- Email: [email protected]
Buthionine sulfoximine prevents the reduction of the genotoxic activity of maleic hydrazide by soil humic substances in Vicia faba seedlings
Mutation Research 438 Ž1999. 89–95
Buthionine sulfoximine prevents the reduction of the genotoxic activity of maleic hydrazide by soil humic substances in Vicia faba seedlings Antonio De Marco
a,)
, Claudio De Simone b, Claudia D’Ambrosio b, Malgorzata Owczarek a
a
b
Centro di Genetica EÕoluzionistica, C.N.R., UniÕersita` La Sapienza, Rome, Italy Istituto Sperimentale per lo Studio e la Difesa del Suolo, Õia Casette 1, P.O. Box 117, 02100 Rieti, Italy Received 16 June 1998; revised 20 October 1998; accepted 21 October 1998
Abstract A significant reduction of the genotoxic effects caused by herbicide maleic hydrazide ŽMH. in Vicia faba seedlings was observed to be induced by a growth step in an organic soil as well as by a pretreatment with highly purified humic substances. In addition, such protective activity was resulted quite similar to that observed when the conditioning pretreatment was carried out with metal salts, so suggesting the involvement of the GSH biosynthesis in determining the protective activity observed. In agreement with this hypothesis, a previous exposure to buthionine sulfoximine ŽBSO., an inhibitor of the phytochelatins production, through the inhibition of GSH synthesis, prevented the reduction of the genotoxic activity of MH. The findings provide evidence for the involvement of the GSH biosynthesis pathway in determining the antigenotoxic activity revealed and suggest a possible involvement of the phytochelatins in this process. However, yet to be clarified is whether the stimulation of GSH production results as a consequence of a nonspecific influence on the protein synthesis by humic substances or of its direct activation due to the presence, as contaminants, of some heavy metals in both organic soil and humic acids extracts. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Humic substance; Vicia faba; Maleic hydrazide; Phytochelatin; GSH
1. Introduction The soil organic matter can play an important role in modulating the behaviour and the biological activity of the pollutants in the environment w1x. In earlier papers, we observed lower clastogenic damage in )
Corresponding author. Istituto Sperimentale per lo Studio e la Difesa del Suolo, Via Casette 1, 02100 Rieti, Italy. Tel.: q39746-256126; Fax: q39-746-256129; E-mail: [email protected]
Vicia faba seedlings when they were treated with some herbicides in an organic soil, compared to those treated in a sandy soil w2,3x. Similar results were obtained also when only a growth step of the seedlings in the organic soil was previously carried out and they were transplanted and treated, in a sandy soil w4x, with maleic hydrazide ŽMH., whose genotoxicity in plants is well known w5x. These findings suggest that some components of the organic soils, such fulvic and humic acids, could be absorbed
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 5 4 - 5
A. De Marco et al.r Mutation Research 438 (1999) 89–95
90
by the plants and exert an anticlastogenic activity against chemical mutagens within the plant. In agreement with this hypothesis, we observed that a pretreatment with different humic substances significantly reduced the clastogenic activity of MH in plants successively treated in a sandy soil w6,7x. Moreover, natural occurring humic substances are recognized to inhibit the mutagenicity of some environmental mutagens in plant cells in vivo, as well as in mammalian cells in vitro w8–10x. Nevertheless, yet to be clarified is what component of the organic matter can exert the observed anticlastogenic activity. Some recent evidences suggest that several heavy metals can confer genotoxic adaptation to MH in different vegetal systems through the induction of phytochelatins w11–13x. Phytochelatins are the functional analogues of metallothione in animal cells and have been implicated in the detoxification mechanisms and homeostasis of heavy metals in plant cells w14,15x. The main objective of this work was to verify whether a pretreatment in a soil with high organic matter content as well as in solutions of humic acids extracted from the same soil induced in V. faba seedlings a reduction of the genotoxic activity of MH. In order to verify the involvement of the reduced glutathione ŽGSH. biosynthesis, as previously observed in metal-induced adaptative response, the effects of a preliminary exposure to buthionine sulfoximine ŽBSO., a specific inhibitor of this biosynthetic pathway, through the inhibition of the gglutamylcysteine synthetase w16x, were evaluated.
analysis by dispersion by sodium hexametaphosphate. Soil pH was obtained by using a glass electrode at 2.5:1 waterrsoil ratio Žvrw.. Total organic carbon was analyzed using the Walkley–Black method and total nitrogen by the Kjeldhal procedure. The cation exchange capacity ŽCEC. was determined by extraction with NH 4 OAc. The extraction as well as the purification procedures of the humic substances from the Vico soil are reported elsewhere w17x. The elemental analysis of the studied humic extracts can be summarized as follows: % C 51.6; % H 2.42; % N 4.14; % Ashes 1.0. Seeds of V. faba Žvar. minor . were allowed to germinate in aluminium basins each containing 485 g of sandy soil added with 130 ml of deionized water, and stored in a climatic chamber at 20 " 18C. After 48 h, the seedlings were taken out and submitted to different conditioning treatments. The protocol consisted of a 2 h conditioning exposure of the seedlings Ž25 seedlings for plate. in Petri plates containing 30 ml of 10y4 M cadmium sulfate ŽCdSO4 ; Sigma-Italy. or 10y4 M nickel chloride ŽNiCl 2 ; Sigma. or 500 mg ly1 humic acids extracted from the organic soil named Vico. In an another experiment, seedlings that were allowed to germinate for 24 h in the sandy soil, were successively submitted to a 48 h conditioning treatment in 240 g of Vico soil added with 150 ml of deionized water. In parallel experiments, all the conditioning treatments were preceded by an exposure to 10y3 M buthionine sulfoximine ŽBSO; Sigma. for 2 h followed by a wash in running tap water. All the samples subjected to different conditioning exposures were then transplanted in the sandy soil Ž485 g of soil and 130 ml of water, 25 seedlings for basin. for 24 h; successively they were again
2. Materials and methods The main chemical characteristics of the soils used in this work are displayed in Table 1. Particle size distribution of soils was determined by pipette
Table 1 Classification and physico-chemical characteristics of the two soils used Soil
Soil classification ŽUSDA Soil Taxonomy.
Horizon
pH
OM Ž%.
Hs a Ž%.
CEC b Žcmol kgy1 .
Clay Ž%.
Silt Ž%.
Sand Ž%.
Vico Sand
Typic hapludand Typic udifluvent
Bw c Ap d
6.2 7.6
6.95 0.10
4.86 nd
21.7 nd
5.8 4.4
20.8 5.3
73.4 90.3
a
Humic substances; b cation exchange capacity; c subsurface weathered horizon; d plowed surface horizon.
A. De Marco et al.r Mutation Research 438 (1999) 89–95
91
Fig. 1. Influence of BSO on genotoxic adaptation, as evaluated by micronucleate cells ŽMC. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 mg ly1 . against MH challenge carried out in water Ž10y4 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
transplanted in the sandy soil and treated with 5 = 10y5 M maleic hydrazide ŽMH; Sigma. for 48 h. In further experiment, the exposure to a higher dose Ž5 = 10y4 M. of MH was carried out for
shorter time Ž40 min. in Petri plates Ž30 ml of solution; 25 seedlings for plate., and the seedlings were successively transplanted for 48 h in the sandy soil.
Fig. 2. Influence of BSO on genotoxic adaptation, as evaluated by micronucleate cells ŽMC. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 mg ly1 . against MH challenge carried out in the sandy soil Ž10y5 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
92
A. De Marco et al.r Mutation Research 438 (1999) 89–95
Fig. 3. Influence of BSO on genotoxic adaptation, as evaluated by aberrant anatelophases ŽAA. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 ml ly1 . against MH challenge carried out in water Ž10y4 Mg.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
At the end of all the experiments, the seedlings were removed, their primary root length measured and the root tips fixed in ethyl alcohol and glacial acetic acid 3:1 Žvrv., Feulgen stained and squashed, according to the technique previously described w2x.
Appropriate negative as well as positive controls for each experimental step were simultaneously carried out. The genotoxic effects were assessed by following the frequency of micronucleated cells and aberrant anatelophases Žfragments, lagging chromo-
Fig. 4. Influence of BSO on genotoxic adaptation, as evaluated by aberrant anatelophases ŽAA. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 mg ly1 . against MH challenge carried out in the sandy soil Ž10y5 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
A. De Marco et al.r Mutation Research 438 (1999) 89–95
some and chromosomic bridges. in 30,000 cells Ž15 root tips, 2000 cells for each root tip.. Micronuclei were taken into consideration only when their diameter did not exceed 1r3 of the main nucleus and when they were localized within the cell wall in the cytoplasmatic area surrounding the main nucleus. Each experiment was carried out in duplicate. Data were analyzed by a one-way variance analysis and the means were compared using the Duncan’s multirange test at p s 0.01 level. In order to obtain a greater homogeneity among variances ŽCochran’s test., the data on micronucleated cells frequency were submitted to square-root transformation before the analysis. However, in figures, the original data are displayed.
3. Results A 2-h conditioning treatment with cadmium ŽCdSO4 ., or nickel ŽNiCl 2 ., or with a solution of humic acids Ž500 mg ly1 ., extracted from the Vico organic soil, reduced significantly Ž p - 0.01. the frequency of micronucleated cells ŽFigs. 1 and 2. and aberrant anatelophases ŽFigs. 3 and 4. in V. faba seedlings, treated with the herbicide MH at a dose
93
that resulted strongly genotoxic when added alone Ž10y4 M in water for 40 min or 5 = 10y5 M in the sandy soil for 48 h.. The genotoxic effects induced by MH alone resulted similar for both procedure of exposure. No significant differences in the reduction of the genotoxicity was observed when the challenge exposure with MH was carried out in water ŽFigs. 1 and 3. or in the sandy soil ŽFigs. 2 and 4.: these results indicate that the time of contact between the mutagen and the V. faba seedlings did not influence the effect of the conditioning exposure. A significant reduction of the frequency of micronucleated cells and aberrant anatelophases was obtained when the seedlings were submitted to a 48 h conditioning treatment in Vico soil and then they were transplanted in the sandy soil and here treated with MH Ž10y5 M. for 48 h ŽFig. 5.. Moreover, the data reported show that the conditioning treatments carried out in Vico soil or in humic acids solutions failed to protect the seedlings from the genotoxic effects induced by MH challenge when a treatment with BSO was carried out before the conditioning treatment. Similarly, a pre-exposition of the seedlings to 10y3 M buthionine sulfoximine ŽBSO. for 2 h also prevented the reduction of genotoxic effects of MH induced by a pretreatment
Fig. 5. Influence of BSO on genotoxic adaptation, as evaluated by micronucleated cells ŽMC. and aberrant anatelophases ŽAA. frequency, induced by a growth step in an organic soil ŽVico. against MH challenge carried out in the sandy soil Ž10y5 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
94
A. De Marco et al.r Mutation Research 438 (1999) 89–95
with cadmium and nickel, as indicated by the data on cells with micronuclei or aberrant anatelophases ŽFigs. 1–5.. BSO, when used alone, failed to reduce significantly the genotoxic activity of MH. No significant increases with respect to the controls in micronucleated cells and aberrant anatelophases frequency was found to be induced by all the conditioning treatments assayed ŽCdSO4 , NiCl 2 , humic acids and organic soil.. 4. Discussion Several authors found that some metals such as cadmium, nickel, chromium and zinc, when administered at low doses, are capable of exhibiting a protective activity against different chemical mutagens in both animal and vegetal systems w18,19x. Inducible adaptative responses have been widely attributed to the involvement of DNA repair processes as well as to induction of some antioxidant enzymes Ži.e., catalase, peroxidase and superoxide dismutase. or small molecules Ži.e., glutathione and a-tocopherol.. In particular, the involvement of the metallothioneins in animal cells, and of the phytochelatins in plant cells Žboth synthesized from glutathione., has been suggested in the adaptation process to heavy metals w15,20x. The data reported in this work show that a pretreatment with both organic soil Vico and humic acids extracted from the same soil, significantly reduced the genotoxic effects induced by MH. Similar results were also obtained when the conditioning pretreatment was carried out with no genotoxic doses of CdSO4 and NiCl 2 , so suggesting that similar processes could underlie reduction of the genotoxicity observed. Furthermore, several heavy metals have been widely reported to induce a genotoxic adaptation through mechanisms that involve the phytochelatins synthesis w12,13,20x. This finding underscores the involvement of the GSH biosynthesis in determining the antigenotoxic effects induced by soil humic substances. In agreement with this hypothesis, the buthionine sulfoximine ŽBSO., that is a specific inhibitor of the GSH synthesis, not only prevented the genotoxic adaptation induced by some metallic salts but also inhibited the protective activity carried out by both the growth step in Vico soil and the
humic acids extracted from the same soil. On the other hand, GSH, whose antigenotoxic features are well known w14,21x, is a precursor of the phytochelatins synthesis and the involvement of these molecules in the protective processes observed in this research could be the basis of further studies. This observation suggests that some components of soil organic matter, particularly the humic acids, can play a role in the stimulation of the GSH biosynthesis, even if yet to be clarified is whether this stimulation is a consequence of a nonspecific influence on the protein synthesis by humic substances, that are well known to influence a wide range of enzymatic activities w22,23x, or of the presence, as contaminants, of some heavy metals in both organic soil and humic acids extracts. However, it is to be noted that the possible contaminant presence of heavy metals in humic extracts is drastically reduced by extraction procedure because of the treatment with dilute solutions of HClrHF that dissolves hydrated clay minerals and forms complexes with di and trivalent cations w1x. However, in our experiments, the presence of residual fractions of heavy metals in purified humic materials cannot be left out at all. As a conclusion, we consider that further studies on this topic could be commended in order to clarify the role played by humic substances in determining the antimutagenic processes in soil environment and the mechanisms that underlie this protective activity. References w1x E.J. Stevenson, Humus Chemistry: Genesis, Composition and Reactions, 2nd edn., Wiley, New York, 1994. w2x A. De Marco, P. Boccardi, C. De Simone, A. Piccolo, M. Raglione, A. Testa, S. Trinca, Induction of micronuclei in Vicia faba root tips treated in different soils with herbicide alachlor, Mutat. Res. 241 Ž1990. 1–6. w3x A. De Marco, C. De Simone, M. Raglione, A. Testa, S. Trinca, Importance of the type of the soil for the induction of micronuclei and the growth of primary roots of Vicia faba treated with herbicides atrazine, glyphosate and maleic hydrazide, Mutat. Res. 279 Ž1992. 9–13. w4x A. De Marco, C. De Simone, M. Raglione, P. Lorenzoni, Influence of soil characteristics on the clastogenic activity of maleic hydrazide in root tips of Vicia faba, Mutat. Res. 344 Ž1995. 5–12. w5x B.A. Kihlman, S. Sturelid, Effects of caffeine on frequencies of chromosomal aberrations and sister chromatid exchanges induced by chemical mutagens in root tips of Vicia faba, Hereditas 88 Ž1978. 35–41.
A. De Marco et al.r Mutation Research 438 (1999) 89–95 w6x C. De Simone, A. Piccolo, A. De Marco, Effects of humic acids on the genotoxic activity of maleic hydrazide, Fresenius’ Environ. Bull. 2 Ž1993. 157–161. w7x C. De Simone, A. Piccolo, A. De Marco, C. D’Ambrosio, Antimutagenic activity of humic acids of different origin, 8th Meeting of the International Humic Substances Society, Wroclaw, Poland, 1996. w8x R. Cozzi, M. Nicolai, P. Perticone, R. De Salvia, F. Spuntarelli, Desmutagenic activity of natural humic acids: inhibition of MMC and MH mutagenicity, Mutat. Res. 299 Ž1993. 37–44. w9x T. Gichner, S.A. Badaev, F. Pospisil, J. Valeminsky, Effects of humic acids, para-aminobenzoic acid and ascorbic acid on the N-nitrosation of the carbamate insecticide propoxur and on mutagenicity of nitroso propoxur, Mutat. Res. 229 Ž1990. 37–41. w10x T. Sato, Y. Ose, H. Nagase, K. Hayase, Mechanism of desmutagenic effect of humic acid, Mutat. Res. 176 Ž1987. 199–204. w11x K.K. Panda, A.C.V. Subhadra, B.B. Panda, Effect of buthionine sulfoximine on cadmium induced adaptative response to maleic hydrazide in root meristematic cells of Allium cepa L., Ind. J. Exp. Biol. 32 Ž1994. 584–587. w12x J.C. Steffens, The heavy metal-binding peptides of plants, Annu. Rev. Plant Physiol. Mol. Biol. 41 Ž1990. 553–575. w13x A.V. Subhadra, B.B. Panda, Metal-induced genotoxic adaptation in barley Ž Hordeum Õulgare . to maleic hydrazide and methyl mercuric chloride, Mutat. Res. 321 Ž1994. 93–102. w14x R. Howden, C.R. Andersen, P.R. Goldsbrough, C.S. Cobbett, A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana, Plant Physiol. 107 Ž1995. 1067–1073. w15x W.E. Rauser, Phytochelatins and related peptides, Plant Physiol. 109 Ž1995. 1141–1149. w16x J. Patra, A.V. Subhadra, B.B. Panda, Cycloheximide and
w17x
w18x
w19x
w20x
w21x
w22x
w23x
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buthionine sulfoximine prevent genotoxic adaptation by cadmium salt against methyl mercuric chloride in embryonic shoot cells of Hordeum Õulgare L., Mutat. Res. 348 Ž1995. 13–18. A. Piccolo, S. Nardi, G. Concheri, Structural characteristics of humic substances as related to nitrate uptake and growth regulation in plant system, Soil. Biol. Biochem. 24 Ž1992. 373–380. W. Burkart, B. Ogorek, Genotoxic action of cadmium and mercury in cell cultures and modulation of radiation effects, Toxicol. Environ. Chem. 12 Ž1986. 173–183. R.A. Rieger, A. Michaelis, S. Takehisa, Involvement of phytochelatins in NiCl 2 -triggered protection against induction of chromatid aberrations by TEM and MH in Vicia faba root tips meristems, Mutat. Res. 244 Ž1990. 31–35. E. Grill, E.L. Winnacker, M.H. Zenk, Phytochelatins, a class of heavy metal binding peptides from plants are functionally analogous to metallothioneines, Proc. Natl. Acad. Sci. USA 84 Ž1987. 439–443. D. Anderson, Antioxidants defences against reactive oxygen species causing genetic and other damage, Mutat. Res. 350 Ž1996. 103–108. G. Concheri, S. Nardi, A. Piccolo, G. Dell’Agnola, N. Rascio, Effects of humic fractions on morphological changes related to invertase and peroxidase activities in wheat seedlings roots, in: N. Senesi, T. Miano ŽEds.., Humic Substances in the Global Environment and Implications for Human Health, Elsevier, Amsterdam, 1994, pp. 257–262. D. Vaughan, R. Malcolm, B.G. Ord, Influence of humic substances on biochemical processes in plants, in: D. Vaughan, R. Malcolm ŽEds.., Soil Organic Matter and Biological Activity, Martinus Nijhoff, Dordrecht, 1985, pp. 78– 108.
Buthionine sulfoximine prevents the reduction of the genotoxic activity of maleic hydrazide by soil humic substances in Vicia faba seedlings Antonio De Marco
a,)
, Claudio De Simone b, Claudia D’Ambrosio b, Malgorzata Owczarek a
a
b
Centro di Genetica EÕoluzionistica, C.N.R., UniÕersita` La Sapienza, Rome, Italy Istituto Sperimentale per lo Studio e la Difesa del Suolo, Õia Casette 1, P.O. Box 117, 02100 Rieti, Italy Received 16 June 1998; revised 20 October 1998; accepted 21 October 1998
Abstract A significant reduction of the genotoxic effects caused by herbicide maleic hydrazide ŽMH. in Vicia faba seedlings was observed to be induced by a growth step in an organic soil as well as by a pretreatment with highly purified humic substances. In addition, such protective activity was resulted quite similar to that observed when the conditioning pretreatment was carried out with metal salts, so suggesting the involvement of the GSH biosynthesis in determining the protective activity observed. In agreement with this hypothesis, a previous exposure to buthionine sulfoximine ŽBSO., an inhibitor of the phytochelatins production, through the inhibition of GSH synthesis, prevented the reduction of the genotoxic activity of MH. The findings provide evidence for the involvement of the GSH biosynthesis pathway in determining the antigenotoxic activity revealed and suggest a possible involvement of the phytochelatins in this process. However, yet to be clarified is whether the stimulation of GSH production results as a consequence of a nonspecific influence on the protein synthesis by humic substances or of its direct activation due to the presence, as contaminants, of some heavy metals in both organic soil and humic acids extracts. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Humic substance; Vicia faba; Maleic hydrazide; Phytochelatin; GSH
1. Introduction The soil organic matter can play an important role in modulating the behaviour and the biological activity of the pollutants in the environment w1x. In earlier papers, we observed lower clastogenic damage in )
Corresponding author. Istituto Sperimentale per lo Studio e la Difesa del Suolo, Via Casette 1, 02100 Rieti, Italy. Tel.: q39746-256126; Fax: q39-746-256129; E-mail: [email protected]
Vicia faba seedlings when they were treated with some herbicides in an organic soil, compared to those treated in a sandy soil w2,3x. Similar results were obtained also when only a growth step of the seedlings in the organic soil was previously carried out and they were transplanted and treated, in a sandy soil w4x, with maleic hydrazide ŽMH., whose genotoxicity in plants is well known w5x. These findings suggest that some components of the organic soils, such fulvic and humic acids, could be absorbed
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 5 4 - 5
A. De Marco et al.r Mutation Research 438 (1999) 89–95
90
by the plants and exert an anticlastogenic activity against chemical mutagens within the plant. In agreement with this hypothesis, we observed that a pretreatment with different humic substances significantly reduced the clastogenic activity of MH in plants successively treated in a sandy soil w6,7x. Moreover, natural occurring humic substances are recognized to inhibit the mutagenicity of some environmental mutagens in plant cells in vivo, as well as in mammalian cells in vitro w8–10x. Nevertheless, yet to be clarified is what component of the organic matter can exert the observed anticlastogenic activity. Some recent evidences suggest that several heavy metals can confer genotoxic adaptation to MH in different vegetal systems through the induction of phytochelatins w11–13x. Phytochelatins are the functional analogues of metallothione in animal cells and have been implicated in the detoxification mechanisms and homeostasis of heavy metals in plant cells w14,15x. The main objective of this work was to verify whether a pretreatment in a soil with high organic matter content as well as in solutions of humic acids extracted from the same soil induced in V. faba seedlings a reduction of the genotoxic activity of MH. In order to verify the involvement of the reduced glutathione ŽGSH. biosynthesis, as previously observed in metal-induced adaptative response, the effects of a preliminary exposure to buthionine sulfoximine ŽBSO., a specific inhibitor of this biosynthetic pathway, through the inhibition of the gglutamylcysteine synthetase w16x, were evaluated.
analysis by dispersion by sodium hexametaphosphate. Soil pH was obtained by using a glass electrode at 2.5:1 waterrsoil ratio Žvrw.. Total organic carbon was analyzed using the Walkley–Black method and total nitrogen by the Kjeldhal procedure. The cation exchange capacity ŽCEC. was determined by extraction with NH 4 OAc. The extraction as well as the purification procedures of the humic substances from the Vico soil are reported elsewhere w17x. The elemental analysis of the studied humic extracts can be summarized as follows: % C 51.6; % H 2.42; % N 4.14; % Ashes 1.0. Seeds of V. faba Žvar. minor . were allowed to germinate in aluminium basins each containing 485 g of sandy soil added with 130 ml of deionized water, and stored in a climatic chamber at 20 " 18C. After 48 h, the seedlings were taken out and submitted to different conditioning treatments. The protocol consisted of a 2 h conditioning exposure of the seedlings Ž25 seedlings for plate. in Petri plates containing 30 ml of 10y4 M cadmium sulfate ŽCdSO4 ; Sigma-Italy. or 10y4 M nickel chloride ŽNiCl 2 ; Sigma. or 500 mg ly1 humic acids extracted from the organic soil named Vico. In an another experiment, seedlings that were allowed to germinate for 24 h in the sandy soil, were successively submitted to a 48 h conditioning treatment in 240 g of Vico soil added with 150 ml of deionized water. In parallel experiments, all the conditioning treatments were preceded by an exposure to 10y3 M buthionine sulfoximine ŽBSO; Sigma. for 2 h followed by a wash in running tap water. All the samples subjected to different conditioning exposures were then transplanted in the sandy soil Ž485 g of soil and 130 ml of water, 25 seedlings for basin. for 24 h; successively they were again
2. Materials and methods The main chemical characteristics of the soils used in this work are displayed in Table 1. Particle size distribution of soils was determined by pipette
Table 1 Classification and physico-chemical characteristics of the two soils used Soil
Soil classification ŽUSDA Soil Taxonomy.
Horizon
pH
OM Ž%.
Hs a Ž%.
CEC b Žcmol kgy1 .
Clay Ž%.
Silt Ž%.
Sand Ž%.
Vico Sand
Typic hapludand Typic udifluvent
Bw c Ap d
6.2 7.6
6.95 0.10
4.86 nd
21.7 nd
5.8 4.4
20.8 5.3
73.4 90.3
a
Humic substances; b cation exchange capacity; c subsurface weathered horizon; d plowed surface horizon.
A. De Marco et al.r Mutation Research 438 (1999) 89–95
91
Fig. 1. Influence of BSO on genotoxic adaptation, as evaluated by micronucleate cells ŽMC. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 mg ly1 . against MH challenge carried out in water Ž10y4 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
transplanted in the sandy soil and treated with 5 = 10y5 M maleic hydrazide ŽMH; Sigma. for 48 h. In further experiment, the exposure to a higher dose Ž5 = 10y4 M. of MH was carried out for
shorter time Ž40 min. in Petri plates Ž30 ml of solution; 25 seedlings for plate., and the seedlings were successively transplanted for 48 h in the sandy soil.
Fig. 2. Influence of BSO on genotoxic adaptation, as evaluated by micronucleate cells ŽMC. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 mg ly1 . against MH challenge carried out in the sandy soil Ž10y5 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
92
A. De Marco et al.r Mutation Research 438 (1999) 89–95
Fig. 3. Influence of BSO on genotoxic adaptation, as evaluated by aberrant anatelophases ŽAA. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 ml ly1 . against MH challenge carried out in water Ž10y4 Mg.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
At the end of all the experiments, the seedlings were removed, their primary root length measured and the root tips fixed in ethyl alcohol and glacial acetic acid 3:1 Žvrv., Feulgen stained and squashed, according to the technique previously described w2x.
Appropriate negative as well as positive controls for each experimental step were simultaneously carried out. The genotoxic effects were assessed by following the frequency of micronucleated cells and aberrant anatelophases Žfragments, lagging chromo-
Fig. 4. Influence of BSO on genotoxic adaptation, as evaluated by aberrant anatelophases ŽAA. frequency, induced by CdSO4 Ž10y4 M., NiCl 2 Ž10y4 M. or humic substances Ž500 mg ly1 . against MH challenge carried out in the sandy soil Ž10y5 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
A. De Marco et al.r Mutation Research 438 (1999) 89–95
some and chromosomic bridges. in 30,000 cells Ž15 root tips, 2000 cells for each root tip.. Micronuclei were taken into consideration only when their diameter did not exceed 1r3 of the main nucleus and when they were localized within the cell wall in the cytoplasmatic area surrounding the main nucleus. Each experiment was carried out in duplicate. Data were analyzed by a one-way variance analysis and the means were compared using the Duncan’s multirange test at p s 0.01 level. In order to obtain a greater homogeneity among variances ŽCochran’s test., the data on micronucleated cells frequency were submitted to square-root transformation before the analysis. However, in figures, the original data are displayed.
3. Results A 2-h conditioning treatment with cadmium ŽCdSO4 ., or nickel ŽNiCl 2 ., or with a solution of humic acids Ž500 mg ly1 ., extracted from the Vico organic soil, reduced significantly Ž p - 0.01. the frequency of micronucleated cells ŽFigs. 1 and 2. and aberrant anatelophases ŽFigs. 3 and 4. in V. faba seedlings, treated with the herbicide MH at a dose
93
that resulted strongly genotoxic when added alone Ž10y4 M in water for 40 min or 5 = 10y5 M in the sandy soil for 48 h.. The genotoxic effects induced by MH alone resulted similar for both procedure of exposure. No significant differences in the reduction of the genotoxicity was observed when the challenge exposure with MH was carried out in water ŽFigs. 1 and 3. or in the sandy soil ŽFigs. 2 and 4.: these results indicate that the time of contact between the mutagen and the V. faba seedlings did not influence the effect of the conditioning exposure. A significant reduction of the frequency of micronucleated cells and aberrant anatelophases was obtained when the seedlings were submitted to a 48 h conditioning treatment in Vico soil and then they were transplanted in the sandy soil and here treated with MH Ž10y5 M. for 48 h ŽFig. 5.. Moreover, the data reported show that the conditioning treatments carried out in Vico soil or in humic acids solutions failed to protect the seedlings from the genotoxic effects induced by MH challenge when a treatment with BSO was carried out before the conditioning treatment. Similarly, a pre-exposition of the seedlings to 10y3 M buthionine sulfoximine ŽBSO. for 2 h also prevented the reduction of genotoxic effects of MH induced by a pretreatment
Fig. 5. Influence of BSO on genotoxic adaptation, as evaluated by micronucleated cells ŽMC. and aberrant anatelophases ŽAA. frequency, induced by a growth step in an organic soil ŽVico. against MH challenge carried out in the sandy soil Ž10y5 M.. Means followed by the same letters are resulted not significantly different as assayed with the Duncan’s test at p F 0.01 level. Standard errors are also displayed.
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with cadmium and nickel, as indicated by the data on cells with micronuclei or aberrant anatelophases ŽFigs. 1–5.. BSO, when used alone, failed to reduce significantly the genotoxic activity of MH. No significant increases with respect to the controls in micronucleated cells and aberrant anatelophases frequency was found to be induced by all the conditioning treatments assayed ŽCdSO4 , NiCl 2 , humic acids and organic soil.. 4. Discussion Several authors found that some metals such as cadmium, nickel, chromium and zinc, when administered at low doses, are capable of exhibiting a protective activity against different chemical mutagens in both animal and vegetal systems w18,19x. Inducible adaptative responses have been widely attributed to the involvement of DNA repair processes as well as to induction of some antioxidant enzymes Ži.e., catalase, peroxidase and superoxide dismutase. or small molecules Ži.e., glutathione and a-tocopherol.. In particular, the involvement of the metallothioneins in animal cells, and of the phytochelatins in plant cells Žboth synthesized from glutathione., has been suggested in the adaptation process to heavy metals w15,20x. The data reported in this work show that a pretreatment with both organic soil Vico and humic acids extracted from the same soil, significantly reduced the genotoxic effects induced by MH. Similar results were also obtained when the conditioning pretreatment was carried out with no genotoxic doses of CdSO4 and NiCl 2 , so suggesting that similar processes could underlie reduction of the genotoxicity observed. Furthermore, several heavy metals have been widely reported to induce a genotoxic adaptation through mechanisms that involve the phytochelatins synthesis w12,13,20x. This finding underscores the involvement of the GSH biosynthesis in determining the antigenotoxic effects induced by soil humic substances. In agreement with this hypothesis, the buthionine sulfoximine ŽBSO., that is a specific inhibitor of the GSH synthesis, not only prevented the genotoxic adaptation induced by some metallic salts but also inhibited the protective activity carried out by both the growth step in Vico soil and the
humic acids extracted from the same soil. On the other hand, GSH, whose antigenotoxic features are well known w14,21x, is a precursor of the phytochelatins synthesis and the involvement of these molecules in the protective processes observed in this research could be the basis of further studies. This observation suggests that some components of soil organic matter, particularly the humic acids, can play a role in the stimulation of the GSH biosynthesis, even if yet to be clarified is whether this stimulation is a consequence of a nonspecific influence on the protein synthesis by humic substances, that are well known to influence a wide range of enzymatic activities w22,23x, or of the presence, as contaminants, of some heavy metals in both organic soil and humic acids extracts. However, it is to be noted that the possible contaminant presence of heavy metals in humic extracts is drastically reduced by extraction procedure because of the treatment with dilute solutions of HClrHF that dissolves hydrated clay minerals and forms complexes with di and trivalent cations w1x. However, in our experiments, the presence of residual fractions of heavy metals in purified humic materials cannot be left out at all. As a conclusion, we consider that further studies on this topic could be commended in order to clarify the role played by humic substances in determining the antimutagenic processes in soil environment and the mechanisms that underlie this protective activity. References w1x E.J. Stevenson, Humus Chemistry: Genesis, Composition and Reactions, 2nd edn., Wiley, New York, 1994. w2x A. De Marco, P. Boccardi, C. De Simone, A. Piccolo, M. Raglione, A. Testa, S. Trinca, Induction of micronuclei in Vicia faba root tips treated in different soils with herbicide alachlor, Mutat. Res. 241 Ž1990. 1–6. w3x A. De Marco, C. De Simone, M. Raglione, A. Testa, S. Trinca, Importance of the type of the soil for the induction of micronuclei and the growth of primary roots of Vicia faba treated with herbicides atrazine, glyphosate and maleic hydrazide, Mutat. Res. 279 Ž1992. 9–13. w4x A. De Marco, C. De Simone, M. Raglione, P. Lorenzoni, Influence of soil characteristics on the clastogenic activity of maleic hydrazide in root tips of Vicia faba, Mutat. Res. 344 Ž1995. 5–12. w5x B.A. Kihlman, S. Sturelid, Effects of caffeine on frequencies of chromosomal aberrations and sister chromatid exchanges induced by chemical mutagens in root tips of Vicia faba, Hereditas 88 Ž1978. 35–41.
A. De Marco et al.r Mutation Research 438 (1999) 89–95 w6x C. De Simone, A. Piccolo, A. De Marco, Effects of humic acids on the genotoxic activity of maleic hydrazide, Fresenius’ Environ. Bull. 2 Ž1993. 157–161. w7x C. De Simone, A. Piccolo, A. De Marco, C. D’Ambrosio, Antimutagenic activity of humic acids of different origin, 8th Meeting of the International Humic Substances Society, Wroclaw, Poland, 1996. w8x R. Cozzi, M. Nicolai, P. Perticone, R. De Salvia, F. Spuntarelli, Desmutagenic activity of natural humic acids: inhibition of MMC and MH mutagenicity, Mutat. Res. 299 Ž1993. 37–44. w9x T. Gichner, S.A. Badaev, F. Pospisil, J. Valeminsky, Effects of humic acids, para-aminobenzoic acid and ascorbic acid on the N-nitrosation of the carbamate insecticide propoxur and on mutagenicity of nitroso propoxur, Mutat. Res. 229 Ž1990. 37–41. w10x T. Sato, Y. Ose, H. Nagase, K. Hayase, Mechanism of desmutagenic effect of humic acid, Mutat. Res. 176 Ž1987. 199–204. w11x K.K. Panda, A.C.V. Subhadra, B.B. Panda, Effect of buthionine sulfoximine on cadmium induced adaptative response to maleic hydrazide in root meristematic cells of Allium cepa L., Ind. J. Exp. Biol. 32 Ž1994. 584–587. w12x J.C. Steffens, The heavy metal-binding peptides of plants, Annu. Rev. Plant Physiol. Mol. Biol. 41 Ž1990. 553–575. w13x A.V. Subhadra, B.B. Panda, Metal-induced genotoxic adaptation in barley Ž Hordeum Õulgare . to maleic hydrazide and methyl mercuric chloride, Mutat. Res. 321 Ž1994. 93–102. w14x R. Howden, C.R. Andersen, P.R. Goldsbrough, C.S. Cobbett, A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana, Plant Physiol. 107 Ž1995. 1067–1073. w15x W.E. Rauser, Phytochelatins and related peptides, Plant Physiol. 109 Ž1995. 1141–1149. w16x J. Patra, A.V. Subhadra, B.B. Panda, Cycloheximide and
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