Exposure to nickel (Ni) at high concentrations can lead to production of reactive oxygen species (ROS) resulting in oxidative damage at the cellular level. We investigated the antioxidative responses of Nicotiana tabacum cv BY-2 cell suspension to Ni stress (0.075 and 0.75 mM NiCl2) over a 72 h period with special attention to potential alterations in isoenzymes of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR). Two main SOD isoenzymes were observed, a Mn-SOD (band I) and a Fe-SOD (band II), as well as one CAT isoenzyme and four GR isoenzymes. Activity staining analysis revealed that CAT activity plays a major role in the early response to Ni-induced oxidative stress, particularly when the Ni concentration used was low, whilst a specific GR isoenzyme appears to respond to the Ni-induced oxidative stress when a much higher Ni concentration was used to induce the stress for the same period of treatment. These results illustrate the importance and advantages of determining individual isoenzyme activities.A exposição ao níquel (Ni), em altas concentrações, pode levar à produção de espécies reativas de oxigênio (EAOs), resultando em danos oxidativos em nível celular. Foram investigadas as respostas antioxidativas de células em suspensão do cultivar BY-2 de Nicotiana tabacum submetidas ao estresse por Ni (0.075 e 0.75 mM de NiCl2) por 72 h, com atenção especial às alterações potencias em isoenzimas de superóxido dismutase (SOD), catalase (CAT) e glutatione redutase (GR). Duas principais isoenzimas de SOD foram observadas, uma Mn-SOD (banda I) e outra Fe-SOD (banda II), bem como uma isoenzima CAT e quatro isoenzimas de GR. As análises revelaram que a atividade de CAT tem papel principal no momento inicial de resposta ao estresse oxidativo induzido por Ni, particularmente, quando sua concentração foi mais baixa, enquanto uma isoenzima específica de GR parece responder a este estresse na concentração mais alta de Ni, no mesmo período de tratamento. Os resultados ilustram a importância e as vantagens de se determinar a atividade de isoenzimas individuais

Azevedo, Ricardo Antunes

Gratão, Priscila Lupino

Pompeu, Georgia Bertoni

Vitorello, Victor Alexandre

English

A exposição ao níquel (Ni), em altas concentrações, pode levar à produção de espécies reativas de oxigênio (EAOs), resultando em danos oxidativos em nível celular. Foram investigadas as respostas antioxidativas de células em suspensão do cultivar BY-2 de Nicotiana tabacum submetidas ao estresse por Ni (0.075 e 0.75 mM de NiCl2) por 72 h, com atenção especial às alterações potencias em isoenzimas de superóxido dismutase (SOD), catalase (CAT) e glutatione redutase (GR). Duas principais isoenzimas de SOD foram observadas, uma Mn-SOD (banda I) e outra Fe-SOD (banda II), bem como uma isoenzima CAT e quatro isoenzimas de GR. As análises revelaram que a atividade de CAT tem papel principal no momento inicial de resposta ao estresse oxidativo induzido por Ni, particularmente, quando sua concentração foi mais baixa, enquanto uma isoenzima específica de GR parece responder a este estresse na concentração mais alta de Ni, no mesmo período de tratamento. Os resultados ilustram a importância e as vantagens de se determinar a atividade de isoenzimas individuais.Exposure to nickel (Ni) at high concentrations can lead to production of reactive oxygen species (ROS) resulting in oxidative damage at the cellular level. We investigated the antioxidative responses of Nicotiana tabacum cv BY-2 cell suspension to Ni stress (0.075 and 0.75 mM NiCl2) over a 72 h period with special attention to potential alterations in isoenzymes of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR). Two main SOD isoenzymes were observed, a Mn-SOD (band I) and a Fe-SOD (band II), as well as one CAT isoenzyme and four GR isoenzymes. Activity staining analysis revealed that CAT activity plays a major role in the early response to Ni-induced oxidative stress, particularly when the Ni concentration used was low, whilst a specific GR isoenzyme appears to respond to the Ni-induced oxidative stress when a much higher Ni concentration was used to induce the stress for the same period of treatment. These results illustrate the importance and advantages of determining individual isoenzyme activities

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Pompeu et al.548Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.548-552, September/October 2008NoteANTIOXIDANT ISOENZYME RESPONSES TONICKEL-INDUCED STRESS IN TOBACCO CELLSUSPENSION CULTUREGeorgia Bertoni Pompeu1; Priscila Lupino Gratão2; Victor Alexandre Vitorello3; RicardoAntunes Azevedo2*1USP/CENA - Lab. de Ecologia Aplicada, Av. Centenário, 303 - 13416-700 - Piracicaba, SP - Brasil.2USP/ESALQ - Depto. de Genética, C.P. 83 - 13418-900 - Piracicaba, SP - Brasil.3USP/CENA - Lab. de Biologia Celular e Molecular.*Corresponding author <raazeved@esalq.usp.br>ABSTRACT: Exposure to nickel (Ni) at high concentrations can lead to production of reactive oxygenspecies (ROS) resulting in oxidative damage at the cellular level. We investigated the antioxidativeresponses of Nicotiana tabacum cv BY-2 cell suspension to Ni stress (0.075 and 0.75 mM NiCl2) overa 72 h period with special attention to potential alterations in isoenzymes of superoxide dismutase(SOD), catalase (CAT) and glutathione reductase (GR). Two main SOD isoenzymes were observed, aMn-SOD (band I) and a Fe-SOD (band II), as well as one CAT isoenzyme and four GR isoenzymes.Activity staining analysis revealed that CAT activity plays a major role in the early response to Ni-induced oxidative stress, particularly when the Ni concentration used was low, whilst a specific GRisoenzyme appears to respond to the Ni-induced oxidative stress when a much higher Ni concentrationwas used to induce the stress for the same period of treatment. These results illustrate the importanceand advantages of determining individual isoenzyme activities.Key words: antioxidant enzymes, catalase, nickel, superoxide dismutaseRESPOSTA DE ISOENZIMAS ANTIOXIDANTES AO ESTRESSEINDUZIDO POR NÍQUEL EM CULTURA DE CÉLULASEM SUSPENSÃO DE FUMORESUMO: A exposição ao níquel (Ni), em altas concentrações, pode levar à produção de espéciesreativas de oxigênio (EAOs), resultando em danos oxidativos em nível celular. Foram investigadas asrespostas antioxidativas de células em suspensão do cultivar BY-2 de Nicotiana tabacum submetidasao estresse por Ni (0.075 e 0.75 mM de NiCl2) por 72 h, com atenção especial às alterações potencias emisoenzimas de superóxido dismutase (SOD), catalase (CAT) e glutatione redutase (GR). Duas principaisisoenzimas de SOD foram observadas, uma Mn-SOD (banda I) e outra Fe-SOD (banda II), bem comouma isoenzima CAT e quatro isoenzimas de GR. As análises revelaram que a atividade de CAT tempapel principal no momento inicial de resposta ao estresse oxidativo induzido por Ni, particularmente,quando sua concentração foi mais baixa, enquanto uma isoenzima específica de GR parece respondera este estresse na concentração mais alta de Ni, no mesmo período de tratamento. Os resultadosilustram a importância e as vantagens de se determinar a atividade de isoenzimas individuais.Palavras-chave: enzimas antioxidantes, catalase, níquel, superóxido dismutaseINTRODUCTIONIt has been widely reported, particularly inmore recent years, the problems related to contami-nation of the environment with heavy metals. Suchcontamination is mainly due to anthropogenic activi-ties (Gratão et al., 2005). As a consequence, severalaspects of heavy metal research has gained importanceand special attention, resulting in a several fold increasein papers published in the literature describing the ef-fect of heavy metal-induced oxidative stress in plants,a trend that is being followed by the study of otherimportant elements (Vitória et al., 2001; Garcia et al.,2006; Gomes-Júnior et al., 2006a, 2006b; Lea &Azevedo, 2006; Gomes-Júnior et al., 2007; Lea &Azevedo, 2007), the effect on ultrastructure alterations(Vitória et al., 2006) and other aspects involving tech-niques of phytoremediation and heavy metal-soil-plantNickel-induced stress in tobacco 549Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.548-552, September/October 2008interactions (Fontes & Alleoni, 2006; Gonzaga et al.,2006; Mendes et al., 2006; Nascimento, 2006; Sonmezet al., 2006; Pereira et al., 2007).Plant responses to heavy metal exposure var-ies depending on plant species, tissue, stage of devel-opment, metal concentration and type of metal, trig-gering a series of defence mechanisms which involveenzymatic and non-enzymatic components (Gratão etal., 2005). In addition to the use of plant seedlings oradult plants, another interesting approach has been theuse of in vitro cell cultures (Gomes-Júnior et al., 2007),since some of these factors can be controlled better.Most of the published reports have presenteddata on total enzyme activity and isoenzyme activityprofiles using non-denaturing PAGE. Our group hasconcentrated on the study of antioxidant enzyme re-sponses to several distinct heavy metals in a series ofplant species. We now report the effect of Ni on theactivity of three important antioxidant enzymes in to-bacco cell suspension culture. Such a study allows thebetter understanding of specific isoenzyme responseswhich is not possible if only total enzyme activity isdetermined.MATERIAL AND METHODSTobacco BY-2 (Nicotiana tabacum L. cv.Bright Yellow 2) cells were cultured as described byVitorello & Haug (1996). Cultures were grown in thedark on a refrigerated rotary shaker (MA830/A,MARCONI, Brazil) at 160 rpm at 27ºC and growth wasevaluated by packed cell volume following centrifuga-tion at 500 rpm for 5 min. BY-2 cells were maintainedin liquid medium for seven days and 3 mL of cell in-oculum were subcultured into new medium (50 mL).Two-day-old cell cultures (beginning of log phase ofgrowth) were submitted to preliminary trials on the ef-fect of several NiCl2 concentrations (0, 0.05, 0.075,0.1, 0.15, 0.2, 0.5, 0.75, 1 and 2 mM) for up to 72 h(beginning of stationary phase). Two concentrationswere chosen for the main experiments, 0.075 mMNiCl2, which exhibited growth and cell viability levelssimilar to the control, and 0.75 mM NiCl2, which ex-hibited about half the growth and cell viability at 72 hof treatment when compared to the control (data notshown). Two-day-old cell cultures were submitted tothe treatments and cells were harvested at differentperiods following exposure (12, 36 and 72 h), weresuction-dried and weighed for cell mass determinationand storage at -80°C for further analyses.The following steps were carried out at 4°Cunless stated otherwise. The BY-2 cells were homog-enized (2:1 buffer volume: fr. wt) in a mortar with apestle with 100 mM potassium phosphate buffer (pH7.5) containing 1 mM ethylene-diaminetetra-acetic acid(EDTA), 3 mM DL-dithiothreitol and 5% (w/v) in-soluble PVPP (Azevedo et al., 1998). The homogenatewas centrifuged at 15,000 rpm for 30 min and the su-pernatant was kept stored in separate aliquots at -80°C,prior to non-denaturing PAGE and SOD, CAT and GRanalyses.SOD, CAT and GR non-denaturing PAGE ac-tivity determinations, electrophoresis buffers and gels,and SOD isoenzyme classification were carried out asdescribed by Gomes-Júnior et al. (2007). Briefly, elec-trophoresis was carried out in 8% polyacrylamide non-denaturing gels and a constant current of 20 mA/gelwas applied for 8 h (CAT gel) or 4h (SOD and GRgels) and the temperature maintained at 4°C. Equalamounts (60 mg) of protein were loaded on to eachgel lane.Isoenzyme profiles were also subjected to adensitometric analysis for band intensity through theuse of Kodak Digital Science - 1D - Image AnalysisSoftware version 3.0.1. in a Power Look 1120 imag-ine system (Umax Technology, Texas, U.S.A).Protein concentration for all samples was de-termined by the method of Bradford (1976) using bo-vine serum albumin as a standard in a Lambda 40 spec-trophotometer (Perkin-Elmer Corporation, USA).The experimental design was randomized withthree replicates for each flask/treatment/time interval.The results of the densitometric analysis were ex-pressed as mean of three independent replicates ofCAT, GR and SOD for one of the experiments. Thegel images shown are from one of the replicates.RESULTS AND DISCUSSIONIn this study, SOD, CAT and GR isoenzymebands were observed following staining of native PAGEgels for enzyme activity of tobacco BY-2 cells sub-jected to Ni-induced oxidative stress (Figure 1). Bandintensity changes were also assessed based on a den-sitometry analysis (Table 1). Tobacco BY-2 cell cul-tures revealed two SOD, four GR and one CAT isoen-zymes (Figure 1). SOD bands were classified, accord-ing to the inhibition patterns to hydrogen peroxide andKCN, as Mn-SOD (SOD I - resistant to both inhibi-tors) and Fe-SOD (SOD II - inactivated by one of theinhibitors) (Figure 2), which have been shown to belocated in distinct cell compartments in plants species.The tobacco SOD I is likely to be located in the mito-chondria whereas SOD II in the plastid.SOD activity staining did not reveal any spe-cific isoenzyme alteration, but there were changes inactivity, particularly at 36 h of exposure to 0.075 mMNiCl2 when both SOD bands exhibited increases in ac-Pompeu et al.550Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.548-552, September/October 2008emyzneosIlCiNMm570.02lCiNMm57.02h21 h63 h27 h21 h63 h27IDOS 54.1 13.29 5.2- 76.1 95.9- 19.1IIDOS 90.5 87 65.3 65.2 59.1 8.2-ITAC 63.04 53.95 * 81.93- 9.14- *IRG * * * * 07.3-IIRG * * * ** ** *IIIRG 14.2 72.3- 63.4- 09.1 58.1 24.3-VIRG 56.4- 51.1- 39.4- 57.5 21.1- 34.6-Table 1 - SOD, CAT and GR densitometry analysis of non-denaturing PAGE presented in Figure 1. The pixel intensity of eachband was used to calculate the relative change (%) with respect to the control (zero Ni) of each exposure period.*band not was identified and **band was identified.tivity. Apart from these increases, SOD activity wasvery similar to control levels in both concentrationstested (Figure 1A). SOD is widely distributed amongO2-consuming organisms and is responsible for thedismutation of O2•- into H2O2, and therefore influenc-ing the concentration of O2•- and H2O2. SOD isoen-zymes are compartmentalized in higher plants and threeisoenzymes have been detected in plants, which areclassified according to their metal cofactor; Mn, Feand Cu/Zn (Gratão et al., 2005). Mn-SODs are locatedin the mitochondria and peroxisomes, Fe-SOD hasbeen shown to be associated with the chloroplasts(Gratão et al., 2005), while the Cu/Zn-SODs are lo-cated in the cytosol, chloroplasts and peroxisomes(Gratão et al., 2005). SOD activity responses to NiFigure 2 - SOD isoenzyme classification of tobacco BY-2 cellcultures. Lane 1, control SOD activity; lane 2, plus2 mM potassium cyanide and lane 3, plus 5 mMhydrogen peroxide.Figure 1 - Activity staining for (A) superoxide dismutase (SOD), (B) catalase (CAT) and (C) glutathione reductase (GR) following non-denaturing PAGE of extracts of tobacco BY-2 cells. Lane 1, bovine SOD, bovine liver CAT and Saccharomyces cerevisiae GRstandards for (A), (B) and (C), respectively; lane 2, control (zero Ni), 12 h; lane 3, control, 36 h; lane 4, control 72 h; lane 5,0.075 mM NiCl2, 12 h; lane 6, 0.075 mM NiCl2, 36 h; lane 7, 0.075 mM NiCl2, 72 h; lane 8, 0.75 mM NiCl2, 12 h; lane 9, 0.75mM NiCl2, 36 h; lane 10, 0.75 mM NiCl2, 72 h.Nickel-induced stress in tobacco 551Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.548-552, September/October 2008stress have been shown to vary considerably depend-ing on plant species, tissue and duration of exposure.We identified and classified two SOD isoenzymes inBY-2 cells, Mn-SOD and Fe-SOD, but up to nine SODisoforms have been reported in cell suspension culturesof C. arabica, two bands corresponding to Mn-SODand three bands to Fe-SOD isoenzymes (Gomes-Júnioret al., 2006a, 2006b, 2007).CAT activity staining (Figure 1B) revealed onlyone CAT isoenzyme in BY-2 cell culture, which wasalso shown to vary in response to NiCl2, exhibiting anincrease in activity in the 0.075 mM NiCl2 treatmentat 6 h and 36 h of treatment, whereas in the 0.75 NiCl2treatment CAT activity was reduced when comparedto their respective controls. Interestingly, no CAT ac-tivity was detected at 72 h in both control and Ni treat-ments (Figure 1B). More than one CAT isoenzyme isnormally observed in plant species (Azevedo et al.,1998), however, in the present study only one majorCAT isoenzyme could be detected following non-de-naturing PAGE activity staining. CAT is directly regu-lated by H2O2 levels and the increase in CAT activityparticularly during the first 36 h of Ni treatment indi-cates that CAT is possibly acting in the dismutation ofthe excess H2O2 produced as a result of the Ni-inducedoxidative stress. Nevertheless the participation of otherperoxidases to dismutate the H2O2 cannot be ruled out,particularly in the highest NiCl2 concentration tested,since CAT activity was even slightly reduced whencompared to the controls.The possibility of other enzymes being in-volved in the response to Ni stress under certain con-centrations or periods of treatment was further con-firmed by the results observed for GR activity (Fig-ure 1C). Overall, GR activity did not vary much sincethe isoenzymes III and IV, which accounted for themajority of GR activity, exhibited similar levels of ac-tivities among Ni concentrations and controls duringthe duration of the experiment, however, GR I onlyappeared after 72 h treatment, but not as a responseto Ni-induced stress, since this isoenzyme was alsoobserved in the control at 72 h (Figure 1C). On theother hand, the appearance of GR II isoenzyme at 12h and 36 h of 0.75 mM NiCl2 treatments is clearlydue to the Ni-induced oxidative stress, confirming theimportance of non-denaturing PAGE analysis in theidentification of such specific changes. In plants, cy-tosolic and plastidic GR isoenzymes have been iden-tified (Xiang & Oliver, 1998), however in BY-2 cellscytosolic GR isoenzymes are likely to be predominantsince the cells were grown in the dark. 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Resposta de isoenzimas antioxidantes ao estresse induzido por níquel em cultura de células em suspensão de fumo

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Antioxidant isoenzyme responses to nickel-induced stress in tobacco cell suspension culture

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