Jita Patra

A comparison of biochemical responses to oxidative and metal stress in seedlings of barley, Hordeum vulgare L.

Biochemical responses on the bases of activities of antioxidant enzymes; peroxidase, catalase, superoxide dismutase and glutathione reductase as well as estimations of total protein, lipid peroxidation and thiols in the form of protein, non-protein, glutathione and phytochelatin measured in growing seedlings of barley, Hordeum vulgare L., from Day 2 through 8 were compared following treatment of seeds for 2 h with oxidative agents, paraquat 5 x 10(-5), 10(-4), 10(-3) M, H2O2 10(-3), 5 x 10(-3), 10(-2) M and a metal salt, CdSO4 10(-5), 10(-4), 10(-3) M. A significant induction of all antioxidant enzymes along with an increase in the levels of protein, lipid peroxidation and glutathione was noted in response to oxidative stress, CdSO4 induced significant peroxidase and catalase activities but not superoxide dismutase. In a marked contrast from oxidative stress, CdSO4 decreased glutathione reductase activity as well as glutathione levels but increased phytochelatin level. The differential biochemical responses thus underlined the crucial involvement of glutathione and phytochelatin in the oxidative and metal-induced adaptive responses, respectively.

Differential induction of adaptive responses by paraquat and hydrogen peroxide against the genotoxicity of methyl mercuric chloride, maleic hydrazide and ethyl methane sulfonate in plant cells in vivo

Induction of adaptive response by conditioning doses of paraquat (PQ) and hydrogen peroxide (H2O2) in embryonic shoot cells of Hordeum vulgare and root meristem cells of Allium cepa was tested against the genotoxicity of challenge doses of methyl mercuric chloride (MMCl), maleic hydrazide (MH) or ethylmethane sulfonate (EMS). Plant tissue fixed at different recovery hours following the challenge treatments was analysed for cells with genotoxicity markers that include spindle or chromosome aberrations and micronuclei. The results provided clear-cut evidence that whereas H2O2 induced adaptive response for the chromosome damage caused by MMCl and MH, PQ induced the same for MMCl and EMS, but not for damage caused by MH. The findings pointed to the differences in the underlying mechanisms of oxidative responses induced by H2O2 and O2-.

Persistence and prevention of aluminium- and paraquat-induced adaptive response to methyl mercuric chloride in plant cells in vivo

Induction and persistence of adaptive response by aluminium (Al), 1 or 10 microM, and paraquat (PQ), 5 or 10 microM, against genotoxicity of methyl mercuric chloride (MMCl), 1.26 microM, a standard environmental genotoxin, was investigated in root meristem cells of Allium cepa. Subsequently, three metabolic inhibitors, namely, 3-aminobezamide (3-AB, 10 or 100 microM), an inhibitor of poly(ADP-ribose) polymerase (PARP) implicated in DNA repair and/or apoptosis, cycloheximide (CH, 0.1 or 1 microM), an inhibitor of protein synthesis, and buthionine sulfoximine (BSO, 100 microM or 1mM), an inhibitor of glutathione synthesis were tested for their ability to prevent the adaptive response induced by conditioning doses of Al, 10 or 100 microM; and PQ, 5 or 100 microM, against MMCl-challenge, 1.26 or 100 microM, in root meristems of A. cepa or embryonic shoots of Hordeum vulgare, respectively. The findings demonstrated that once triggered, the Al- or PQ-adaptive response to MMCl could persist for at least 48h in root meristems of A. cepa. Furthermore, the adaptive response could effectively be prevented by 3-AB, to a lesser degree by CH, and the least by BSO, suggesting primarily the involvement of PARP and implicating DNA repair in the underlying mechanisms of adaptive response in plant cells in vivo.

Persistence of cadmium-induced adaptive response to genotixicity of maleic hydrazide and methyl mercuric chloride in root meristem cells of Allium cepa L.: Differential inhibition by cycloheximide and buthionine sulfoximine

With an objective to determine the period of persistence of the metal-induced adaptive response to chemical mutagens and heavy metals, growing root meristems of Allium cepa were conditioned by cadmium sulfate (CdSO4), 4 x 10(-7) and 4 x 10(-6) M for 1 h and subsequently challenged by maleic hydrazide (MH), 5 x 10(-3) M or methyl mercuric chloride (MMCl), 1.26 x 10(-6) M for 3 h at different time intervals ranging from a few minutes to several hours following the conditioning dose. Root meristems, fixed at regular intervals during recovery from 6 to 48 h, were cytologically analysed for cells with micronuclei (MNC). The adaptive responses to MH and MMCl were observed as early as 5 min after the Cd-conditioning that persisted for at least 48 h. Metabolic inhibitors, cycloheximide (CH). 10(-7) M and buthionine sulfoximine (BSO), 10(-4) M administered either prior to or simultaneous with Cd-conditioning effectively prevented the adaptive response to MH. Whereas BSO, an inhibitor of phytochelatin synthesis, prevented the adaptive responses from 15 min to 8 h after the conditioning dose, CH an inhibitor of cytoplasmic protein synthesis prevented the same from 6 to 48 h. The findings underscored the differential roles of phytochelatins and proteins underlying the foregone metallo-adaptive response.

Cycloheximide and buthionine sulfoximine prevent induction of genotoxic adaptation by cadmium salt against methyl mercuric chloride in embryonic shoot cells of Hordium vulgare L

Presoaked seeds of barley Hordeum vulgare L. pretreated with cycloheximide (CH), 10(-6) M or bythionine sulfoximine (BSO), 10(-4) M, were exposed to methyl mercuric chloride (MMCl), 10(-4) M, with or without prior conditioning with cadmium sulfate (CdSO4), 10(-4) M. Subsequently as the seeds germinated the endpoints measured were mitotic index, cells with mitotic aberrations and micronuclei (MNC) in embryonic shoot cells fixed at 40, 44, 48 and 52 h of recovery. Indicated by the significant reduction (p < or = 0.05) of the yield of cells with aberrations or MNC, the results confirmed that CdSO4-conditioning triggered an adaptive response to MMCl-challenge. Pretreatments of CH and BSO, whereas they potentiated the genotoxicity of MMCl, significantly prevented (p < or = 0.05) the Cd-induced genotoxic adaptation. That underscores a possible involvement of proteins in addition to phytochelatins in the underlaying mechanisms.

Aluminium triggers genotoxic adaptation to methyl mercuric chloride and ethyl methane sulfonate, but not to maleic hydrazide in plant cells in vivo.

Non-toxic, conditioning doses of aluminium chloride were tested for induction of adaptive response to the genotoxic challenge doses of methyl mercuric chloride (MMCl), maleic hydrazide (MH) and ethyl methane sulfonate (EMS). Embryonic shoot cells of Hordeum vulgare and root meristem cells of Allium cepa were employed as the assay systems. Plant tissues fixed at different recovery hours following the challenge treatments with or without prior Al-conditioning were analyzed for cells with genotoxicity markers that include spindle and/or chromosome aberrations and micronuclei (MNC). The results provided evidence that Al(3+) triggered adaptive response that protected the plant cells from the genotoxicity of MMCl and EMS. Al(3+), however, failed to induce adaptive response against the genotoxicity of MH. A comparison of Al-induced adaptive response with that induced by heavy metals: Cd(2+), Cu(2+), Hg(2+), Ni(2+), Pb(2+), Zn(2+) and oxidative agents: hydrogen peroxide (H(2)O(2)) and paraquat (PQ) pointed to the similarity of Al-adaptive response to that of PQ rather than to other heavy metals or H(2)O(2). Al-induced adaptive response demonstrated in the present study to MMCl and EMS possibly involved antioxidant defense and DNA repair systems, respectively.

Induction of sister chromatid exchanges by heavy metal ,salts in root meristem cells ofAllium cepa L.

Four heavy metal salts, nickel sulphate, mercuric chloride, cadmium sulphate and zinc sulphate, were tested for induction of sister chromatid exchange (SCE) in root meristem cells ofAllium cepa. A simple modified Feulgen staining procedure was employed for SCE-analysis. Maleic hydrazide and paraquat were included for comparison. An evaluation of genotoxicity of the above test chemicals made on the basis of SCE-assay was found positive for all the test chemicals with exception of zinc sulphate which gave a weak positive result.

Differential antioxidant enzyme and thiol responses of tolerant and non-tolerant clones of Chloris barbata to cadmium-stress

Tolerant and non-tolerant clones of Chloris barbata Sw. obtained, respectively, from an erstwhile mercury contaminated solid waste dump site near a chloralkali plant and a non-contaminated (control) site were subjected to cadmium-stress by growing the rooted cuttings in water containing CdSO4, 13 and 130 µM. Differences between the two clones in their response to cadmium-stress were noted in root growth, and also with respect to certain biochemical parameters. Whereas catalase activity decreased and non protein-thiol levels increased in the non-tolerant clone, the level of protein-thiol alone increased significantly in the tolerant clone in response to cadmium-stress. No remarkable differences between the clones, however, were noted with respect to total soluble protein, peroxidase activity and lipid peroxidation. Remarkably the two clones responded differently to buthionine sulfoximine, an inhibitor of glutathione and/or phytochelatin synthesis, which inhibited root growth significantly in non-tolerant clone but not in the tolerant clone. Buthionine sulfoximine, nonetheless, could potentate cadmium toxicity in either of the clones, but more effectively in the tolerant clone. The high sensitivity of tolerant-clone to the combined treatment of BSO and Cd in the present study could, therefore, be attributed to the cumulative oxidative stress generated synergistically by BSO and Cd.