Behavioral, Physiological and Biochemical Hormetic Responses to the Autoxidizable Dye Methylene Blue

Abstract

The goals of this review were to identify methylene blue (MB) as a compound that follows hormetic behavior for a wide range of effects and to address the question of what is unique about MB that could account for its wide applicability and hormetic behavior as a drug. The MB hormetic dose-response relationship is exemplified by an increase in various behavioral, physiological and biochemical responses with increasing dose, followed by a decrease in the same responses with an even higher dose, until the responses are equal to control responses. With MB doses increasing beyond the hormetic zone, the responses decrease even further, until they are below the control responses. At doses spanning its hormetic zone, MB can increase select responses until they are 130-160% of control. For example, low doses of MB produce maximum behavioral and biochemical responses with averages of approximately 140% of control. As MB dose is raised outside the hormetic zone the response decreases below the control response, as exemplified by MB's ability to increase cytochrome oxidase activity at intermediate doses, while decreasing cytochrome oxidase activity at higher doses. It is proposed that MB's autoxidizable chemical property may be responsible for its unique biological action as both a metabolic energy enhancer and antioxidant that is frequently characterized by hormetic dose-response relationships.

Figures

Fig. 1

Autoxidizable dye methylene blue. a) In its oxidized form methylene blue can accept electrons from an electron donor. In its reduced form, leukomethylene blue (MBH2) is colorless, acts as an electron donor, and it can transfer electrons to oxygen to form water. Thus methylene blue is capable of autoxidizing. At low concentrations in vivo methylene blue and leukomethylene blue are at an equilibrium, so that they form a reversible reduction-oxidation system. This autoxidazing property provides a mechanisms for electron transfer to oxygen, and may be responsible for various hormetic dose-response effects of methylene blue at the biochemical, physiological and behavioral levels. b) Chlorpromazine hydrochloride is a methylene blue derivative which shares a common phenothiazine ring system, as well as certain hormetic effects, with methylene blue. However, unlike methylene blue chlorpromazine hydrocholoride has a reduced (non-aromatic) ring system, and is not capable of autoxidizing. Therefore its applications are more circumscribed than the numerous therapeutic uses of methylene blue

Fig. 2

Methylene blue can act as an artificial electron donor to the electron transport chain complexes of the mitochondria. Embedded in the inner mitochondrial membrane are several protein complexes capable of shuttling electrons and pumping protons against a concentration gradient into the intermembrane space, thus generating the proton gradient (energy) required for the ATP synthesis. These electron transport chain complexes can receive electrons from reduced cosubstrates such as NADH and FADH2 and transfer them to oxygen. Coenzyme Q is shared by complexes I, II and III, while cytochrome c is shared by complexes III and IV. Reduced methylene blue can donate electrons to coenzyme Q and possibly to the cytochrome c, thus increasing cytochrome oxidase (complex IV) activity. In addition, at low doses methylene blue can also enter a reversible oxidation and reduction cycle and interact with oxygen to form water, which would decrease the superoxide radicals produced during the process of oxidative phosporylation. This way, methylene blue can act as an antioxidant inside the mitochondria where most of the oxygen reactive species are formed. At higher doses, methylene blue can take electrons away from the electron transport chain complexes, thereby decreasing activity of these complexes

Fig. 3

Hormetic effects of methylene blue on brain cytochrome oxidase activity[8]. This is an example of in vitro hormetic dose-response relationships of methylene blue on a biochemical measure. Cytochrome oxidase is a terminal enzyme in the electron transport chain of the mitochondria, and as such acts on its substrate, cytochrome c in order to provide energy for ATP synthase. At low doses methylene blue can act as an electron donor and increase the amount of the reduced cytochrome c, thus elevating cytochrome oxidase activity, as measured spectrophotometrically. Maximum increase of 138% of control was achieved at 0.5 μM methylene blue dose, while a 5 μM dose did not differ from control. When 10 μM methylene blue was added to the rat brain homogenates, the cytochrome oxidase activity decreased bellow the baseline response, following principles of hormetic dose-response relationships outlined by Calabrese et al. (2007)

Fig. 4

Hormetic effects of methylene blue on spontaneous locomotor activity[10]. This is an example of in vivo hormetic dose-response relationships of methylene blue on a behavioral measure. Rats injected intraperitoneally with increasing doses of methylene blue were subjected to a running wheel test to examine general locomotor activity. The maximum wheel-running response (average 142% of control) was observed for the intermediate 4 mg kg−1 dose, while lower (1 mg/kg) and higher (10 mg kg−1) methylene blue doses did not differ from the baseline. Even higher doses (≥50 mg kg−1) decreased the wheel-running response below control levels. Therefore, methylene blue exerts hormetic dose-response effects on general locomotor activity in rats, with a hormetic zone between doses of 1 mg kg−1 and 10 mg kg−1