Ramón Soto-Otero

Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson's disease.

Abstract: 6‐Hydroxydopamine (6‐OHDA) is a dopaminergic neurotoxin putatively involved in the pathogenesis of Parkinsons disease (PD). Its neurotoxicity has been related to the production of reactive oxygen species. In this study we examine the effects of the antioxidants ascorbic acid (AA), glutathione (GSH), cysteine (CySH), and N‐acetyl‐CySH (NAC) on the autoxidation and neurotoxicity of 6‐OHDA. In vitro, the autoxidation of 6‐OHDA proceeds rapidly with the formation of H2O2 and with the participation of the H2O2 produced in the reaction. The presence of AA induced a reduction in the consumption of O2 during the autoxidation of 6‐OHDA and a negligible presence of the p‐quinone, which demonstrates the efficiency of AA to act as a redox cycling agent. The presence of GSH, CySH, and NAC produced a significant reduction in the autoxidation of 6‐OHDA. In vivo, the presence of sulfhydryl antioxidants protected against neuronal degeneration in the striatum, which was particularly remarkable in the case of CySH and was attributed to its capacity to remove the H2O2 produced in the autoxidation of 6‐OHDA. These results corroborate the involvement of oxidative stress as the major mechanism in the neurotoxicity of 6‐OHDA and the putative role of CySH as a scavenger in relation to PD.

Autoxidation and MAO-mediated metabolism of dopamine as a potential cause of oxidative stress: role of ferrous and ferric ions

The autoxidation and monoamine oxidase (MAO)-mediated metabolism of dopamine (3-hydroxytyramine; DA) cause a continuous production of hydroxyl radical (*OH), which is further enhanced by the presence of iron (ferrous iron, Fe(2+) and ferric ion, Fe(3+)). The accumulation of hydrogen peroxide (H2O2) in the presence of Fe(2+) appears to discard the involvement of the Fenton reaction in this process. It has been found that the presence of DA significantly reduces the formation of thiobarbituric acid reagent substances (TBARS), which under physiological conditions takes place in mitochondrial preparations. The presence of DA is also able to reduce TBARS formation in mitochondrial preparations even in the presence of iron (Fe(2+) and Fe(3+)). However, DA boosted the carbonyl content of mitochondrial proteins, which was further increased in the presence of iron (Fe(2+) and Fe(3+)). This latter effect is also accompanied by a significant reduction in thiol content of mitochondrial proteins. It has also been observed how the pre-incubation of mitochondria with pargyline, an acetylenic MAO inhibitor, reduces the production of *OH and increases the formation of TBARS. Although, the MAO-mediated metabolism of DA increases MAO-B activity, the presence of iron inhibits both MAO-A and MAO-B activities. Consequently, DA has been shown to be a double-edged sword, because it displays antioxidant properties in relation to both the Fenton reaction and lipid peroxidation and exhibits pro-oxidant properties by causing both generation *OH and oxidation of mitochondrial proteins. Evidently, these pro-oxidant properties of DA help explain the long-term side effects derived from l-DOPA treatment of Parkinsons disease and its exacerbation by the concomitant use of DA metabolism inhibitors.

Effects of (-)-nicotine and (-)-cotinine on 6-hydroxydopamine-induced oxidative stress and neurotoxicity: relevance for Parkinson's disease

In view of the apparent controversial properties of (-)-nicotine (NIC) in relation to both oxidative stress and neuroprotection, we studied the effects of NIC on hydroxyl radical (*OH) formation, oxidative stress production by 6-hydroxydopamine (6-OHDA) autoxidation in the presence and absence of ascorbate, and 6-OHDA neurotoxicity. Both NIC and (-)-cotinine (COT) exhibited increased *OH production during 6-OHDA autoxidation. Although the same effect was observed in *OH generation by the Fenton reaction (H2O2 + Fe2+), this reaction was completely prevented with the previous incubation of Fe2+ with NIC or COT. Furthermore, both NIC and COT demonstrated a capacity to be able to reduce the TBARS formation provoked in rat brain mitochondrial preparations by 6-OHDA autoxidation. This effect is assumed as a consequence of the action of NIC and COT on lipid peroxidation propagation. We treated with NIC (1mg/kg, i.p.) two 6-OHDA-induced rat models of Parkinsons disease. However, only in one of these models did we obtain clear evidence of a neuroprotective effect of NIC on nigrostriatal terminals, as revealed by immunohistochemistry against tyrosine hydroxylase. Thus, the antioxidant properties of both NIC and COT in relation to the lipid peroxidation induced by 6-OHDA autoxidation, together with their reported capacity to prevent the Fenton reaction, probably by sequestration of Fe2+, may contribute to an understanding of its neuroprotective properties. In addition, the reported capacity of both NIC and COT to increase the production of *OH by 6-OHDA autoxidation might help explain the controversial observation found under different experimental conditions.

Angiotensin type-1-receptor antagonists reduce 6-hydroxydopamine toxicity for dopaminergic neurons

Angiotensin II activates (via type 1 receptors) NAD(P)H-dependent oxidases, which are a major source of superoxide, and is relevant in the pathogenesis of several cardiovascular diseases and certain degenerative changes associated with ageing. Given that there is a brain renin-angiotensin system and that oxidative stress is a key contributor to Parkinsons disease, we investigated the effects of angiotensin II and angiotensin type 1 (AT(1)) receptor antagonists in the 6-hydroxydopamine model of Parkinsons disease. Rats subjected to intraventricular injection of 6-hydroxydopamine showed bilateral reduction in the number of dopaminergic neurons and terminals. Injection of angiotensin alone did not induce any significant effect. However, angiotensin increased the toxic effect of 6-hydroxydopamine. Rats treated with the AT(1) receptor antagonist ZD 7155 and then 6-hydroxydopamine (with or without exogenous administration of angiotensin) showed a significant reduction in 6-hydroxydopamine-induced oxidative stress (lipid peroxidation and protein oxidation) and dopaminergic degeneration. Dopaminergic degeneration was also reduced by the NAD(P)H inhibitor apocynin. Angiotensin may play a pivotal role, via AT(1) receptors, in increasing the oxidative damage of dopaminergic cells, and treatment with AT(1) antagonists may reduce the progression of Parkinsons disease.

Effects of aluminum and zinc on the oxidative stress caused by 6-hydroxydopamine autoxidation: relevance for the pathogenesis of Parkinson's disease.

Aluminum and zinc have been related to the pathogenesis of Parkinsons disease (PD), the former for its neurotoxicity and the latter for its apparent antioxidant properties. 6-Hydroxydopamine (6-OHDA) is an important neurotoxin putatively involved in the pathogenesis of PD, its neurotoxicity often being related to oxidative stress. The potential effect of these metals on the oxidative stress induced by 6-OHDA autoxidation and the potential of ascorbic acid (AA), cysteine, and glutathione to modify this effect were investigated. Both metals, particularly Al3+, induced a significant reduction in *OH production by 6-OHDA autoxidation. The combined action of AA and a metal caused a significant and sustained increase in *OH generation, particularly with Al3+, while the effect of sulfhydryl reductants was limited to only the first few minutes of the reaction. However, both Al3+ and Zn2+ provoked a decrease in the lipid peroxidation induced by 6-OHDA autoxidation using mitochondrial preparations from rat brain, assessed by TBARS formation. In the presence of AA, only Al3+ induced a significant reduction in lipid peroxidation. After intrastriatal injections of 6-OHDA in rats, tyrosine hydroxylase immunohistochemistry revealed that Al3+ reduces 6-OHDA-induced dopaminergic lesion in the striatum, which corroborates the involvement of lipid peroxidation in 6-OHDA neurotoxicity and appears to discard the participation of this mechanism on PD by Al3+ accumulation. The previously reported antioxidant properties of Zn2+ appear to be related to the induction of Zn2+-containing proteins and not to the metal per se.

Discovery of a novel class of potent coumarin monoamine oxidase B inhibitors: development and biopharmacological profiling of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one methanesulfonate (NW-1772) as a highly potent, selective, reversible, and orally active monoamine oxidase B inhibitor.

In an effort to discover novel selective monoamine oxidase (MAO) B inhibitors with favorable physicochemical and pharmacokinetic profiles, 7-[(m-halogeno)benzyloxy]coumarins bearing properly selected polar substituents at position 4 were designed, synthesized, and evaluated as MAO inhibitors. Several compounds with MAO-B inhibitory activity in the nanomolar range and excellent MAO-B selectivity (selectivity index SI > 400) were identified. Structure-affinity relationships and docking simulations provided valuable insights into the enzyme-inhibitor binding interactions at position 4, which has been poorly explored. Furthermore, computational and experimental studies led to the identification and biopharmacological characterization of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one methanesulfonate 22b (NW-1772) as an in vitro and in vivo potent and selective MAO-B inhibitor, with rapid blood-brain barrier penetration, short-acting and reversible inhibitory activity, slight inhibition of selected cytochrome P450s, and low in vitro toxicity. On the basis of this preliminary preclinical profile, inhibitor 22b might be viewed as a promising clinical candidate for the treatment of neurodegenerative diseases.

Angiotensin-converting enzyme inhibition reduces oxidative stress and protects dopaminergic neurons in a 6-hydroxydopamine rat model of Parkinsonism.

It is now established that the brain possesses a local renin‐angiotensin system and that angiotensin II exerts multiple actions in the nervous system, including regulation of striatal dopamine release. Furthermore, angiotensin activates NADPH‐dependent oxidases, which are a major source of superoxide, and angiotensin‐converting enzyme inhibitors, commonly used in the treatment of hypertension and chronic heart failure, have shown antioxidant properties in several tissues. Oxidative stress is a key contributor to the pathogenesis and progression of Parkinsons disease. In the present study, we treated rats with intraventricular injections of the dopaminergic neurotoxin 6‐hydroxydopamine and subcutaneous injections of the angiotensin‐converting enzyme inhibitor Captopril to study the possible neuroprotective effect of the latter on the dopaminergic system and on 6‐hydroxydopamine‐induced oxidative stress. Rats treated with Captopril and 6‐hydroxydopamine showed significantly less reduction in the number of dopaminergic neurons (i.e., immunoreactive to tyrosine hydroxylase) in the substantia nigra and in the density of striatal dopaminergic terminals than 6‐hydroxydopamine‐lesioned rats not treated with Captopril. In addition, Captopril reduced the levels of major oxidative stress indicators (i.e., lipid peroxidation and protein oxidation) in the ventral midbrain and the striatum of 6‐hydroxydopamine‐lesioned rats. Our results suggest that angiotensin‐converting enzyme inhibitors may be useful for treatment of Parkinsons disease and that further investigation should focus on the neuroprotective capacity of these compounds.

Reduction of dopaminergic degeneration and oxidative stress by inhibition of angiotensin converting enzyme in a MPTP model of parkinsonism

There is growing evidence indicating that oxidative stress is a key contributor to the pathogenesis and progression of Parkinsons disease. The brain, and particularly the basal ganglia, possesses a local rennin-angiotensin system. Angiotensin activates NAD(P)H-dependent oxidases, which are a major intracellular source of superoxide, and angiotensin converting enzyme inhibitors (ACEIs) have shown antioxidant properties. We treated mice with MPTP and the ACEI captopril to study the possible neuroprotective and antioxidant effects of the latter on the dopaminergic system. Pre-treatment with captopril induced a significant reduction in the MPTP-induced loss of dopaminergic neurons in the substantia nigra and a significant reduction in the loss of dopaminergic terminals in the striatum. Furthermore, captopril reduced the MPTP-induced increase in the levels of major oxidative stress indicators (i.e. lipid peroxidation and protein oxidation) in the ventral midbrain and the striatum. Captopril did not reduce striatal MPP(+) levels, MAO-B activity or dopamine transporter activity, which may reduce MPTP neurotoxicity. Our results suggest that angiotensin-converting enzyme inhibitors may be useful for treatment of Parkinsons disease, and that further investigation should focus on the neuroprotective capacity of these compounds.

Antioxidant properties of dimethyl sulfoxide and its viability as a solvent in the evaluation of neuroprotective antioxidants

INTRODUCTION Dimethyl sulfoxide is an amphiphilic compound whose miscibility with water and its ability to dissolve lipophilic compounds make it an appreciated solvent in biomedical research. However, its reported antioxidant properties raise doubts about its use as a solvent in evaluating new antioxidants. The goal of this investigation was to evaluate its antioxidant properties and carry out a comparative study on the antioxidant properties of some known neuroprotective antioxidants in the presence and absence of dimethyl sulfoxide. METHODS The antioxidant properties of dimethyl sulfoxide were studied in rat brain homogenates by determining its ability to reduce both lipid peroxidation (TBARS formation) and protein oxidation (increase in protein carbonyl content and decrease in free thiol content) induced by ferrous chloride/hydrogen peroxide. Its ability to reduce the production of hydroxyl radicals by 6-hydroxydopamine autoxidation was also estimated. The same study was also performed with three known antioxidants (α-phenyl-N-tert-butylnitrone; 2-methyl-2-nitrosopropane; 5,5-dimethyl-1-pyrroline N-oxide) in the presence and absence of dimethyl sulfoxide. RESULTS Our results showed that dimethyl sulfoxide is able to reduce both lipid peroxidation and protein carbonyl formation induced by ferrous chloride/hydrogen peroxide in rat brain homogenates. It can also reduce the production of hydroxyl radicals during 6-hydroxydopamine autoxidation. However, it increases the oxidation of protein thiol groups caused by ferrous chloride/hydrogen peroxide in rat brain homogenate. DISCUSSION Despite the here reported antioxidant and pro-oxidant properties of dimethyl sulfoxide, the results obtained with α-phenyl-N-tert-butylnitrone, 2-methyl-2-nitrosopropane, and 5,5-dimethyl-1-pyrroline N-oxide corroborate the antioxidant properties attributed to these compounds and support the potential use of dimethyl sulfoxide as a solvent in the study of the antioxidant properties of lipophilic compounds. CONCLUSION Dimethyl sulfoxide is a very useful solvent that may be used at relatively low concentrations in the development of new antioxidants with neuroprotective properties.

Brain oxidative stress and selective behaviour of aluminium in specific areas of rat brain: potential effects in a 6-OHDA-induced model of Parkinson’s disease

J. Neurochem. (2009) 109, 879–888.