Neurochem Res DOI 10.1007/s11064-014-1309-z

ORIGINAL PAPER

Minocycline Increases the Activity of Superoxide Dismutase and Reduces the Concentration of Nitric Oxide, Hydrogen Peroxide and Mitochondrial Malondialdehyde in Manganese Treated Drosophila melanogaster Marylu´ Mora • Ernesto Bonilla • Shirley Medina-Leendertz Yanauri Bravo • Jose´ Luis Arcaya



Received: 5 February 2014 / Revised: 2 April 2014 / Accepted: 11 April 2014 Ó Springer Science+Business Media New York 2014

Abstract The toxicity caused by high concentrations of manganese (Mn) could be due to a production of free radicals. Minocycline is an effective antioxidant with a high potential to capture free radicals. We investigated the effect of minocycline in the activities of superoxide dismutase (SOD) and catalase, and in the concentrations of nitric oxide (NO), hydrogen peroxide (H2O2) and mitochondrial malondialdehyde (MDA) in manganese-treated Drosophila melanogaster. Five groups of flies were used: (1) control: not treated; (2) continuously treated with minocycline (0.05 mM); (3) treated with 30 mM Mn for 6 days and then no additional treatment; (4) continuously treated with Mn; (5) treated only with Mn for 6 days and then treated with minocycline; (6) simultaneously treated with Mn and minocycline. On the 6th day, Mn treatment caused 50 % mortality; in the surviving flies increased levels of MDA (67.93 %), NO (11.04 %), H2O2 (14.62 %) and SOD and catalase activity (165.34 and 71.43 %, respectively) were detected. All the flies continuously treated with Mn died by the 21st day. On day 40, MDA levels were decreased in groups two, three and five (43.04, 29.67, and 34.72 % respectively), as well as NO in group two (29.21 %) and H2O2 in groups two and five (53.94 % and 78.69 %,

M. Mora  E. Bonilla (&)  S. Medina-Leendertz  Y. Bravo Laboratorio de Neurobiologı´a, Centro de Investigaciones Biome´dicas, Instituto Venezolano de Investigaciones Cientı´ficas (IVIC), Hospital Universitario de Maracaibo, 9no Piso, Maracaibo 4002, Estado Zulia, Venezuela e-mail: [email protected] E. Bonilla  J. L. Arcaya Facultad de Medicina, Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’, Universidad del Zulia, Maracaibo 4002, Estado Zulia, Venezuela

respectively), while in group three the concentration of H2O2 was increased (408.25 %). In conclusion, Mn exerted a prooxidant effect on the 6th day as shown by the increased levels of oxidative markers. Minocycline extended the lifespan, increased the activity of SOD and reduced the levels of NO, H2O2 and mitochondrial MDA. Keywords Minocycline  Manganese  Drosophila melanogaster  Superoxide dismutase

Introduction The fruit fly Drosophila melanogaster has been intensively used in basic and applied research on human neurological diseases because of advantages over mammalian model systems such as ease of laboratory maintenance and genetic manipulations. The Drosophila genome is simpler than that of mammals, in terms of gene and chromosome number, but nonetheless it demonstrates extraordinary phylogenetic conservation of gene structure and function, especially among the genes whose mutations cause neurodevelopmental, neuropsychiatric, or neurodegenerative disorders [1]. Manganese (Mn) is an essential trace element that is required for maintaining proper function and regulation of numerous biochemical and cellular reactions [2]. Manganese may cross the blood brain barrier, and it is delivered to different brain regions, via an axonal transport system. The unidirectional transport of manganese across the choroid plexus seems to provide the anatomic-functional basis linking the systemic exposure to manganese with the spreading pattern of manganese accumulation observed in brain imaging, and explains the polarized sensitivity of choroid epithelial cells to manganese toxicity [3]. Various transport mechanisms have been identified, including active transport, facilitated

123

Neurochem Res

diffusion, and high affinity metal transporters such as calcium and iron transporters (ferritin). Ferritin may be involved in the homeostasis of other divalent metals (iron and manganese), and that overexpression of ferritin, sometimes employed to rescue neurodegenerative models of disease, serves to limit divalent metal bio-availability in cells [4]. The Mn-induced disruption of the glutamine/glutamate -c-aminobutyric acid cycle between astrocytes and neurons contributes to changes in Glu-ergic and/or GABA-ergic transmission, and is associated with several neuropathological conditions, including Mn toxicity [5]. Manganese is a well-established neurotoxin associated with specific damage to the basal ganglia in humans [6]. MnCl2 has been shown to induce apoptosis via endoplasmic reticulum stress and mitochondria dysfunction [7]. In fact, it has recently been reported that treatment with Mn resulted in a dose-dependent increase in neuronal apoptosis, reactive oxygen species (ROS) levels, and a decrease in superoxide dismutase (SOD) activity as well as oxidative damage in cell lipids and proteins [8]. However, little is known of the role that ROS play in protein aggregation resulting from Mn exposure. Minocycline is a semi-synthetic second-generation tetracycline. Its antioxidant efficacy has been demonstrated [9]. It also exerts ameliorative effects in neurodegenerative disease models, including Drosophila [10]. Minocycline prevented NMDA excitotoxicity of PC12 cells, partly mediated by the inhibition of lipooxigenase [11]. More recently, it was also shown that minocycline significantly inhibits H2O2-induced activation of JNK, p38MAPK and caspase and protects melanocytes against H2O2-induced apoptosis in vitro [12]. Minocycline was found to be able to block nitric oxideinduced neurotoxicity in cerebellar granule neurons treated with nitric oxide (NO) by inhibiting NO-induced phosphorylation of p38 mitogen-activated protein kinase. These findings may explain the neuroprotective mechanism of minocycline in models of global and focal cerebral ischemia, the R6/2 model of Huntington´s disease as well as glutamateinduced neurotoxicity in mixed neuronal/glial cultures [13]. We have previously shown that minocycline increased the life span and motor activity and decreased MDA formation of manganese treated D. melanogaster [14]. In the present study we determined the effect of minocycline in the activities of SOD and catalase, and in the concentrations of NO, hydrogen peroxide and mitochondrial MDA of D. melanogaster treated with manganese.

dark cycle at 25 °C. Standard corn meal contained: agar– agar (0.3 g), corn flour (5 g), yeast (1.5 g), 100 % ethanol (1.25 mL), brown sugar solution (5 mL), methyl phydroxybenzoate (Sigma Chemistry Co. MO. USA) (0.065 g) and 43.75 mL of distilled water [15]. Longevity Two days old males were transferred to glass vials (Pyrex culture 9.6 9 100 mm) containing 1 mL of the test food and closed with cotton stoppers. The flies were held in groups of five per vial. Fresh solutions of minocycline and manganese chloride, both obtained from Sigma-Aldrich Co. LLC. USA, were prepared daily at a concentration of 0.05 and 30 mM, respectively, in standard corn meal. Every day at 10 A.M., the dead flies were counted and survivors were transferred to freshly prepared food. Three replicates of each treatment and control were done. Treatment of the Flies with Manganese and Minocycline Two days after emerging from the pupa flies were divided in five groups: (1) Control: not treated; (2) continuously treated with minocycline (0.05 mM); (3) treated with 30 mM manganese (Mn) for 6 days and then no additional treatment; (4) continuously treated with Mn; (5) treated only with Mn for 6 days and then treated with minocycline; (6) simultaneously treated with Mn and minocycline. To give continuity to our previous work we decided to assay the pro-oxidant effect of Mn by increasing its concentration to 30 mM. We therefore had to change the number of days of intoxication from 13 to 6 days, since at the 6th day the dose–response curve showed that the population of flies reached 50 % of survival, and the concentration of MDA was increased. For this reason the first sample was taken on day 6. The second sample was taken on day 40, when the population of flies reached 75 % of mortality. All biochemical assays were performed on days 6 and 40 in flies not treated or treated with minocycline after being exposed to Mn for 6 days.

Biochemical Assays Isolation of Mitochondria

Materials and Methods Experimental Animals Stock Male wild-type flies of D. melanogaster (Oregon wild strain) were used. Flies were maintained in 12 h/12 h light/

123

Mitochondria from whole body of Drosophila melanogaster were prepared by differential centrifugation. Briefly, a homogenate was prepared with one hundred and fifty flies in 990 lL ice–cold Tris–sucrose buffer (0.32 M sucrose, 1 mM EDTA and 10 mM Tris–HCl at pH 7.4) containing 1 % Butylated hydroxytoluene (BHT), all obtained from Sigma-

Neurochem Res

Aldrich Co. LLC USA, using a glass–glass grinder (Pyrex 2 mL) obtained from Thermo Fisher Scientific Inc. USA. The homogenate was centrifuged 15,000g (IEC CENTRA MP4R International Equipment Company) for 2 min at 4 °C. The supernatant was removed carefully and the pellet, which contains the mitochondria, was centrifuged 15,000g (IEC CENTRA MP4R International Equipment Company) for 2 min at 4 °C three times in ice–cold Tris–sucrose buffer obtained from Sigma-Aldrich Co. LLC. U.S.A. [16]. Determination of Oxidative Markers Determination of Malondialdehyde Lipid peroxidation determination was made by measuring MDA-TBA adducts formed from malondialdehyde (MDA) reaction in samples with thiobarbituric acid (TBA), using OxiSelectTM TBARS Assays kit (MDA Quantitation) (CELL BIOLABS, Inc., USA). ‘‘MDA forms a 1:2 adduct with thiobarbituric acid. The MDA-TBA adduct formed from the reaction of MDA in samples with TBA can be measured colorimetrically or fluorometrically. TBARS levels are determined from a malondialdehyde equivalence standard’’. The supernatant obtained during the isolation of mitochondria was removed and the pellet was resuspended in phosphate-buffered saline (PBS), following the protocol described in the kit. The absorbance was read at 532 nm (Synergy HT, BioTeck, USA). The results were calculated and expressed in nmoles MDA/mg protein. The Bicinchoninic Acid Protein Assay Kit BCA1 obtained from Sigma-Aldrich Co. LLC. USA was used to determine soluble protein concentrations. ‘‘The principle of the bicinchoninic acid (BCA) assay is similar to the Lowry procedure, in that both rely on the formation of a Cu2? -protein complex under alkaline conditions, followed by reduction of the Cu2?–Cu1?. The amount of reduction is proportional to the protein present. It has been shown that cysteine, cystine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2?–Cu1?. BCA forms a purple-blue complex with Cu1? in alkaline environments, thus providing a basis to monitor the reduction of alkaline Cu2? by proteins’’. The supernatant obtained during the isolation of mitochondria was removed and the pellet was resuspended in deionized water, following the protocol described in the kit. The absorbance was measured at 562 nm (Synergy HT, BioTeck, USA). The results were calculated and expressed in mg protein/mL. Hydrogen Peroxide and Nitric Oxide Determinations A homogenate of whole body of Drosophila melanogaster was prepared with 50 flies in 500 lL PBS, using a glass– glass grinder (Pyrex 2 mL) obtained from Thermo Fisher Scientific Inc. USA. The homogenate was centrifuged at

10,000g (IEC CENTRA MP4R International Equipment Company) for 5 min at 4 °C. The supernatant was removed to determine H2O2 and NO. Hydrogen Peroxide The OxiSelectTM hydrogen peroxide Assay Kit (CELL BIOLABS, INC. USA) was used to measure the H2O2 present in the samples. ‘‘This is a quantitative assay for measuring hydrogen peroxide in aqueous and lipid samples. For aqueous samples, sorbitol first converts peroxide to a peroxyl radical, which oxidizes Fe2? into Fe3?. For lipid samples, peroxide converts Fe2? into Fe3? directly. Then Fe3? reacts with an equal molar amount of xylenol orange in the presence of acid to create a purple product that absorbs maximally between 540 and 600 nm’’. 90 lL of the supernatant were removed to determine H2O2 following the protocol described in the kit. The absorbance was read at 540 nm (Synergy HT, BioTeck, USA). The results were calculated and expressed in nmol H2O2/mg protein. Nitric Oxide The QuantiChromTM Nitric oxide assay kit (D2NO-100) was used to measure the nitric oxide present in the samples. ‘‘Since NO is oxidized to nitrite and nitrate, it is common practice to 2 quantitate total NO2 2 /NO3 as a measure for NO level. BioAssay Systems QuantiChromTM Nitric Oxide Assay Kit is designed to accurately measure NO production following reduction of nitrate to nitrite using improved Griess method’’. 100 lL of the supernatant were removed to determine NO following the protocol described in the kit. The absorbance was read at 540 nm (Synergy HT, BioTeck, USA). The results were calculated and expressed in lM nitrite. Catalase Activity Determination A homogenate of whole body of Drosophila melanogaster was prepared with 10 flies in ice–cold PBS with 1 mM EDTA per gram of tissue (500 lL), using a glass–glass grinder (Pyrex 2 mL). The homogenate was centrifuged at 10,000g (IEC CENTRA MP4R International Equipment Company) fory 15 min at 4 °C. The supernatant was removed to determine catalase activity. The OxiSelectTM Catalase activity Assay Kit colorimetric (CELL BIOLABS, INC, USA) was used to measure the catalase activity present in the samples. ‘‘This involves two reactions; the first reaction is the catalase-induced decomposition of hydrogen peroxide H2O2 into water and oxygen. The rate of disintegration of hydrogen peroxide into water and oxygen is proportional to the concentration of catalase. A catalase-containing sample can be incubated in a known amount of hydrogen peroxide. The reaction

123

Neurochem Res

proceeds for exactly 1 min, at which time the catalase is quenched with sodium azide. The remaining hydrogen peroxide in the reaction mixture facilitates the coupling reaction of DHBS and AAP in conjunction with an HRP catalyst (reaction 2). The quinoneimine dye-coupling product is measured at 520 nm’’. 20 lL of the supernatant were removed to determine catalase activity following the protocol described in the kit. The absorbance was read at 520 nm (Synergy HT, BioTeck, USA). The results were calculated and expressed in units/mg protein. Superoxide Dismutase Activity A homogenate of whole body of Drosophila melanogaster was prepared with 10 flies in 400 lL of ice-cold lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.5 % triton-100), using a glass–glass grinder (Pyrex 2 mL). The homogenate was centrifuged at 12,000g (IEC CENTRA MP4R International Equipment Company) by 10 min. at 4 °C. The supernatant was removed to determine superoxide dismutase activity. The OxiSelectTM Superoxide Dismutase activity Assay Kit (CELL BIOLABS, INC. USA) was used to measure the superoxide dismutase activity present in the samples. ‘‘Superoxide dismutase activity assay uses a xanthine/xanthine oxidase (XOD) system to generate superoxide anions. The included chromagen produces a water-soluble formazan dye upon reduction by superoxide anions. The activity of SOD is determined as the inhibition of chromagen reduction’’. 70 lL of the supernatant were removed to determine superoxide activity following the protocol described in the kit. The absorbance was read at 490 nm (Synergy HT, BioTeck, USA). The results were calculated and expressed in Units/mg protein. Only citosolic SOD (Cu/ZnSOD) was determined, since only the supernatant was used. Statistical Analysis Data are expressed as mean ± SEM and the significance between specific means was determined by one-way ANOVA test, and differences among experimental groups were tested for significance using the test of Dunnett for comparisons. Differences were considered statistically significant when p \ 0.05.

Results Manganese Induced Lethality Response: LC50 Determination Data on the incidence of lethality among flies exposed to manganese over 6 days is presented in Fig. 1. The

123

Fig. 1 Effect of manganese (30 mM) and minocycline on mortality of male D. melanogaster after 6 days of treatment

50 ± 0.57 % mortality was observed after 6 days of treatment with manganese (30 mM), while only 2 ± 0.58 and 1 ± 0.17 % dead were found in control and minocycline treated flies. Effect on Oxidative Markers and Antioxidant Enzymes Data on the levels of oxidative markers in flies after 6 days of treatment are presented in Table 1. The flies treated with manganese showed enhanced level of MDA (67.93 %), NO (11.04 %), H2O2 (14.62 %) with respect to control, while in the animals treated with minocycline it was observed a significant decrease in the concentrations of mitochondrial MDA (9.99 %), NO (21.97 %) and H2O2 (50.97 %) with respect to control. The effect on endogenous antioxidant enzymes after 6 days of treatment is presented in Table 2. The activity of SOD was significantly increased in groups two (continuously treated with minocycline) and four (continuously treated with manganese) (24.23 and 165.34 % respectively) with respect to control, whereas catalase activity was enhanced in group four (71.43 %). Observations on the levels of oxidative markers in flies after 40 days of treatment are presented in Table 3. Malondialdehyde levels were decreased in the groups two (continuously treated with minocycline), three (Mn-no treatment) and five (treated only with Mn for 6 days and then treated with minocycline) (43.04, 29.67, and 34.72 %, respectively) with respect to control. A decrease in NO was detected in group two (29.21 %). On the other hand, H2O2 levels were decreased in groups two and five (53.94 and 78.69 % respectively) with respect to control. In contrast, a highly significant increase was observed in the concentration of H2O2 (408.25 %) in group three, with respect to control. The effect on endogenous antioxidant enzymes after 40 days of treatment is presented in Table 4. A significant increased of SOD activity in groups two (continuously treated with minocycline) and three (Mn- no treatment) was

Neurochem Res Table 1 Oxidative stress markers in adult male Drosophila melanogaster after 6 days of treatment Treatments (groups)

Oxidative stress markers Mitochondrial MDA

Nitric oxide

H2O2

Control (1)

6.61 ± 0.0586

39.23 ± 0.6936

0.773 ± 0.0088

Continuously treated with minocycline (2)

5.95 ± 0.0441**

30.61 ± 0.3496**

0.379 ± 0.0069**

11.10 ± 0.1530**

43.56 ± 0.3482**

0.886 ± 0.0088

Continuously treated manganese (4)

Data was analyzed by one-way ANOVA test and differences among experimental groups were tested for significance using the test of Dunnett for comparisons; **p \ 0.01. Values are expressed as nmol malondialdehyde/mg protein, lM nitrite, nmol hydrogen peroxide/mg protein. Three replicates of each treatment and control were done. MDA n = 150 flies; nitric oxide and H2O2 n = 50 Table 2 Antioxidant enzymes in adult male Drosophila melanogaster after 6 days of treatment

Table 4 Antioxidant enzymes in adult male Drosophila melanogaster after 40 days of treatment

Treatments (groups)

SOD

Catalase

Treatments (groups)

SOD

Catalase

Control (1)

186.97 ± 1.01

0.14 ± 0.0021

Control (1)

160.13 ± 0.75

0.157 ± 0.0009

Continuously treated with minocycline (2)

232.64 ± 1.38**

0.15 ± 0.0058

Continuously treated with minocycline (2)

321.36 ± 0.89**

0.163 ± 0.0009

Continuously treated with manganese (4)

494.28 ± 5.77**

0.24 ± 0.0058**

Mn- no treatment (3) Mn-minocycline (5)

179.27 ± 0.62** 168.48 ± 0.81

0.240 ± 0.0058** 0.157 ± 0.0009

Data was analyzed by one-way ANOVA test and differences among experimental groups were tested for significance using the test of Dunnett for comparisons; **p \ 0.001. Enzymatic values are expressed in Units/mg protein. Three replicates of each treatment and control were done. SOD n = 10 flies; catalase n = 10

observed (100.69, and 11.95 %, respectively). In the flies treated only with Mn for 6 days and then treated with minocycline (group five) no variations in SOD were detected. Furthermore, the activity of catalase was enhanced in group three (52.87 %), with respect to control, but no changes in catalase activity were found in groups two and five. As shown in Fig. 2, the lifespan of control untreated flies was 74.6 ± 1.06 days. Flies continuously treated with minocycline (group two) had a significant longer life span (110.3 ± 1.33 days, 47.86 % more than control). On the other hand, the flies treated only with Mn for 6 days and then treated with minocycline (group five) reached a life span that was similar to that of flies in group two (109.00 ± 2.56 days, 46.11 % more than control). In contrast, the lifespan of the Mn-no treatment flies (group

Data was analyzed by one-way ANOVA test and differences among experimental groups were tested for significance using the test of Dunnett for comparisons; **p \ 0.01. Enzymatic values are expressed in Units/mg protein. Three replicates of each treatment and control were done. SOD n = 10 flies; Catalase n = 10

three) was 87 ± 1.20 days, 16.62 % more than control. All the flies continuously treated with Mn were death on the 21th day. It was interesting to note that the simultaneously treatment with manganese and minocycline (group six) resulted in a decrease in life span to 18, 3 days less than the flies that received manganese continuously (group four). All groups showed a significant difference (p \ 0.001) in longevity with respect to the control flies.

Discussion In this study, we show that feeding of D. melanogaster with 30 mM manganese induced 50 % mortality after 6 days of

Table 3 Oxidative stress markers in adult male Drosophila melanogaster after 40 days of treatment Treatments (groups)

Oxidative stress markers Mitochondrial MDA

Nitric oxide

H2O2

6.97 ± 0.0491

40.09 ± 0.2585

0.812 ± 0.0072

Continuously treated with minocycline (2)

3.97 ± 0.0426**

28.38 ± 0.3221**

0.374 ± 0.0044**

Mn-no treatment (3)

4.93 ± 0.0788**

39.07 ± 0.6212

4.127 ± 0.0754**

Mn-minocycline (5)

4.55 ± 0.0786**

40.26 ± 0.3557

0.173 ± 0.0059**

Control (1)

Data was analyzed by one-way ANOVA test and differences among experimental groups were tested for significance using the test of Dunnett for comparisons; **p \ 0.01. Values are expressed as nmol malondialdehyde/mg protein, lM nitrite and nmol hydrogen peroxide/mg protein. Three replicates of each treatment and control were done. MDA n = 150 flies; nitric oxide and H2O2 n = 50

123

Neurochem Res Fig. 2 Life span in male D. melanogaster treated with minocycline and manganese

treatment. This result agrees with previous studies showing that exposure to Mn decreases the lifespan of the fly [14, 17]. The mechanisms of Mn induced toxicity are not completely understood. However, previous works suggest that oxidative stress plays a key role in Mn toxicity. For instance, Cordova et al. [18] reported that Mn exposure of young rats increased oxidative stress markers in the brain and induced neurologic disturbances. Xu et al. [8] also reported that Mn exposure was associated to oxidative damage to lipids and proteins and to a decrease in the activity of SOD. Taka et al. [19] demonstrated that MnCl2 when added to rat PC12 cells induced a dose dependent loss of cell viability, which was associated with enhanced production of H2O2 concomitant to elevation of gene expression for some antioxidant enzymes. Similar results, in terms of increased H2O2 levels, have been reported for Mn by Zhang et al. [20]. In cultured human astrocytes, MnCl2 (1–100 lM MnCl2) induced a slight increase of cellular reactive oxygen and nitrogen species levels and a slight decrease of ATP levels [21]. Manganese strongly inhibited H2O2stimulated poly(ADP-ribosyl)ation at low, completely noncytotoxic concentrations starting at 1 lM [22]. Katyal et al. [23] showed that manganese, under conditions of either overload due to high exposure or disturbed homeostasis can disturb the cellular response to DNA strand breaks. Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP); this enzyme is strongly activated by DNA strand breaks and apparently plays a role in DNA repair and other cellular responses to DNA damage [24]. Whereas

123

activation of PARP-1 by mild genotoxic stimuli may facilitate DNA repair and cell survival, irreparable DNA damage triggers apoptotic or necrotic cell death. In most severe oxidative stress situations, excessive DNA damage causes over activation of PARP-1, which incapacitates the apoptotic machinery and switches the mode of cell death from apoptosis to necrosis [25]. We hypothesized that the lifespan decreasing effect of Mn treatment could be mediated by oxidative stress induced damage. In the present study, we found that the activity of SOD and catalase were enhanced as were the levels of MDA and NO in flies exposed to 30 mM Mn for 6 days. These findings suggest that oxidative stress was induced in Mn treated flies. It is important to note that the simultaneous treatment with minocycline during manganese exposure did not prevent the decrease in life span induced by Mn. This observation contrasts with our previous study where we observed that minocycline was able to ameliorate the life span decrease induced by a lower manganese concentration (15 mM) [14]. Kreutzmann et al. [26] reported that the life span increase observed with the simultaneous manganese and minocycline treatment is due to minocycline chelation of divalent manganese cation. We cannot rule out that the chelating activity of minocycline contributes to its protective action in Mn toxicity. However we consider that this is not the most important mechanism to explain minocycline´s effect. A number of studies have shown that minocycline can exert its protective effects in several ways including: (1) Its direct free radical scavenging activity [9]; (2) the inhibition of the c-Jun N-terminal kinase (JNK) and Akt/Protein kinase B mediated cell

Neurochem Res

death [10]; (3) minocycline inhibits microglial activation and ameliorates the inflammatory process associated to Mn neurotoxicity [27]. Our observation of elevated activities of SOD and the enhanced MDA and NO levels (markers of oxidative stress) in flies exposed to Mn suggests that Mn-treated flies were exposed to elevated oxidative stress in vivo. A growing body of evidence supports this view. For instance, exposure of rat microglia to manganese chloride resulted in a rapid activation of the extracellular signal-regulated kinase (ERK) and p38-MAPK that appeared to precede the MnCl2-induced H2O2 release, suggesting that ERK and p38-MAPK influenced the MnCl2-induced H2O2 release in microglia [20]. Yoon et al. [28] found that MnCl2 induced intracellular ROS production and also induced a neurotoxicity that significantly dissipated mitochondria membrane potential. It has also been shown that the Mncatalyzed autoxidation of dopamine (DA) involves redox cycling of Mn2? and Mn3? in a reaction that generates ROS and DA-o-quinone, thereby leading to oxidative damage [29]. Oxidative stress generated by high Mn concentrations leads to the induction and opening of the mitochondrial permeability pore (MPT), a Ca2?-dependent process, resulting in increased solubility to protons, ions and solutes, loss of the mitochondrial inner membrane potential, impairment of oxidative phosphorylation and ATP synthesis and mitochondrial swelling [30]. On the other hand, it has also been observed that exposure of SH-SY5Y cells to Mn promotes both the accumulation of oxidative DNA damage, and that the accumulated damage to DNA is ameliorated by chemical antioxidants, which confirms the potential of antioxidants as a therapeutic strategy for protection against Mn(2?)induced oxidative DNA damage [31]. Marreijha et al. [32] also showed that a significant decrease in Mn cytotoxicity was observed in cells co-exposed to N-acetylcisteine or trolox during Mn treatment, confirming that oxidative stress plays a critical role in the mechanism of Mn toxicity. In our study, after the administration of minocycline for 40 days it was observed a significant increase in SOD activity and a decrease in MDA, NO and H2O2. Similar results have been reported by Inamdar et al. [10], who showed that minocycline blocked the generation of reactive oxygen species in Drosophila. The reduction in H2O2 levels in animals treated with minocycline was not accompanied by an increment of catalase activity, due possibly to a direct scavenging effect of minocycline of the radicals generated by the pro-oxidant activity of Mn [9, 33]. Bonilla et al. [14] also found that minocycline increased the life span and motor activity and decreased MDA formation of manganese treated D. melanogaster. The increased activity of SOD and catalase after the 6 days

treatment with Mn may be interpreted as a compensatory mechanism intending to protect the cell from Mn-induced oxidative damage. This mechanism seems to cooperate with the minocycline antioxidant effects. In this regard, it is interesting to note that the activity of SOD was still increased after 40 days in the flies treated with Mn for 6 days. However, in the flies treated with minocycline after Mn poisoning the activity of SOD and catalase returned to normal values. In addition, it has been shown that treatment of microglia with minocycline attenuates microglial activation and decreases the production of IL-1beta, TNF-alpha, and iNOS by these cells while the neurotoxic effect of manganese on dopamine neurons is reduced [34]. Treatment of activated neutrophils with either the chemotactic tripeptide or the phorbol ester resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H2O2. Similar effects of Mn with respect to superoxide reactivity and H2O2 formation were observed with activated macrophages, while generation of NO was unaffected. Manganese can cause microglia activation, which can up-regulate the level of tumor necrosis factor-alpha, interleukin-1beta and nitric oxide synthase, and these inflammatory factors can cause dopaminergic neuronal injury, which can be prevented by minocycline [27]. The inflammatory response mediated by macrophages induced by intranigral Mn microinjection, is fully attenuated by minocycline treatment, suggesting that suppression of macrophage infiltration provides neuroprotection to dopaminergic neurons [35]. Minocycline is a caspase inhibitor, and also inhibits the inducible nitric oxide synthase, which are important for apoptotic cell death. Furthermore, minocycline has been shown to block microglial activation of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned parkinsonism animal models and protect against nigrostriatal dopaminergic neurodegeneration [36]. In conclusion, 30 mM Mn exerted a pro-oxidant effect on D. melanogaster after 6 days of treatment as shown by the increased levels of oxidative markers (MDA and nitric oxide). In the flies continuously treated with minocycline the activity of SOD was increased and the levels of nitric oxide, H2O2 and mitochondrial MDA were reduced. Besides, flies treated with minocycline (groups two and five) had significant longer lifespan than control flies. But, Mn (30 mM) was highly toxic as revealed by the 50 % mortality detected after 6 days of treatment. In flies treated only with Mn for 6 days and them treated with minocycline (group five) the decrease in H2O2 levels was due possibly to a direct scavenging effect of minocycline of the radicals generated by the pro-oxidant effect of Mn.

123

Neurochem Res

References 1. Soon-Il K, Je-Won J, Young-Joon A, Linda LR, Hyung-Wook K (2011) Drosophila as a model system for studying lifespan and neuroprotective activities of plant-derived compounds. J Asia Pac Entomol 14:509–517. doi:10.1016/j.aspen.2011.07.001 2. Sidoryk-Wegrzynowicz M, Aschner M (2013) Role of astrocytes in manganese mediated neurotoxicity. BMC Pharmacol Toxicol 18:14–23. doi:10.1186/2050-6511-14-23 3. Schmitt C, Strazielle N, Richaud P, Bouron A, Ghersi-Egea JF (2011) Active transport at the blood-CSF barrier contributes to manganese influx into the brain. J Neurochem 117:747–756. doi:10.1111/j.1471-4159.2011.07246 4. Gutierrez L, Zubow K, Nield J, Gambis A, Mollereau B, La´zaro F, Missirlis F (2013) Biophysical and genetic analysis of iron partitioning and ferritin function in Drosophila melanogaster. Metallomics 5:997–1005. doi:10.1039/c3mt00118k 5. Sidoryk-Wegrzynowicz M, Aschner M (2013) Manganese toxicity in the central nervous system: the glutamine/glutamate-caminobutyric acid cycle. J Intern Med 273:466–477. doi:10.1111/ joim.12040 6. Racette BA, Aschner M, Guilarte TR, Dydak U, Criswell SR, Zheng W (2012) Pathophysiology of manganese-associated neurotoxicity. Neurotoxicology 33:881–886. doi:10.1016/j.neuro.2011.12.010 7. Hyonok Y, Do-Sung K, Geum-Hwa L, Kee-Won K, HyungRyong K, Han-Jung C (2011) Apoptosis induced by manganese on neuronal SK-N-MC cell line: endoplasmic reticulum (ER) stress and mitochondria dysfunction. Environ Health Toxicol 26:1–17. doi:10.5620/eht.2011.26.e2011017 8. Xu B, Wu SW, Lu CW, Deng Y, Liu W, Wei YG, Yang TY, Xu Z-F (2013) Oxidative stress involvement in manganese-induced alpha-synuclein oligomerization in organotypic brain slice cultures. Toxicology 305:71–78. doi:10.1016/j.tox.2013.01.006 9. Kraus RL, Pasieczny R, Lariosa-Willingham K, Turner MS, Jiang A, Trauger JWJ (2005) Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radicalscavenging activity. Neurochem 94:819–827. doi:10.1111/j.14714159.2005.03219.x 10. Inamdar A, Chaudhuri A, O’Donnell J (2012) The protective effect of minocycline in a paraquat-induced Parkinson’s disease model in Drosophila is modified in altered genetic backgrounds. Parkinson’s Dis 2012:1–16. doi:10.1155/2012/938528 11. Song Y, Wei EQ, Zhang WP, Ge QF, Liu JR, Wang ML, Huang XJ, Hu X, Chen Z (2006) Minocycline protects PC12 cells against NMDA-induced injury via inhibiting 5-lipoxygenase activation. Brain Res 1085:57–67 www.ncbi.nlm.nih.gov/ pubmed/16574083 12. Song X, Xu A, Pan W, Wallin B, Kivlin R, Lu S, Cao C, Bi Z, Wan Y (2008) Minocycline protects melanocytes against H2O2induced cell death via JNK and p38 MAPK pathways. Int J Mol Med 22:9–16 www.ncbi.nlm.nih.gov/pubmed/18575770 13. Lin S, Zhang Y, Dodel R, Farlow MR, Paul SM, Du Y (2001) Minocycline blocks nitric-oxide induced neurotoxicity by inhibition of p38 MAP kinase in rat cerebellar granule neurons. Neurosci Lett 315: 61–64 www.ncbi.nlm.nih.gov/pubmed/ 11711215 14. Bonilla E, Contreras R, Medina-Leendertz S, Mora M, Villalobos V, Bravo Y (2012) Minocycline increases the life span and motor activity and decreases lipid peroxidation in manganese treated Drosophila melanogaster. Toxicology 294:50–53. doi:10.1016/j. tox.2012.01.016 15. Bonilla E, Medina S, Dı´az S (2002) Extension of life span and stress resistance of drosophila melanogaster by long-term supplementation with melatonin. Exp Gerontol 37:629–638 www. ncbi.nlm.nih.gov/pubmed/11909680

123

16. Ferna´ndez-Vizarra E, Ferrı´n G, Pe´rez-Martos A, Ferna´ndez-Silva P, Zeviani M, Enrı´quez JA (2009) Isolation of mitochondria for biogenetical studies: an update. Mitochondrion 10:253–262. doi:10.1016/j.mito.2009.12.148 17. Bonilla-Ramirez L, Jimenez-Del-Rio M, Velez-Pardo C (2011) Acute and chronic metal exposure impairs locomotion activity in Drosophila melanogaster: a model to study Parkinsonism. Biometals 24:1045–1057. doi:10.1007/s10534-011-9463-0 18. Cordova FM, Aguiar AS Jr, Peres TV, Lopes MW, Gonc¸alves FM, Remor AP, Lopes SC, Pilati C, Latini AS, Prediger RD, Erikson KM, Aschner M, Leal RB (2012) In vivo manganese exposure modulates Erk, Akt and Darpp-32 in the striatum of developing rats, and impairs their motor function. PLoS One 7:e33057. doi:10.1371/journal.pone.0033057 19. Taka E, Mazzio E, Soliman KFA, Reams RR (2012) Microarray genomic profile of mitochondrial and oxidant response in manganese chloride treated PC12 cells. Neurotoxicology 33:162–168. doi:10.1016/j.neuro.2012.01.001 20. Zhang P, Hatter A, Liu B (2007) Manganese chloride stimulates rat microglia to release hydrogen peroxide. Toxicol Lett 173:88–100 www.ncbi.nlm.nih.gov/pubmed/17669604 21. Bornhorst J, Meyer S, Weber T, Bo¨ker C, Marschall T, Mangerich A, Beneke S, Bu¨rkle A, Schwerdtle T (2013) Molecular mechanisms of Mn induced neurotoxicity: RONS generation, genotoxicity, and DNA-damage response. Mol Nutr Food Res 57:1255–1269. doi:10.1002/mnfr.201200758 22. Bornhorst J, Ebert F, Hartwig A, Michalke B, Schwerdtle T (2010) Manganese inhibits poly(ADP-ribosyl)ation in human cells: a possible mechanism behind manganese-induced toxicity? J Environ Monit 12:2062–2069. doi:10.1039/c0em00252f 23. Katyal S, McKinnon PJ (2008) DNA strand breaks, neurodegeneration and aging in the brain. Mech Ageing Dev 129:483–491. doi:10.1016/j.molcel.2013.06.018 24. Bu¨rkle A, Grube K, Ku¨pper JH (1992) Poly(ADP-ribosyl)ation: its role in inducible DNA amplification, and its correlation with the longevity of mammalian species. Exp Clin Immunogenet 9:230–240 www.ncbi.nlm.nih.gov/pubmed/1307244 25. Vira´g L (2005) Structure and function of poly(ADP-ribose) polymerase-1: role in oxidative stress-related pathologies. Curr Vasc Pharmacol 3:209–214 www.ncbi.nlm.nih.gov/pubmed/1602 6317 26. Kreutzmann P, Franz C, Scho¨nfeld P (2012) Minocycline forms complexes with manganese in vitro: explaining reported beneficial effect in manganese treated Drosophila melanogaster. Toxicology 300:100–101. doi:10.1016/j.tox.2012.04.010 27. Liu M, Cai T, Zhao F, Zheng G, Wang Q, Chen Y, Huang C, Luo W, Chen J (2009) Effect of microglia activation on dopaminergic neuronal injury induced by manganese, and its possible mechanism. Neurotox Res 16:42–49. doi:10.1007/s12640-009-9045-x 28. Yoon H, Kim DS, Lee GH, Kim KW, Kim HR, Chae HJ (2011) Apoptosis induced by manganese on neuronal SK-N-MC cell line: endoplasmic reticulum (ER) stress and mitochondria dysfunction. Environ Health Toxicol 26:e2011017. doi:10.5620/eht. 2011.26.e2011017 29. Reaney SH, Smith DR (2005) Manganese oxidation state mediates toxicity in PC12 cells. Toxicol Appl Pharmacol 205:271–281 www.ncbi.nlm.nih.gov/pubmed/15922012 30. Yin Z, Aschner JL, Dos Santos AP, Aschner M (2008) Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures. Brain Res 1203:1–11. doi:10.1016/j.brainres. 2008.01.079 31. Stephenson AP, Schneider JA, Nelson BC, Atha DH, Jain A, Soliman KF, Aschner M, Mazzio E, Renee R (2013) Manganeseinduced oxidative DNA damage in neuronal SH-SY5Y cells: attenuation of thymine base lesions by glutathione and N-

Neurochem Res acetylcysteine. Toxicol Lett 218:299–307. doi:10.1016/j.toxlet. 2012.12.024 32. Marreilha dos Santos AP, Santos D, Au C, Milatovic D, Aschner M, Batore´u MC (2008) Antioxidants prevent the cytotoxicity of manganese in RBE4 cells. Brain Res 1236:200–205. doi:10.1016/ j.brainres.2008.07.125 33. Sl Schildknecht, Pape R, Mu¨ller N, Robotta M, Marquardt A, Bu¨rkle A, Drescher M, Leist M (2011) Neuroprotection by minocycline caused by direct and specific scavenging of peroxynitrite. J Biol Chem 286(7):4991–5002. doi:10.1074/jbc.M110. 169565

34. Zhao F, Cai T, Liu M, Zheng G, Luo W, Chen J (2009) Manganese induces dopaminergic neurodegeneration via microglial activation in a rat model of manganism. Toxicol Sci 107:156–164. doi:10.1093/toxsci/kfn213 35. Ponzoni S (2012) Macrophages-mediated neurotoxic effects of intranigral manganese administration are attenuated by minocycline. Neurosci Lett 506:136–140. doi:10.1016/j.neulet.2011.10.066 36. Thomas M, Le WD (2004) Minocycline: neuroprotective mechanisms in Parkinson’s disease. Curr Pharm Des 10(6): 679–686 www.ncbi.nlm.nih.gov/pubmed/14965330

123

Minocycline increases the activity of superoxide dismutase and reduces the concentration of nitric oxide, hydrogen peroxide and mitochondrial malondialdehyde in manganese treated Drosophila melanogaster.

The toxicity caused by high concentrations of manganese (Mn) could be due to a production of free radicals. Minocycline is an effective antioxidant wi...
412KB Sizes 0 Downloads 4 Views