Methylene blue prevents methylmalonate-induced seizures and oxidative damage in rat striatum

https://doi.org/10.1016/j.neuint.2006.07.012Get rights and content

Abstract

Methylene blue (MB) is a thiazine dye with cationic and lipophilic properties that acts as an electron transfer mediator in the mitochondria. Due to this metabolic improving activity and free radicals scavenging effects, MB has been used in the treatment of methemoglobinemia and ifosfamide-induced encephalopathy. Considering that methylmalonic acidemia consists of a group of inherited metabolic disorders biochemically characterized by impaired mitochondrial oxidative metabolism and reactive species production, we decided to investigate whether MB, protects against the behavioral and neurochemical alterations elicited by the intrastriatal injection of methylmalonate (MMA). In the present study we showed that intrastriatal injection of MB (0.015–1.5 nmol/0.5 μl) protected against seizures (evidenced by electrographic recording), protein carbonylation and Na+,K+-ATPase inhibition ex vivo induced by MMA (4.5 μmol/1.5 μl). Furthermore, we investigated whether convulsions elicited by intrastriatal MMA administration are accompanied by striatal protein carbonyl content increase and changes in Na+,K+-ATPase activity in rat striatum. The effect of MB (0.015–1.5 nmol/0.5 μl) and MMA (4.5 μmol/0.5 μl) on striatal NOx (NO2 plus NO3) content was also evaluated. Statistical analysis revealed that the MMA-induced NOx content increase was attenuated by intrastriatal injection of MB and the duration of convulsive episodes correlated with Na+,K+-ATPase inhibition, but not with MMA-induced total protein carbonylation. In view of that MB decreases MMA-induced neurotoxicity assessed by behavioral and neurochemical parameters, the authors suggest that MB may be of value to attenuate neurological deficits of methylmalonic acidemic patients.

Introduction

Methylene blue (MB) is a redox compound (thiazine dye) with cationic and lipophilic properties that acts as an electron transfer mediator, stimulating of mitochondrial respiration (Visarius et al., 1997, Visarius et al., 1999). Experimental findings have demonstrated that low concentrations of MB improve mitochondrial respiration by shuttling electrons to oxygen in the electron transport chain, thereby increasing cellular oxygen consumption (Visarius et al., 1997). Due to these metabolic improving activities and radical scavenging, MB has been used prophylactically or an antidote for encephalopathy induced by ifosfamide (IFO) in chemotherapeutic treatment (Küpfer et al., 1994, Küpfer et al., 1996, Pelgrims et al., 2000, Turner et al., 2003, Raj et al., 2004, Pavarana et al., 2005).

Methylene blue also acts as a potent inhibitor of superoxide and hydroxyl radical production by serving as an artificial electron acceptor, diverting electron flow away from the enzyme sites of various oxidases, where molecular oxygen is converted to superoxide radicals (Fridovich, 1997, Aeschlimann et al., 1998, Kelner et al., 1988). In this context, a significant amount of work has suggested that MB represents a free radical scavenger which competitively inhibit the reduction of molecular oxygen to superoxide, acting as an alternative electron acceptor for enzymes either in heme or non-heme forms such as xantine oxidase and nitric oxide synthase (NOS) (Salaris et al., 1991, Mayer et al., 1992, Mayer et al., 1993, Volke et al., 1999).

Methylmalonic acidemia is biochemically characterized by a primary accumulation of methylmalonate (MMA) and a secondary accumulation of other metabolites, such as succinate, propionate, 3-hydroxypropionate, and 2-methylcitrate (Fenton et al., 2001, Okun et al., 2002, Kölker et al., 2003) in tissue and body fluids. They are caused by an inherited deficiency of the mitochondrial enzyme methylmalonil-CoA mutase (MCM, EC 5.4.99.2) or by defects in the synthesis of 5′-deoxyadenosylcobalamin, the cofactor of MCM (Fenton et al., 2001). Although the mechanisms of brain damage caused by MMA accumulation are not yet fully established, experimental findings in vitro and ex vivo support that MMA causes mitochondrial dysfunction (Brusque et al., 2002, Okun et al., 2002, Marisco et al., 2003, Fleck et al., 2004, Schuck et al., 2004, Maciel et al., 2004), leading to increased lactate production (Royes et al., 2003), ATP depletion (McLaughlin et al., 1998), decreased CO2 production (Wajner et al., 1992) and O2 utilization (Toyoshima et al., 1995).

Furthermore, a growing body of evidence has suggested that organic acids, which accumulate in some organic acidurias cause reactive species generation (Wajner et al., 2004) and behavioral manifestations, such as seizures (de Mello et al., 1996, Royes et al., 2003). In this context, it has been shown that the intrastriatal administration of MMA induces convulsions, reactive species generation and inhibits Na+,K+-ATPase activity in rat striatum (de Mello et al., 1996, Fighera et al., 1999, Fighera et al., 2003, Marisco et al., 2003, Wyse et al., 2000, Malfatti et al., 2003, Royes et al., 2005). Although it is difficult at present to determine a cause effect relationship between reactive species and seizures induced by MMA, recent studies from our laboratory have shown that while the convulsions induced by MMA are attenuated by antioxidant treatment (ascorbate, α-tocopherol and GM1 ganglioside) (Fighera et al., 1999, Fighera et al., 2003), ammonia, a pro-oxidant agent, increases MMA-induced convulsions (Marisco et al., 2003), suggesting that reactive species are involved in the genesis of convulsions elicited by MMA. Therefore, considering that reactive species generation have been implicated in the convulsive behavior elicited by MMA it is rather possible that drugs, such as MB, which increase brain mitochondrial respiration and act as scavenger of superoxide radical may protect against the acute behavioral and neurochemical effects induced by MMA. In this study we investigate whether the intrastriatal injection of MB attenuates MMA-induced behavioral, electrographic and neurochemical deleterious effects.

Section snippets

Animals and reagents

Adult male Wistar rats (270–300 g) maintained under controlled light and environment (12 h light–12 h dark cycle, 24 ± 1 °C, 55% relative humidity) with free access to food (Guabi, Santa Maria, Brazil) and water were used. Animal utilization protocols followed the Official Government Ethics guidelines and were approved by the University Ethics Committee. All reagents were purchased from Sigma (St. Louis, MO, USA) and all solutions were prepared with type I ultra pure water. MB was prepared in 100 mM

Statistical analysis

The latency for convulsion and total time spent convulsioning data were analyzed by one or two-way analysis of variance (ANOVA), depending on the experimental design. Post hoc analysis was carried out by the Student–Newman–Keuls test, when appropriate. Biochemical data were analyzed by a two (PBS or MB) × two (saline or MMA) × two (injected or contralateral side) factorial ANOVA, with the hemisphere factor treated as a within-subject factor. Post hoc analyses were carried out by the F-test for

Effect of MB on MMA-induced convulsive behavior

The intrastriatal injection of MMA (4.5 μmol/1.5 μl) caused the appearance of clonic convulsions (Fig. 1), which were confirmed by electrographic recording (Fig. 2). Electrographically recorded seizures were accompanied by clonus of the left forelimb and/or hindlimb and head, rotational behavior and full lateralization toward the left side of the body. Seizures elicited by MMA were defined by the occurrence of episodes consisting of the simultaneous occurrence of at least two of the following

Discussion

In the current work we studied the MB intrastriatal injection in the behavioral, eletrographic and neurochemical alterations elicited by MMA administration. Therefore, we confirm and extend our previous findings that MMA elicits behavioral and eletrographic seizures and rises reactive species generation ex vivo and show, for the first time, that: (a) MB affords significant protection against MMA-induced protein carbonylation, Na+,K+-ATPase inhibition and electrographic convulsions; (b) the

Acknowledgements

Work supported by CNPq (grant: 500120/2003-0), FAPERGS, and FIPE/UFSM. C.F. Mello, L.F.F. Royes, A.F. Furian, M.S. Oliveira and N.G. Fiorenza are the recipients of CNPq fellowships (grant: 500120/2003-0). R.C. Coelho, A.P. de O. Ferreira are the recipients of FAPERGS fellowships and J.C. Petry is the recipient of FIPE/UFSM fellowships.

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