FSW with 5x105 M luminol and 10 µg/ml horseradish peroxidase. The cuvette was placed in a chemiluminometer (Lumi-Aggregometer; Chronolog, USA) and LDCL intensity was recorded continuously with mixing at room temperature. After a 5 min incubation, the maximum LDCL intensity was measured (spontaneous LDCL) and 10 µl of PMA solution in dimethylsulphoxide was added, at a final PMA concentration of 10 ng/ml. The amplitude of the LDCL response to PMA was recorded (PMA-activated LDCL). The results were expressed as mV/103 embryos or as per cent of the control value in an appropriate blank culture.
Preparation of samples for enzyme and GSH analysis
The embryos were disrupted at 2, 24 and 48 h after fertilization by sonication at the temperature of thawing ice (three pulses for 20 s each) and the cell debris was sedimented by centrifugation at 7000 g for 20 min. Then 10 ml of clear supernatant was divided into two samples for GSH and enzymatic assays. The samples for GSH analysis were deproteinized by adding trichloroacetic acid (10% solution, 2:1 v/v) and centrifugation at 3000 g for 10 min. The samples for enzyme analysis were mixed with ethanol and chloroform (8:2:1 v/v/v) and shaken vigorously for 5 min. Then, the upper clear wateralcohol layer was collected and stored at 20°C until analysis.
Enzyme and GSH determination
Total glutathione [GSH + oxidized glutathione (GSSG)] was measured as described previously (30). Then, 1015 µl aliquots of the samples were incubated with 1 ml of 0.2 M phosphate-buffered saline (pH 7.5) containing 6 mM 5,5'-dithio-bis-2-nitrobenzoic acid, 0.2 mM NADPH and 1 U/ml glutathione reductase for 30 min at 37°C and the absorbance at 412 nm was measured. Standard GSH samples prepared in 10% trichloroacetic acid were measured simultaneously. The lower limit of GSH + GSSG detection by this method was reported to be 110 ng/ml assay mixture.
The MnSOD activity was determined by measuring the suppression of superoxide-mediated lucigenin-amplified chemiluminescence produced during xanthine oxidation by xanthine oxidase in the presence of 1 mM NaCN (3133). The catalase activity was determined by the Aebi method (34). The enzyme activity and GSH content were determined using appropriate calibration curves. The levels of MnSOD and catalase were expressed in ng and µg/mg protein, respectively, and GSH content in µmol/mg protein. The protein content in the ethanolchloroform extract was determined by the Lowry method (35) using protein kits.
Statistical analyses
Differences in the distributions of actual frequencies observed for each larval class (N, R, P1, P2 and D) between controls and experimental groups or among experimental groups were tested for significance by the G procedure (adapted from the log-likelihood ratio test) for 2xk contingency tables (36).
Differences among the data concerning a single class of developmental defects induced by different concentrations of a given agent were tested for significance using the Tukey multiple comparison test (36). Significance level was always set at
= 0.05.
 |
Results
|
---|
DEB- and MMC-associated developmental toxicity
Both agents exerted dose-related developmental toxicity towards sea urchin embryos. As shown in Figure 1A, 3
x105M DEB significantly increased pre-pluteus arrest (P2 = 16.5 ± 3.6%) and larval malformations (P1 = 19.3 ± 4.8%) in S.granularis but did not affect embryogenesis in P.lividus. The frequency of developmental defects in both sea urchin species was increased as a function of DEB concentration (Figure 1A and B
) up to 3x104 M DEB, resulting in 100% embryonic mortality for both species studied. When exposed to MMC, developmental defects were observed at 105 M MMC in S.granularis and at 3x105 M MMC in P.lividus (Figure 2A and B
). There was no embryonic mortality caused by MMC in the range of concentrations studied. The embryotoxic effects of both DEB and MMC were significantly affected by increased O2 levels, as shown in Figure 3
. The frequency of DEB-induced developmental defects in P.lividus embryos increased steadily (P < 0.0006) as a function of O2 level (from ~50% embryonic malformations at 5% O2 up to 100% at 40% O2). No significant changes in the frequency of MMC-induced defects were observed between 5 and 20% O2, yet they were significantly increased (P < 0.006) up to ~80% of embryonic malformations at 40% O2. As observed in Figure 3
, neither hypoxic nor hyperoxic conditions in control cultures had any significant effects on the normal development of sea urchin embryos as compared with normoxic conditions.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1. DEB-induced developmental defects and mortality in sea urchin embryos following exposure from zygote to pluteus stage. (A) S.granularis; (B) P.lividus. P1, per cent larval malformations; P2, per cent developmental arrest at blastula/gastrula stage; D2, per cent embryonic mortality. Results are expressed as means ± SEM from a six replicate experiment.
|
|

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 3. Oxygen-dependent developmental toxicity in P.lividus larvae reared in either DEB (104 M) or MMC (3x106 M). Statistical analysis for the observed differences by the Tukey test gave P = 0.002 for DEB and P = 0.02 for MMC. In the control series, no significant differences were observed (P > 0.32). Regression analysis with replicates gave P = 0.0006 for DEB and P = 0.006 for MMC. Data are from two experiments for a total of 10 replicate lots.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2. MMC-induced developmental defects and mortality. Conditions as in Figure 1 . No mortality was observed up to 3x105 M MMC.
|
|
DEB- and MMC-induced cytogenetic defects
The evaluation of quantitative cytogenetic parameters in P.lividus embryos showed a strong dose-related mitotoxic action of DEB (at levels ranging from 3x105 to 5x104 M), causing a significant decrease (P < 0.0001) in the mean MPE; concurrently, the frequency of IE, i.e. lacking active mitoses, was steadily increased by DEB, as shown in Table I
. Unlike DEB, MMC resulted in a transient mitogenic effect at 5x106M (P < 0.005), whereas higher concentrations, up to 5x105 MMC, caused only a minor, non-significant decrease in MPE, and no changes in IE. The M/A was in no case significantly changed by either DEB or MMC.
Analysis of mitotic aberrations showed two distinct dose-related patterns for DEB versus MMC. Increasing DEB levels, up to 5x104 M, revealed a shift from anaphase aberrations (bridges and lagging chromosomes), observed at the lower DEB levels, to the more severe pattern of scattered chromosomes, which were significantly increased as a function of DEB concentration, along with the occurrence of mitotoxic effects. The frequency of TMA followed a non-monotonic trend, the maximum being reached at 3x104 M DEB. E(Ab+) was steadily increased by DEB. MMC failed to show any significant increase in mitotic aberrations, however, both TMA and E(Ab+) were maximal at MMC levels ranging from 5x106 to 105 M, then decreased up to 5x105 M MMC.
DEB and MMC effects on ROS production and antioxidant systems
The intensity of ROS production by sea urchin embryos, measured as HRP-enhanced LDCL, was sensitive to the two chemicals tested by different modes of action. It was shown that DEB co-incubated with S.granularis embryos slightly (510%) increased the LDCL response to PMA at DEB concentrations ranging from 107 to 105 M (Figure 4
). Likewise, DEB affected LDCL in P.lividus, however, the range of activating concentrations was shifted to the higher DEB levels (up to 3x105 M; data not shown). On the other hand, MMC induced a sharp concentration-dependent increase in LDCL (Figure 4
). The inducing effect of MMC was higher in the latest stages of embryogenesis (gastrula/pluteus stage) than in early embryogenesis (morula/blastula stage; data not shown).
DEB in the concentration range 107106 M significantly increased catalase activity, which was then inhibited at higher concentrations (>105 M) (Figure 5
). With a similar trend, 107 M MMC slightly increased catalase activity, with a subsequent inhibition at higher concentrations (Figure 5
).
The analyses of MnSOD activity in both S.granularis and P.lividus embryos showed that exposure to DEB did not affect the enzyme activity in the concentration range 1073x105 M (data not shown). In contrast, MMC resulted in a sharp dose-related enhancement of MnSOD activity (Figure 6
).
As shown in Figure 7
, GSH levels dropped in S.granularis embryos exposed to DEB in a dose-dependent manner; a less inhibitory effect was observed in P.lividus (data not shown). MMC (3x105 M) only induced non-significant deviations around normal GSH levels (data not shown).
 |
Discussion
|
---|
The occurrence of ROS-mediated mechanisms in DEB- and MMC-induced toxicity has been investigated in sea urchin embryogenesis, by comparing developmental and cytogenetic abnormalities with changes in the state of oxidative stress induced by each of the toxins. The two agents are often referred to as `cross-linkers' due to their recognized ability to form covalent bonds with DNA (12,13,37,38). Nevertheless, a consistent body of literature points to a redox imbalance as a major event involved in MMC toxicity, through quinone reduction coupled with ROS formation (1618,23,24,3941). Together, these studies showed that: (i) MMC-induced toxicity was dependent on O2 levels, consistent with the occurrence of a redox cycling mechanism, not to DNA cross-linking (18); (ii) MMC toxicity was removed by antioxidant enzymes (23,24,41,43), low molecular weight antioxidants (43), hypoxia (18) and overexpression of the thioredoxin gene (44).
DEB toxicity is also associated with oxidative stress, although through different mechanisms. First, the epoxide structure of DEB implies redox-mediated catalysis in the rearrangement of oxygen bonds (45). Similarly to MMC, DEB-induced cytogenetic damage is both decreased by low molecular weight antioxidants and by overexpression of the thioredoxin gene (44,46). A major role in modulating DEB toxicity has recently been reported for GSH. Thus, DEB-induced testicular toxicity in mice was enhanced by preliminary GSH depletion (9) and an increase in DEB-induced micronuclei was observed in GSST1-null mutant cells (defective in the glutathione S-transferase T1 gene) (10). On the other hand, expression of GSST1 in erythrocytes was shown to be a protective factor against DEB toxicity (15). Unlike MMC, however, no data were available as to any dependence on oxygen levels nor about any ad hoc study of DEB effects on cellular GSH level and catalase induction.
This background made it worthwhile to investigate DEB and MMC for their effects in the sea urchin bioassay system, with a major focus on: (i) oxygen-modulated toxicity; (ii) ROS formation; (iii) catalase and MnSOD activities; (iv) GSH levels.
Among the embryological and cytogenetic effects of DEB, the data showed: (i) a dose-related shift from developmental defects (malformations and developmental arrest) to embryonic mortality at DEB levels
104 M; (ii) a highly significant correlation of DEB-induced developmental toxicity and O2 levels; (iii) the occurrence of a severe dose-related mitotoxic effect; (iv) a grading in morphological abnormalities from anaphase aberrations to prevailing scattered chromosome figures concurrent with mitotoxic DEB levels.
The results of the redox activity markers showed that DEB (1073x105 M) modulated catalase activity and led to substantial suppression of GSH levels (Figures 5 and 7
). These data reflect a substantial inhibition of redox metabolic systems as part of a general effect on embryonic metabolism by embryotoxic DEB levels. A very intriguing fact is that before any visible developmental and/or cytogenetic abnormalities appear, subtoxic DEB levels, such as 106 M, induced an adaptive response in the main cellular antioxidant systems, such as catalase activity. Collectively, these findings support the involvement of oxidative stress in DEB toxicity, mainly by affecting the hydrogen peroxide balance through catalase and GSH (or rather ovothiols for sea urchin embryos), both being involved (enzymatically or non-enzymatically) in H2O2 detoxification. The possible scheme of DEB-induced oxidative stress in sea urchin embryos is as follows.
Normal embryogenesis:
hydrogen peroxide formation (47)
O2
H2O2 (embryonic oxidoreductase)
detoxification (47,48)
H2O2 + 2GSH (or ovothiols)
GSSG + H2O (ovoperoxidase)
DEB toxicity:
DEB detoxification by conjugation (1,10)
DEB + GSH
DEBGSH (glutathione S-transferase)
Excess H2O2
HRP-amplified LDCL elevation
catalase induction
Both morphological and biochemical effects were induced by MMC with patterns which differed substantially from DEB-induced effects. Developmental defects were induced in both S.granularis and P.lividus embryos reared at MMC levels ranging from 3x106 to 3x105 M, the latter concentration resulting in 100% developmental arrest (P2). Oxygen-dependent toxicity was also exerted by MMC, however, with a less sharp correlation than observed for DEB, in that 5 and 20% O2 displayed superimposable patterns and a significant increase in larval abnormalities was only observed when O2 levels were raised to 40%. Cytogenetic analysis of MMC-exposed embryos showed a bell curve for mitotic activity; an increased number of MPE was observed in the lower MMC level range (5x106105 M), along with a 4-fold, yet non-significant, increase in anaphase aberrations. Unlike DEB, no change was detected in the frequency of scattered chromosomes.
The effects of MMC on the markers of redox activity followed doseresponse trends that were mostly different compared with DEB. Unlike for DEB, MMC induced a dose-dependent increase in LDCL, up to 3-fold the control values. A similar trend was found for MnSOD: while DEB did not influence this enzyme activity, MMC induced it in a dose-dependent manner. This seems to be a consequence of embryonic superoxide overproduction due to redox cycling of MMC, first reduced to the semiquinone form by some as yet unknown embryonic oxidoreductase (47,48), according to the following reactions:
Where, Q, Q· and QH are the MMC quinone, semiquinone and hydroquinone forms.
Then, due to either enzymatic or spontaneous superoxide dismutation, additional hydrogen peroxide is formed:
This flux of H2O2 is responsible for the dose-related MMC-induced increase in HRP-amplified LDCL, for which H2O2 is a substrate, as well as for the slight transient reaction involving catalase. The primary step of O2· formation might be responsible for the adaptive increase in the activity of inducible MnSOD. The marginal increase in superoxide levels in embryos might cause the observed transient MMC-related increase in mitotic activity (5). No significant decrease in GSH was induced by MMC, unlike DEB, which resulted in a substantial depletion of embryonic GSH.
Altogether, the redox balance in sea urchin embryos was affected by both agents, but by entirely different metabolic pathways.
It is noteworthy that sea urchin sperm (from both species) were affected by neither DEB nor by MMC, even at concentrations causing acute effects on embryos (e.g. 100% mortality). The lack of effects on sperm included both fertilization success and offspring quality. Thus, one could infer that both DEB and MMC were unable to exert any direct effect on either sperm cell membranes or DNA, but required preliminary enzymatic bioactivation. Ovoperoxidase and glutathione S-transferase, which are present in the embryos but not in sperm cells, seem to be good candidates for bioactivation of the two toxins (47,48).
It should be noted that both DEB and MMC are utilized in diagnosing Fanconi's anaemia, as they act as specific clastogens in Fanconi's anaemia cells at concentrations which are ineffective in non-Fanconi's anaemia cells. It is debatable whether the primary defects in the different Fanconi's anaemia genetic subtypes are mainly related to alterations in DNA repair, cell cycle control or redox balance (37,38,4954). Previous reports provided consistent evidence for a major role of oxidative stress in Fanconi's anaemia phenotype (4244,46,5560), as reviewed by Pagano et al. (53). A recent paper by Mian and Moser (61) reported a peroxidase domain in the protein encoded by the Fanconi's anaemia group A gene. Moreover, our unpublished data demonstrated a number of alterations in redox balance consistent with GSH-modulated DEB-induced toxicity. The present study of DEB and MMC mechanisms of action corroborates the theory associating Fanconi's anaemia defects with a redox state imbalance.
In conclusion, the present study provides evidence for the involvement of redox mechanisms in DEB- and MMC-induced developmental and cytogenetic abnormalities in sea urchin embryos. In particular, the results confirm previous data pointing to a redox cycling mechanism in MMC embryotoxicity and provide novel evidence for the relevance of oxygen- and GSH-modulated DEB toxicity.
 |
Acknowledgments
|
---|
Thanks are due to Pasquale Sansone and his co-workers at the Zoological Station, Naples, for providing sea urchins. The help of Floriana Pagano in revising the English style is gratefully acknowledged. M.W. is a Research Associate of the National Fund for Scientific Research (Belgium). This study was supported by the European Commission, DGXII through the BIOMAR II Project (ENV4-CT96-0300) and by the Italian Association for Fanconi's Anaemia Research.
 |
Notes
|
---|
4 To whom correspondence should be addressed Email: gbpagano{at}tin.it

 |
References
|
---|
-
Pinkus,R., Weiner,L.M. and Daniel,V. (1995) Role of quinone-mediated generation of hydroxyl radicals in the induction of glutathione S-transferase gene expression. Biochemistry, 34, 8188.[ISI][Medline]
-
Sies,H. (1991) Oxidative stress: from basic research to clinical application. Am. J. Med., 91, 31S38S.[Medline]
-
Meister,A. (1994) Glutathione, ascorbate and cellular protection. Cancer Res., 54 (suppl.), 1969s1975s.[Medline]
-
Weiner,L.M. (1994) Oxygen radical generation and DNA scission by anticancer and synthetic quinones. Methods Enzymol., 233, 92102.[ISI][Medline]
-
Afanas'ev,I.B. (1991) Superoxide Ion: Chemistry and Biological Implications. CRC Press, Boca Raton, FL, Vol. 2.
-
Saranko,C.J. and Recio,L. (1998) The butadiene metabolite, 1,2:3,4-diepoxybutane, induces micronuclei but is only weakly mutagenic at lacI in the Big Blue Rat2 lacI transgenic cell line. Environ. Mol. Mutagen., 31, 3240.[ISI][Medline]
-
Seaton,M.J., Follansbee,M.H. and Bond,J.A. (1995) Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse and rat liver microsomes. Carcinogenesis, 16, 22872293.[Abstract]
-
Pacchierotti,F., Tiveron,C., Ranaldi,R., Bassani,B., Cordelli,E., Leter,G. and Spanò,M. (1998) Reproductive toxicity of 1,3-butadiene in the mouse: cytogenetic analysis of chromosome aberrations in first-cleavage embryos and flow cytometric evaluation of spermatogonal cell killing. Mutat. Res., 397, 5566.[ISI][Medline]
-
Spanò,M., Cordelli,E., Leter,G. and Pacchierotti,F. (1998) Diepoxybutane cytotoxicity on mouse germ cells is enhanced by in vivo glutathione depletion: a flow cytometric approach. Mutat. Res., 397, 3743.[ISI][Medline]
-
Vlachodimitropoulos,D., Norppa,H., Autio,K., Catalan,J., Hirvonen,A., Tasa,G., Uuskula,M., Demopoulos,N.A. and Sorsa,M. (1997) GSTT1-dependent induction of centromere-negative and -positive micronuclei by 1,2:3,4-diepoxybutane in cultured human lymphocytes. Mutagenesis, 12, 397403.[Abstract]
-
Auerbach,A.D. and Wolman,S.R. (1976) Susceptibility of Fanconi's anaemia fibroblasts to chromosome damage by carcinogens. Nature, 261, 494496.[ISI][Medline]
-
Ishida,R. and Buchwald,M. (1982) Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents. Cancer Res., 42, 40004006.[Abstract]
-
Vock,E.H., Lutz,W.K., Ilinskaya,O. and Vamvakas,S. (1999) Discrimination between genotoxicity and cytotoxicity for the induction of DNA double-strand breaks in cells treated with aldehydes and diepoxides. Mutat. Res., 441, 8593.[ISI][Medline]
-
Tretyakova,N., Sangaiah,R., Yen,T.Y., Gold,A. and Swenberg,J.A (1997) Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA. Chem. Res. Toxicol., 10, 11711179.[ISI][Medline]
-
Kligerman,A.D., DeMarini,D.M., Doerr,C.L., Hanley,N.M., Milholland,V.S. and Tennant,A.H. (1999) Comparison of cytogenetic effects of 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane in mouse, rat and human lymphocytes following in vitro G0 exposures. Mutat. Res., 439, 1323.[ISI][Medline]
-
Pritsos,C. and Sartorelli,A.C. (1986) Generation of reactive oxygen radicals through bioactivation of mitomycin antibiotics. Cancer Res., 46, 35283532.[Abstract]
-
Dusre,L., Covey,J.M., Collins,C. and Sinha,B.K. (1989) DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells. Chem. Biol. Interact., 71, 6378.[ISI][Medline]
-
Clarke,A.A., Philpott,N.J., Gordon-Smith,E.C. and Rutherford,T.R. (1997) The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking. Br. J. Haematol., 96, 240247.[ISI][Medline]
-
Bligh,H.F.J., Bartoszek,A., Robson,C.N., Hickson,I.D., Kasper,C.B., Beggs,J.D. and Wolf,C.R. (1990) Activation of mitomycin C by NADPH:cytochrome P-450 reductase. Cancer Res., 50, 77897792.[Abstract]
-
Joseph,P., Xu,Y. and Jaiswal,A.K. (1996) Non-enzymatic and enzymatic activation of mitomycin C: identification of a unique cytosolic activity. Int. J. Cancer, 65, 263271.[ISI][Medline]
-
Afanas'ev,I.B., Korkina,L.G., Suslova,T.B. and Soodaeva,S.K. (1990) Are quinones producers or scavengers of superoxide ion in cells? Arch. Biochem. Biophys., 281, 245250.[ISI][Medline]
-
Davies,K.J.A., Doroshow,J.H. and Hochstein,P. (1983) Mitochondrial NADH dehydrogenase-catalyzed oxygen radical production by adriamycin and the relative inactivity of 5-iminodaunorubicin. FEBS Lett., 153, 227230.[ISI][Medline]
-
Suzuki,K., Yamamoto,W., Park,J.S., Hanaoka,H., Okamoto,R., Kirihara,Y., Yorishima,T., Okamura,T., Kumazaki,T. and Nishiyama,M. (1999) Regulatory network of mitomycin C action in human colon cancer cells. Jpn. J. Cancer Res., 90, 571577.[ISI][Medline]
-
Wang,X., Doherty,G.P., Leith,M.K., Curphey,T.J. and Begleiter,A. (1999) Enhanced cytotoxicity of mitomycin C in human tumour cells with inducers of DT-diaphorase. Br. J. Cancer, 80, 12231230.[ISI][Medline]
-
Pagano,G., Esposito,A. and Giordano,G.G. (1982) Fertilization and larval development in sea urchins following exposure of gametes and embryos to cadmium. Arch. Environ. Contam. Toxicol., 11, 4755.[ISI][Medline]
-
Pagano,G., Cipollaro,M., Corsale,G., Esposito,A., Ragucci,E., Giordano,G.G. and Trieff,N.M. (1986) The sea urchin: bioassay for the assessment of damage from environmental contaminants. In Cairns,J.Jr (ed.) Community Toxicity Testing. American Society for Testing and Materials, Philadelphia, PA, pp. 6792.
-
Graillet,C., Pagano,G. and Girard,J.P. (1993) Stage-specific effects of teratogens on sea urchin embryogenesis. Teratog. Carcinog. Mutagen., 13, 114.[ISI][Medline]
-
Pagano,G., His,E., Beiras,R., De Biase,A., Korkina,L.G., Iaccarino,M., Oral,R., Quiniou,F., Warnau,M. and Trieff,N.M. (1996) Cytogenetic, developmental and biochemical effects of aluminum, iron and their mixture in sea urchins and mussels. Arch. Environ. Contam. Toxicol., 31, 466474.[ISI][Medline]
-
Pagano,G., Bonassi,S., DeBiase,A., Degan,P., Deeva,I.B., Doronin,Y.K., Iaccarino,M., Oral,R., Warnau,M. and Korkina,L.G. (1997) L-methionine induces stage-dependent changes of differentiation and oxidative activity in sea urchin embryogenesis. Pharmacol. Toxicol., 81, 134143.[ISI][Medline]
-
Tietze,F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem., 27, 502522.[ISI][Medline]
-
Misra,H. and Fridovich,I. (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for SOD. J. Biol. Chem., 247, 31703175.[Abstract/Free Full Text]
-
Hassan,H.M. and Fridovich,I. (1977) Regulation of the synthesis of superoxide dismutase in Escherichia coli. Induction by methyl viologen. J. Biol. Chem., 252, 76677672.[ISI][Medline]
-
Wong,G.H.W., Elwell,J.H., Oberley,L.W. and Goeddel,D.V. (1989) Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell, 58, 923931.[ISI][Medline]
-
Aebi,H. (1984) Catalase in vitro. Methods Enzymol., 105, 121126.[ISI][Medline]
-
Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265275.[Free Full Text]
-
Zar,J.H. (1996) Biostatistical Analysis, 3rd Edn. Prentice-Hall, Upper Saddle River, NJ.
-
Fujiwara,Y. (1982) Defective repair of mitomycin C crosslinks in Fanconi's anemia and loss in confluent normal human and xeroderma pigmentosum cells. Biochim. Biophys. Acta, 699, 217225.[ISI][Medline]
-
German,J. (1980) Chromosome-breakage syndromes: different genes, different treatments, different cancer. Basic Life Sci., 15, 429439.[Medline]
-
Dusre,L., Rajagopalan,S., Eliot,H.M., Covey,J.M. and Sinha,B.K. (1990) DNA interstrand cross-link and free radical formation in a human multidrug resistant cell line from mitomycin C and its analogues. Cancer Res., 50, 648652.[Abstract]
-
Belcourt,M.F., Hodnick,W.F., Rockwell,S. and Sartorelli,A.C. (1996) Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase. Proc. Natl Acad. Sci. USA, 93, 456460.[Abstract/Free Full Text]
-
Gutteridge,J.M.C., Quinlan,G.J. and Wilkins,S. (1984) Mitomycin C-induced deoxyribose degradation inhibited by superoxide dismutase. A reaction involving iron, hydroxyl and semiquinone radicals. FEBS Lett., 167, 3741.[ISI][Medline]
-
Hayashi,K. and Schmid W. (1985) Effect of oxidants and antioxidants on chromosomal breakage in Fanconi's anemia lymphocytes. Hum. Genet., 69, 6265.[ISI][Medline]
-
Bigelow,S.B., Rary,J.M. and Bender,M.A. (1980) The effect of superoxide dismutase, catalase and L-cysteine on spontaneous and on mitomycin C induced chromosomal breakage in Fanconi's anemia and normal fibroblasts as measured by the micronucleus method. Mutat. Res., 78, 5966.[ISI][Medline]
-
Ruppitsch,W., Meisslitzer,C., Hirsch-Kauffmann,M. and Schweiger,M. (1998) Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane. FEBS Lett., 422, 99102.[ISI][Medline]
-
Bartók,M and Láng,K.L. (1980) Oxiranes. In Patai,S. (ed.) The Chemistry of Functional Groups. Supplement E, Part 2. The Chemistry of Ethers, Crown Ethers, Hydroxyl Groups and their Sulphur Analogues. John Wiley & Sons, Chichester, UK, pp. 609673.
-
Dallapiccola,B., Porfirio,B., Mokini,V., Alimena,G., Isacchi,G. and Gandini,E. (1985) Effect of oxidants and antioxidants on chromosomal breakage in Fanconi's anemia lymphocytes. Hum. Genet., 69, 6265.[ISI][Medline]
-
Shapiro,B.M. (1991) The control of oxidant stress at fertilization. Science, 252, 533536.[ISI][Medline]
-
Turner,E., Hager,L.J. and Shapiro,B.M. (1988) Ovothiol replaces glutathione peroxidase as a hydrogen peroxide scavenger in sea urchin eggs. Science, 242, 939941.[ISI][Medline]
-
Carter,D.M. (1981) Human diseases characterized by heritable DNA instability. Birth Defects, 17, 117128.[ISI][Medline]
-
Carreau,M., Gan,O.I., Liu,L., Doedens,M., McKerlie,C., Dick,J.E. and Buchwald,M. (1998) Bone marrow failure in the Fanconi anemia group C mouse model after DNA damage. Blood, 91, 27372744.[Abstract/Free Full Text]
-
Kruyt,F.A., Dijkmans,L.M., Arwert,F. and Joenje,H. (1997) Involvement of the Fanconi's anemia protein FAC in a pathway that signals to the cyclin B/cdc2 kinase. Cancer Res., 57, 22442251.[Abstract]
-
Kupfer,G.M., Yamashita,T., Naf,D., Suliman,A., Shigetaka,A. and D'Andrea,A.D. (1997) The Fanconi anemia polypeptide, FAC, binds to the cyclin-dependent kinase, cdc2. Blood, 90, 10471054.[Abstract/Free Full Text]
-
Pagano,G., Korkina,L.G., Brunk,U.T., Chessa,L., Degan,P., Del Principe,D., Kelly,F.J., Malorni,W., Pallard',F., Pasquier,C., Scovassi,I., Zatterale,A. and Franceschi,C. (1998) Congenital disorders sharing oxidative stress and cancer proneness as phenotypic hallmarks: prospects for joint research in pharmacology. Med. Hypotheses, 51, 253266.[ISI][Medline]
-
Pagano,G., Zatterale,A. and Korkina,L.G. (1999) Mitomycin C-induced DNA damage in Fanconi anemia: cross-linking or redox-mediated effects? Blood, 93, 11161118.[Free Full Text]
-
Nordenson,I. (1977) Effect of superoxide dismutase and catalase on spontaneously occuring chromosome breaks in patients with Fanconi's anemia. Hereditas, 86, 147150.[ISI][Medline]
-
Takeuchi,T. and Morimoto,K. (1993) Increased formation of 8-hydroxydeoxyguanosine, an oxidative DNA damage, in lymphoblasts from Fanconi's anemia patients due to possible catalase deficiency. Carcinogenesis, 14, 11151120.[Abstract]
-
Degan,P., Bonassi,S., De Caterina,M., Korkina,L.G., Pinto,L., Scopacasa,F., Zatterale,A., Calzone,R. and Pagano,G. (1995) In vivo accumulation of 8-hydroxy-2'-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi's anaemia families. Carcinogenesis, 16, 735742.[Abstract]
-
Poot,M., Gross,O., Epe,B., Pflaum,M. and Hoehn,H. (1996) Cell cycle defect in connection with oxygen and iron sensitivity in Fanconi anemia lymphoblastoid cells. Exp. Cell Res., 222, 262268.[ISI][Medline]
-
Korkina,L.G., Samochatova,E.V., Maschan,A.A., Suslova,T.B., Cheremisina,Z.P. and Afanas'ev,I.B. (1992) Release of active oxygen radicals by leukocytes of Fanconi's anemia patients. J. Leukoc. Biol., 52, 357362.[Abstract]
-
Kruyt,F.A., Hoshino,T., Liu,J.M., Joseph,P., Jaiswal,A.K. and Youssoufian,H. (1998) Abnormal microsomal detoxification implicated in Fanconi anemia group C by interaction of the FAC protein with NADPH cytochrome P450 reductase. Blood, 92, 30503056.[Abstract/Free Full Text]
-
Mian,I.S. and Moser,M.J. (1998) The Fanconi anemia complementation group A protein contains a peroxidase domain. Mol. Genet. Metab., 63, 230234.[ISI][Medline]
Received February 24, 1999;
revised February 24, 1999;
accepted October 20, 1999.