Moreover, free radicals' cigarette smoke is a complex chemical system and there are many potential pathways for these species to interact with one another and with biopolymers in a smoker's lung. There is ample evidence that the free radical system plays a significant role in cigarette smoke toxicology. It is becoming increasingly strong and necessary to try to place some possibilities in perspective [28, 29].

Chronic alcohol abuse appears to be linked to increased serum levels of reactive oxygen species, such as hydroperoxides, with a normal antioxidant capacity. The study performed by Trotti et al. [30] suggested that both alterations in the redox balance and a thrombophilic condition can be observed in heavy drinkers without severe liver diseases, such as cirrhosis, hepatitis, and hepatitis C virus (HCV).

Oxidative stress by impaired cell respiration increased oxidant capacity in strenuous exercise. Physical activity increases the free radicals in several ways, such as oxidative phosphorylation enhances in answer to exercise; therefore, there is a simultaneous production of free radicals. Catecholamines, issued when exercising, can produce free radicals. The free radicals that enhance with exercise are produced by the metabolism of prostanoids, by xanthine oxidase, NADPH (Nicotinamide Adenine dinucleotide Phosphate-Oxidase) oxidase, and other secondary sources, for example, by macrophages hired to fix the injured tissue that can produce free radicals [31].

Antioxidant supplements are sold and used by athletes to counteroxidative stress due to strenuous exercise. It is not yet clearif strenuous exercise increases the need for supplementary antioxidants in the diet. When the rise of free radicals exceeds the capacity of antioxidants to neutralize them, the radicals have the cellular components as targets in particular lipids. The lipid attack starts a chain reaction known as lipid peroxidation, which results in formation of a high number of radicals and reactive species that can damage other cellular components. The organism is able to bear a limited increase of free radicals; indeed, the evidence suggests that an ROS enhancement is needed for a muscle adjustment [32, 33].

In a study of Chen and Kotani [34], oxidative stress in premenopausal women with oral contraceptive therapy, which is now commonly used in millions of women worldwide, was investigated. This treatment is associated with an increased risk of deep vein thrombosis, venous thromboembolism, and stroke, so it is critically important to evaluate risks and benefits of therapies. The results of the study show that use of oral contraceptive therapy may increase oxidative stress levels, independently on traditional cardiovascular risk factors, in premenopausal women, providing new perspectives to the prevention of vascular complications in these subjects.

The cells normally generate reactive oxygen species (ROS) during energy metabolism for the respiratory chain. ROS at low or moderate concentrations have important physiological roles. However, an excessive enhancement of ROS in conditions of oxidative stress may be very harmful. The central nervous system (CNS) is helpless to oxidative stress for its high oxygen consume, for the weak antioxidant systems and terminal differentiation of neurons. Therefore, oxidative stress causes several neurodegenerative illnesses. Also, chemotherapy can cause serious side effects on the CNS and peripheral nervous system (PNS) in treated cancer patients, and then several studies show the involvement of ROS in the neurotoxicity induced by drugs. For this reason, the growth of neuroprotective drugs such as antioxidants can be considered a beneficial strategy for clinical treatment [35].

The worldwide incidence of diabetes mellitus (DM) has recently increased rapidly due to lifestyle changes, with DM projected to affect over 300 million people in the last years. DM is related to several complications and poor quality of life of the affected patients; this also leads to an increase in health spending. DM can lead not only to microangiopathy (related to major complications of diabetes, such as diabetic retinopathy, nephropathy, and neuropathy) but can also be considered a major risk factor for macroangiopathy, like coronary heart and cerebrovascular diseases. Moreover, oxidative stress can be an important factor for the development and progression of diabetic complications, related to insulin resistance and reduced insulin secretion with consequent development of DM. Therefore, the oxidative stress in DM is an important factor involved in the development of diabetic complications and in that of the same DM [36].

Numerous studies have shown that the transition metals could be affected in the pathogenesis of various neurodegenerative diseases for their ability to produce oxidative stress. Alzheimer's disease is the most common cause of dementia in older people. The metals, such as iron and copper, which can catalyze the Fenton's reaction by reactive oxygen species, are highly concentrated within the neuritic plaques that represent the features of the Alzheimer's disease brain.

A large body of experimental and *postmortem* findings indicates that Alzheimer's disease is associated with increased oxidative stress levels in the brain. Despite the current limitations of oxidative stress assessment in living subjects, recent data suggest that oxidative challenge might increase early both in the central nervous system and peripheral fluids [37].

## **7. Antioxidants and disease prevention**

It is known that elevated levels of blood reactive oxygen species are correlated with the severity of periodontitis. An improvement of clinical parameters in chronic periodontitis patients, through a nonsurgical periodontal treatment, is able to determine a decrease in blood reactive

Moreover, free radicals' cigarette smoke is a complex chemical system and there are many potential pathways for these species to interact with one another and with biopolymers in a smoker's lung. There is ample evidence that the free radical system plays a significant role in cigarette smoke toxicology. It is becoming increasingly strong and necessary to try to place

Chronic alcohol abuse appears to be linked to increased serum levels of reactive oxygen species, such as hydroperoxides, with a normal antioxidant capacity. The study performed by Trotti et al. [30] suggested that both alterations in the redox balance and a thrombophilic condition can be observed in heavy drinkers without severe liver diseases, such as cirrhosis,

Oxidative stress by impaired cell respiration increased oxidant capacity in strenuous exercise. Physical activity increases the free radicals in several ways, such as oxidative phosphorylation enhances in answer to exercise; therefore, there is a simultaneous production of free radicals. Catecholamines, issued when exercising, can produce free radicals. The free radicals that enhance with exercise are produced by the metabolism of prostanoids, by xanthine oxidase, NADPH (Nicotinamide Adenine dinucleotide Phosphate-Oxidase) oxidase, and other secondary sources, for example, by macrophages hired to fix the injured tissue that can produce

Antioxidant supplements are sold and used by athletes to counteroxidative stress due to strenuous exercise. It is not yet clearif strenuous exercise increases the need for supplementary antioxidants in the diet. When the rise of free radicals exceeds the capacity of antioxidants to neutralize them, the radicals have the cellular components as targets in particular lipids. The lipid attack starts a chain reaction known as lipid peroxidation, which results in formation of a high number of radicals and reactive species that can damage other cellular components. The organism is able to bear a limited increase of free radicals; indeed, the evidence suggests

In a study of Chen and Kotani [34], oxidative stress in premenopausal women with oral contraceptive therapy, which is now commonly used in millions of women worldwide, was investigated. This treatment is associated with an increased risk of deep vein thrombosis, venous thromboembolism, and stroke, so it is critically important to evaluate risks and benefits of therapies. The results of the study show that use of oral contraceptive therapy may increase oxidative stress levels, independently on traditional cardiovascular risk factors, in premenopausal women, providing new perspectives to the prevention of vascular complications in

The cells normally generate reactive oxygen species (ROS) during energy metabolism for the respiratory chain. ROS at low or moderate concentrations have important physiological roles. However, an excessive enhancement of ROS in conditions of oxidative stress may be very

that an ROS enhancement is needed for a muscle adjustment [32, 33].

oxygen species [27].

128 Free Radicals and Diseases

free radicals [31].

these subjects.

some possibilities in perspective [28, 29].

hepatitis, and hepatitis C virus (HCV).

Numerous studies show that diets high in fruits and vegetables are protective against cardiovascular diseases (CVD), several kinds of cancer, and other chronic diseases. However, although a broad consensus, it is still unclear what their mechanism(s) of action enable the protection against certain diseases. The antioxidant hypothesis connects the high content of antioxidant molecules found in plant foods and their health benefits by a direct impact on the decrease of oxidative stress.

Some clinical studies have shown contrasting results: some showing protective effects, while others do not. Antioxidants do not act just in isolation or in synergistic interactions; it should be taken into account that in part they are involved in the antioxidant regeneration. The data emerged from these studies are that dietary and endogenous antioxidants, with various activities and features, act synergistically contributing to the overall effect of protective plant foods.

The efficacy of the nonenzymatic antioxidant barrier can be evaluated by determining the total antioxidant capacity (TAC), termed as moles of oxidizing neutralized by 1 L of the sample tested. TAC treats the cumulative action of all antioxidants in the matrix, thus providing an integrated parameter instead of the simple sum for measurable antioxidants, giving a view balance among antioxidant molecules. Some experiments have shown that plasma TAC of patients with various chronic diseases such as diabetes, AIDS, ulcerative colitis, Crohn's disease, meningitis, cardiovascular diseases, colorectal, lung and breast cancer, is much lower than in healthy controls, suggesting impairment of antioxidant barrier in the development of these pathologies.

In order to optimize the intake of dietary antioxidants, particular attention should be paid to the possibility that the interaction between foods consumed in a meal might affect the *in vivo* efficiency of dietary antioxidants [38].

Lycopenes, which represent more than 80% of the total tomato carotenoids, can reduce the risk of cardiovascular disease by inhibiting cholesterol synthesis, reducing the expression of cell surface adhesion molecules and the binding of monocytes to endothelial cells, and modulating LDL (low-density lipoprotein) susceptibility to oxidation. *In vitro* studies demonstrated that the highest beneficial effects as a cancer preventive of lycopene in the diet occur when it is associated with other compounds. A recent study suggests that α-tocopherol or whole tomato lipophilic extracts (containing more than 80% lycopene along with other compounds) potentiate the effects of lycopene during oxidative stress [39].

Neurons are particularly prone to oxidative stress. Particularly, ROS were shown implicated in the pathology of a number of neurological disorders. The brain, mostly neuronal plasmamembranes, houses large concentrations of polyunsaturated fatty acids, which may undergo lipid peroxidation in such an oxygen-rich environment. Brain catecholamines easily undergo auto-oxidation phenomena, thus generating reactive oxygen species. Furthermore, brain contains conspicuous amount of iron (a powerful catalyst of free radical generation), although in an inactive form (chelated). Physiologically, brain exhibits low antioxidant defenses (vitamin C, vitamin E, glutathione, and superoxide dismutase); moreover, reduced levels of antioxidants such as vitamins E and C have been reported in many neurological conditions. It was demonstrated that vitamin E supplementation in deficient individuals is able to either prevent or at least halt the progression of many neurological features. However, supplementation of vitamin E in patients suffering from Parkinson's disease had no apparent benefits [40].

The study has compared the activity and bioavailability of some antioxidants, which have been used in doses very close to those of an average daily meal. Three different formulations (F1, F2, and F3) were tried. Each one was prepared both in fluid and dry formulations and given to the same group of subjects for 1 week. The antioxidants provided in combination with a dosage near to one RDA (Recommended Daily Allowance) decreased oxidative stress, and the fluid formulation was found to be more active and bioavailable than the dry one. The antioxidants present in F1 are those with affinity for membranes (vitamin A, vitamin E, and carotene), minerals (selenium and zinc), components of antioxidant enzymes, and L-cysteine, which is needed for the synthesis of glutathione peroxidase. In F2, the antioxidants comprise circulating substances (vitamin C, bioflavonoids, and vitamin B-6) and a cytosol antioxidant (coenzyme Q10). In this study, F1 was significantly more active than F2, and F3 enhances the F1 activity without a true synergism. Nevertheless, especially in healthy subjects, the existence of a "roof" effect of antioxidants cannot be ruled. The antioxidant activity can be much more evident in subjects with a chronic oxidative stress. A group of antioxidants in low doses decreased oxidative stress as highlighted by the values expressed as U CARR. Since oxidative stress is important for the prevention and/or treatment of an illness, the dROMs' test appears to be a suitable instrument with which to identify the type and dose of antioxidants [41].

emerged from these studies are that dietary and endogenous antioxidants, with various activities and features, act synergistically contributing to the overall effect of protective plant

The efficacy of the nonenzymatic antioxidant barrier can be evaluated by determining the total antioxidant capacity (TAC), termed as moles of oxidizing neutralized by 1 L of the sample tested. TAC treats the cumulative action of all antioxidants in the matrix, thus providing an integrated parameter instead of the simple sum for measurable antioxidants, giving a view balance among antioxidant molecules. Some experiments have shown that plasma TAC of patients with various chronic diseases such as diabetes, AIDS, ulcerative colitis, Crohn's disease, meningitis, cardiovascular diseases, colorectal, lung and breast cancer, is much lower than in healthy controls, suggesting impairment of antioxidant barrier in the development of

In order to optimize the intake of dietary antioxidants, particular attention should be paid to the possibility that the interaction between foods consumed in a meal might affect the *in vivo*

Lycopenes, which represent more than 80% of the total tomato carotenoids, can reduce the risk of cardiovascular disease by inhibiting cholesterol synthesis, reducing the expression of cell surface adhesion molecules and the binding of monocytes to endothelial cells, and modulating LDL (low-density lipoprotein) susceptibility to oxidation. *In vitro* studies demonstrated that the highest beneficial effects as a cancer preventive of lycopene in the diet occur when it is associated with other compounds. A recent study suggests that α-tocopherol or whole tomato lipophilic extracts (containing more than 80% lycopene along with other compounds) poten-

Neurons are particularly prone to oxidative stress. Particularly, ROS were shown implicated in the pathology of a number of neurological disorders. The brain, mostly neuronal plasmamembranes, houses large concentrations of polyunsaturated fatty acids, which may undergo lipid peroxidation in such an oxygen-rich environment. Brain catecholamines easily undergo auto-oxidation phenomena, thus generating reactive oxygen species. Furthermore, brain contains conspicuous amount of iron (a powerful catalyst of free radical generation), although in an inactive form (chelated). Physiologically, brain exhibits low antioxidant defenses (vitamin C, vitamin E, glutathione, and superoxide dismutase); moreover, reduced levels of antioxidants such as vitamins E and C have been reported in many neurological conditions. It was demonstrated that vitamin E supplementation in deficient individuals is able to either prevent or at least halt the progression of many neurological features. However, supplementation of vitamin E in patients suffering from Parkinson's disease had no apparent benefits [40].

The study has compared the activity and bioavailability of some antioxidants, which have been used in doses very close to those of an average daily meal. Three different formulations (F1, F2, and F3) were tried. Each one was prepared both in fluid and dry formulations and given to the same group of subjects for 1 week. The antioxidants provided in combination with a dosage near to one RDA (Recommended Daily Allowance) decreased oxidative stress, and the fluid formulation was found to be more active and bioavailable than the dry one. The antiox-

foods.

130 Free Radicals and Diseases

these pathologies.

efficiency of dietary antioxidants [38].

tiate the effects of lycopene during oxidative stress [39].

Ascorbic acid (ascorbate or vitamin C) has a controversial history in cancer treatment. Pharmacologic concentrations of ascorbate, only achievable by intravenous (i.v.) administration, produce H2O2, causing cancer and cell death *in vitro* [42]. Parenteral administration of ascorbate in pharmacologic doses produces millimolar concentrations in blood and extracellular fluid, with preferential generation of Asc•, the product of a loss of one electron from ascorbate, and H2O2 in extracellular fluid but not blood. When ascorbate is given parenterally, Asc• is detected preferentially in extracellular fluid compared with blood. Asc• generation in extracellular fluid depended on the ascorbate dose and the resulting concentrations. These findings are all consistent with the hypothesis that pharmacologic ascorbate concentrations *in vivo* serve as a prodrug for selective delivery of H2O2 to the extracellular space. In humans, these experiments are based on principles of tight control of ascorbate. After oral ingestion, control of intracellular and extracellular ascorbate concentrations is mediated by three mechanisms: intestinal absorption, tissue transport, and renal reabsorption. These three mechanisms work in coordination with each other, ensuring that ascorbate is tightly controlled. Parenteral administration bypasses tight control, which is restored as kidneys excrete ascorbate. The results demonstrate an explanation on why tight control happens. If the tight control is exceeded, H2O2 is formed into the extracellular space. When tight control is reset, the production of H2O2 stops. If there was no tight control, the formation of H2O2 and exposure to it could be steady, with disagreeable impact on division and cell growth. Tight control prevents continued exposure of tissues to high concentrations of H2O2. Bypassing provisionally the tight control with the ascorbate parenteral administration, H2O2 is able to form for only a fair period of time, decreasing the damage, and gives a drug for therapeutic of i.v. use of ascorbate [43].

Also, endothelium performs an important role in the regulation of vascular tone, platelet activity, leukocyte adhesion, and thrombosis, and is also implicated in the development of atherosclerosis. In patients with determined coronary heart disease or coronary risk factor, endothelialdysfunctionwasobserved.Treatmentwithlipidloweringdrugs,ACE(Angiotensin Converting Enzyme inhibitor) inhibitors, physical activity, and antioxidant agents has shown an improvement in endothelial function in the coronary and peripheral vessels. Vitamin C is a very efficient antioxidant, and is a scavenger of several reactive oxygen species, such as superoxide anion and peroxynitrite. Several researches have demonstrated that the beneficial effect of vitamin C (24 mg/min) on endothelial dysfunction in subjects with risk factors or coronary artery disorders is specific, because it was observed neitherin healthy control subjects nor on the endothelium-independent vasodilation induced by nitroglycerin or SNP (Sodium Nitroprusside) [44].

## **8. Conclusions**

An unhealthy diet, alcohol abuse, chronic intake of drugs, cigarette smoking, inadequate exercise, and environmental pollution are just some causes of a particular form of "stress" which experts have called "oxidative stress" [45]. It is very different and definitely more dangerous than the more common "emotional distress" that affects every day much of the population in Western countries with high economic level [46]. Oxidative stress is a form of "chemical stress" induced by the presence, in our organism, of high quantities of harmful substances acting as oxidants, whose members are the most dangerous oxygen free radicals [47].

Oxidative stress is considered responsible for premature and many diseases ranging from hypertension to atherosclerosis, infarction to stroke, from Parkinson's to Alzheimer's disease, from colitis to pancreatitis, from obesity to diabetes, from chronic bronchitis to rheumatoid arthritis, from AIDS to several forms of cancer [48, 49]. Oxidative stress is much more sneaky, because it gives rise to characteristic symptoms, or to a particular clinical picture, because its causes are to be found in "invisible" entities, such as free radicals [50]. Therefore, the clinician could not suspect the existence; oxidative stress does not provide any evidence to suggest a more detailed diagnosis, when performing simple laboratory tests allow to understand immediately the problem, avoiding the patient a series of consequences such compromise the duration and/or the quality of life in the short or medium term. It is not currently provided for the execution of any preliminary laboratory tests, although available for clinical routine, to show—by means of the quantification in the blood of suitable biochemical markers—the objective need for such formulations. While it is now known that a cholesterol-lowering drug is taken only after a test that has documented a high blood level of cholesterol, it is now an increasingly widespread tendency to assume antioxidants even without the documentation in the blood, an increase in the level of free radicals and/or a reduction of its "physiological" antioxidant defenses. It is not yet a good practice to perform a preliminary evaluation of the laboratory of oxidative stress.

However, the scientific evidence supports that only adequate assessment biochemistry may allow the identification and the definition of a state of oxidative stress and make monitoring of a possible antioxidant therapy. Because of these specific tests for the evaluation and determination of free radicals and antioxidant defenses, it makes the initial diagnosis of oxidative stress extremely accurate and reliable, whether the two opposite components, either pro- or antioxidant, are measured separately [51]. It is possible to determine in real time whether oxidative stress is due to an increased production and/or a reduced ability to eliminate free radicals. It would be appropriate to undergo the oxidative stress evaluation, even in good health and, more so, when exposed to pro-oxidant factors (e.g., incorrect lifestyles, excessive aerobic exercise, and pollutants in the workplace) or when affected from chronic degenerative diseases (e.g., diabetes, atherosclerosis, cancer, dementia, and rheumatoid arthritis) or, eventually, when performed specific treatments (e.g., dialysis, by-pass, organ transplant, radiotherapy, and chemotherapy) [52, 53]. For this evaluation it will be possible to use specific therapies and to monitor the real efficacy of antioxidants, too often assumed without a preliminary test able to demonstrate its necessity. The same "prescription" of supplements, finally, will lean—in this sensitive field—on a more solid and leave the empirical phase in which it often finds itself.

The evaluation of a real state of oxidative stress may be covered in the predictive medicine area. Predictive medicine is the emerging field of medicine that entails predicting the probability of disease and taking proactive steps to either prevent the disease altogether or significantly decrease its impact upon the patient (such as by preventing mortality or limiting morbidity). The aim of predictive medicine is to predict the likelihood of disease so that healthcare providers and the patients will have an active role in changing the way of life and increase the medical surveillance, such as complete biannual skin examinations by a dermatologist or internist if their patient is found to have an increased risk of melanoma; an ECG and cardiac examination by a cardiologist if a patient has an increased risk of cardiac arrhythmia or alternating magnetic resonance imaging (MRI); or mammograms if a patient has an increased risk of breast cancer. Predictive medicine is useful for both healthy individuals (predictive health) and those with illnesses (predictive medicine); its aim is to provide information on the possibility of having a disease and to predict the progression and treatment for a specific disease. Aside from genetic testing, predictive medicine utilizes a wide variety of tools to predict health and disease, including assessments of exercise, nutrition, spirituality, quality of life, and oxidative stress.

## **Author details**

coronary artery disorders is specific, because it was observed neitherin healthy control subjects nor on the endothelium-independent vasodilation induced by nitroglycerin or SNP (Sodium

An unhealthy diet, alcohol abuse, chronic intake of drugs, cigarette smoking, inadequate exercise, and environmental pollution are just some causes of a particular form of "stress" which experts have called "oxidative stress" [45]. It is very different and definitely more dangerous than the more common "emotional distress" that affects every day much of the population in Western countries with high economic level [46]. Oxidative stress is a form of "chemical stress" induced by the presence, in our organism, of high quantities of harmful substances acting as oxidants, whose members are the most dangerous oxygen free radicals

Oxidative stress is considered responsible for premature and many diseases ranging from hypertension to atherosclerosis, infarction to stroke, from Parkinson's to Alzheimer's disease, from colitis to pancreatitis, from obesity to diabetes, from chronic bronchitis to rheumatoid arthritis, from AIDS to several forms of cancer [48, 49]. Oxidative stress is much more sneaky, because it gives rise to characteristic symptoms, or to a particular clinical picture, because its causes are to be found in "invisible" entities, such as free radicals [50]. Therefore, the clinician could not suspect the existence; oxidative stress does not provide any evidence to suggest a more detailed diagnosis, when performing simple laboratory tests allow to understand immediately the problem, avoiding the patient a series of consequences such compromise the duration and/or the quality of life in the short or medium term. It is not currently provided for the execution of any preliminary laboratory tests, although available for clinical routine, to show—by means of the quantification in the blood of suitable biochemical markers—the objective need for such formulations. While it is now known that a cholesterol-lowering drug is taken only after a test that has documented a high blood level of cholesterol, it is now an increasingly widespread tendency to assume antioxidants even without the documentation in the blood, an increase in the level of free radicals and/or a reduction of its "physiological" antioxidant defenses. It is not yet a good practice to perform a preliminary evaluation of the

However, the scientific evidence supports that only adequate assessment biochemistry may allow the identification and the definition of a state of oxidative stress and make monitoring of a possible antioxidant therapy. Because of these specific tests for the evaluation and determination of free radicals and antioxidant defenses, it makes the initial diagnosis of oxidative stress extremely accurate and reliable, whether the two opposite components, either pro- or antioxidant, are measured separately [51]. It is possible to determine in real time whether oxidative stress is due to an increased production and/or a reduced ability to eliminate free radicals. It would be appropriate to undergo the oxidative stress evaluation, even in good health and, more so, when exposed to pro-oxidant

Nitroprusside) [44].

132 Free Radicals and Diseases

**8. Conclusions**

laboratory of oxidative stress.

[47].

Simona Tafuri1 , Natascia Cocchia1 , Francesco Landolfi2 , Eugenio Luigi Iorio3 and Francesca Ciani1\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy

2 ASL Na 3 Sud, Castellammare di Stabia, Naples, Italy

3 International Observatory of Oxidative Stress, Salerno, Italy

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#### **The Role of** *Attractin* **in Neurodegeneration Caused by Oxidative Stress The Role of** *Attractin* **in Neurodegeneration Caused by Oxidative Stress**

Ayuka Ehara, Shin-ichi Sakakibara and Shuichi Ueda Ayuka Ehara, Shin-ichi Sakakibara and Shuichi Ueda

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63330

#### **Abstract**

Oxidative stress is linked to dopaminergic (DA) neurodegeneration in Parkinson's disease. Our laboratory reported slowly progressive DA neurodegeneration in the zitter (*zi*) rat, which is *Attractin* (*Atrn*) deficient. However, little is known about the function of *Atrn* in the central nervous system (CNS). Thus, we investigated whether DA neurodegeneration in the *zi* rat was induced by oxidative stress, and how *Atrn* affects oxidative stress. First, we summarize our previous *in vivo* data, which revealed suppression of DA neurodegeneration using antioxidants (vitamin E and melatonin) in *zi* rats. Second, our current *ex vivo* and *in vitro* studies are introduced. Using primary neuronal cultures of *zi* mesencephalon as a model of *Atrn*-deficient neurons or *Atrn*-GFP-overexpressing HEK293 cells, accumulation of reactive oxygen species (ROS) in mitochondria and cell viability was examined under oxidative stress. *Atrn*-deficient neurons accumulated excess ROS in mitochondria, resulting in neurodegeneration, whereas *Atrn*-overexpressing cells showed suppression of ROS accumulation under oxidative stress. These results showed that *Atrn* plays a suppressive role against ROS and that the loss of *Atrn* function induced excess ROS accumulation and led to DA neurodegeneration. This is the first report to show that *Atrn* directly modulates mitochondrial ROS accumulation in the CNS.

**Keywords:** dopaminergic neurodegeneration, *Attractin*, reactive oxygen species, Parkinson's disease, oxidative stress

## **1. Introduction**

Oxidative stress is considered to be the cause of several neurological diseases. In particular, dopaminergic (DA) neurons are vulnerable to oxidative stress, which is normally generated

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

by dopamine metabolism [1, 2]. The progressive DA neurodegeneration that occurs with age in Parkinson's disease (PD) is caused by increased oxidative stress. The neurotoxins 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine and6-hydroxydopamine induce free radicals (H2O2, O2 – ,and OH) that destroy DA neurons [3, 4] and are used to produce acute animal models of PD. Recently, we reported slowly progressive DA neurodegeneration, which is similar to the symptoms of humans with PD, in a mutant rat, the *zitter* (*zi*) rat [5].

The *zi* rat, which occurred in a colony of Sprague-Dawley (SD) rats, is a spontaneous autosomal recessive mutant rat with body tremors. The gene symbol of the mutation is designated *zi* (zitter means shake and tremble in German). The rats develop the tremor at around 15 days of age and progressive flaccid paresis of hind limbs at 6 months of age. The behavioral defects are associated with progressive hypomyelination and vacuolation in the central nervous system (CNS) [6]. *Zi* is homozygous for an 8-bp deletion in intron 12 of *Attractin* (*Atrn*) [7]. *Atrn* is involved in regulating physiological processes such as initial immune cell clustering during an inflammatory response, melanocortin signaling pathways that regulate energy homeostasis, pigmentation, and normal myelination in the CNS. *Atrn* encodes a protein that is secreted or transmembrane form as a result of alternative splicing of the same mRNA. Both forms of the protein have several domains in common such as epidermal growth factor-related domains, a CUB domain, and a C-type lectin. The transmembrane form, which has a transmembrane domain, has a long N-terminal extracellular domain. Rats and humans express both secreted and transmembrane proteins. However, mice express only the transmembrane protein. The *zi* rat expresses neither the secreted nor the transmembrane form of Atrn proteins. The abnormalities in the CNS of *zi* rats are mainly caused by the loss of the transmembrane form [7].

Our laboratory first reported DA neurodegeneration that occurs with age in *zi* rats [5]. In particular, the nigrostriatal DA pathway shows the most severe progressive neurodegeneration. In the substantia nigra pars compacta (SNc), a significant decrease in the number of DA neurons is detected from 2 months of age in *zi* rats, whereas no difference is detected at 1 month of age between *zi* rats and wild-type SD rats [8, 9]. With age, the number of DA neurons decreases significantly [8] (**Figure 1**). Loss of DA neurons in the SNc results in a decrease in the number of DA fibers and terminals in the dorsolateral caudate putamen (CPu), which receives DA input from the SNc. Consequently, the level of dopamine in the CPu of *zi* rats at 12 months of age is decreased to one-seventh of that in age-matched SD rats [8].

**Figure 1.** Progressive dopaminergic neurodegeneration with age in *zi* rats. Tyrosine hydroxylase-immunoreactive cells in the substantia nigra at 1 month (A), 6 months (B), and 12 months (C) of age. Scale bar: 500 μm.

Previous studies have indicated that *zi* rats show abnormal activities of some antioxidant enzymes in the brain and tend to accumulate reactive oxygen species (ROS) [10–13]. Examination of the ultrastructure of DA neurons in the SNc of *zi* rats reveals abnormal mitochondria with disrupted and enlarged cristae [9]. Additionally, *Atrn* null mice show reduced complex IV activity (cytochrome c oxidase activity) [14]. Complex IV is an enzyme in the respiratory electron transport chain in mitochondria. It was suggested that *Atrn* regulated ROS production in mitochondria. Therefore, we hypothesized that the neurodegeneration in *zi* rats is due to oxidative stress, and thus, we investigated whether antioxidants can protect against neurodegeneration in the mutant. Furthermore, to investigate whether *Atrn* is directly involved in regulation of the ROS-producing system in neurons, we established primary neuronal cultures from *zi* rats to represent *Atrn-*deficit neurons, and an *Atrn*overexpressing cell line. Using these cells, we examined the viability and ROS accumulation under several oxidative stress conditions.

## **2. Materials and methods**

#### **2.1. Reagents**

by dopamine metabolism [1, 2]. The progressive DA neurodegeneration that occurs with age in Parkinson's disease (PD) is caused by increased oxidative stress. The neurotoxins 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine and6-hydroxydopamine induce free radicals

models of PD. Recently, we reported slowly progressive DA neurodegeneration, which is

The *zi* rat, which occurred in a colony of Sprague-Dawley (SD) rats, is a spontaneous autosomal recessive mutant rat with body tremors. The gene symbol of the mutation is designated *zi* (zitter means shake and tremble in German). The rats develop the tremor at around 15 days of age and progressive flaccid paresis of hind limbs at 6 months of age. The behavioral defects are associated with progressive hypomyelination and vacuolation in the central nervous system (CNS) [6]. *Zi* is homozygous for an 8-bp deletion in intron 12 of *Attractin* (*Atrn*) [7]. *Atrn* is involved in regulating physiological processes such as initial immune cell clustering during an inflammatory response, melanocortin signaling pathways that regulate energy homeostasis, pigmentation, and normal myelination in the CNS. *Atrn* encodes a protein that is secreted or transmembrane form as a result of alternative splicing of the same mRNA. Both forms of the protein have several domains in common such as epidermal growth factor-related domains, a CUB domain, and a C-type lectin. The transmembrane form, which has a transmembrane domain, has a long N-terminal extracellular domain. Rats and humans express both secreted and transmembrane proteins. However, mice express only the transmembrane protein. The *zi* rat expresses neither the secreted nor the transmembrane form of Atrn proteins. The abnormalities in the CNS of *zi* rats are mainly caused by the loss of the transmembrane

Our laboratory first reported DA neurodegeneration that occurs with age in *zi* rats [5]. In particular, the nigrostriatal DA pathway shows the most severe progressive neurodegeneration. In the substantia nigra pars compacta (SNc), a significant decrease in the number of DA neurons is detected from 2 months of age in *zi* rats, whereas no difference is detected at 1 month of age between *zi* rats and wild-type SD rats [8, 9]. With age, the number of DA neurons decreases significantly [8] (**Figure 1**). Loss of DA neurons in the SNc results in a decrease in the number of DA fibers and terminals in the dorsolateral caudate putamen (CPu), which receives DA input from the SNc. Consequently, the level of dopamine in the CPu of *zi* rats at 12 months of age is decreased to one-seventh of that in age-matched SD rats

**Figure 1.** Progressive dopaminergic neurodegeneration with age in *zi* rats. Tyrosine hydroxylase-immunoreactive cells

in the substantia nigra at 1 month (A), 6 months (B), and 12 months (C) of age. Scale bar: 500 μm.

similar to the symptoms of humans with PD, in a mutant rat, the *zitter* (*zi*) rat [5].

,and OH) that destroy DA neurons [3, 4] and are used to produce acute animal

(H2O2, O2

form [7].

[8].

–

140 Free Radicals and Diseases

Materials for cultures were B27 supplement, B27 supplement minus antioxidants (AO (–)), neurobasal medium, Opti-MEM, and penicillin/streptomycin from Gibco (Life Technologies; Carlsbad, CA); MitoTracker Red CM-H2Xros, MitoTracker green FM, and Lipofectamine Plus Reagent from Invitrogen (Life Technologies); cell proliferation reagent WST-1 from Roche Diagnostics (Basel, Switzerland); cytosine-arabinofuranoside, l-glutamine, and poly-l-lysine from Sigma-Aldrich (St. Louis, MO); pAcGFP1-N1 vector from Clontech Laboratories Inc. (Mountain View, CA). Reagents for the immunocytochemistry were purchased from the following sources: biotinylated horse anti-mouse IgG, normal goat serum, and vectastain ABC kit from Vector (Burlingame, CA); anti-tyrosine hydroxylase (TH) antibody from Incstar (Stillwater, MN); Alexa Fluor 488 goat anti-mouse IgG from Molecular Probes (Life Technologies).

#### **2.2.** *In vivo* **study**

Male *zitter* rats were housed in-groups of two or three in a cage with food and water ad libitum. The room was maintained at a constant temperature and humidity with a 12 h light/dark cycle in the Laboratory Animal Research Center at Dokkyo University School of Medicine. All procedures in this study were certified by the Animal Welfare Committee at Dokkyo University School of Medicine and were conducted in accordance with NIH guidelines. Between 1 and 10 months of age, we fed *zi* rats a diet supplied with vitamin E (500 mg d-, l-alpha-tocopherol/ kg diet) or administered oral melatonin at dose of 0.5 mg/ml in drinking water, as previously reported [13, 15]. Tissue preparation and immunohistochemical procedure were described previously [5].

#### **2.3.** *Ex vivo* **study**

Ventral mesencephalic neurons were prepared from fetal *zi* and SD rats (Charles River Laboratories, Tsukuba, Japan) at gestation day 14. Cells were seeded in 24-well plates at a density of 2 × 105 cells/cm2 . Wells (for the cell viability assay and ROS assay) or cover glasses (for immunofluorescence, 15-mm diameter) were pre-treated with poly-l-lysine (0.1 mg/ml). After 2 h of initial plating, the medium was changed to neurobasal medium (containing penicillin/streptomycin and l-glutamine) supplemented with B27. Cultures were maintained at 37°C in a humidified 5% CO2 incubator (Revco Ultima RCO5000T, Thermo Fisher Scientific; Asheville, NC). To obtain neuron-enriched cultures, cytosine-arabinofuranoside (7.5 μM) was added beginning at 2 days *in vitro* (DIV) to 6 DIV. At 6 DIV, neurons were divided into two groups and cultured in neurobasal medium supplemented with B27 (Cont) or B27 without antioxidants (AO (−)), consisting of B27 supplement without vitamin E, vitamin E acetate, superoxide dismutase (SOD), catalase, or glutathione (GSH). Cultures were used for experiments at 11 DIV. Over 95% of the cells in the cultures were neurons as determined by immunostaining for the neuron-specific marker microtubule-associated protein-2 (data not shown).

The cell viability assay was performed by using the cell proliferation reagent WST-1 according to the manufacturer's protocol. Cell viability was expressed as a percent of the values of SD-Cont.

ROS accumulation was monitored with a plate reader using MitoTracker Red CM-H2Xros (MTR). Neurons were co-stained with 0.5 μM MTR and 0.2 μM MitoTracker green (MTG: mitochondria marker) in Opti-MEM for 30 min at 37°C and then assessed (MTR, excitation 535 nm, barrier filter 595 nm; MTG, excitation 485 nm, barrier filter 535 nm). The MTR/MTG ratio of each group was normalized to that of SD-Cont, which was used as an index of ROS content.

Immunofluorescence procedure was described previously [16].

#### **2.4.** *In vitro* **study**

*Atrn* cDNA, which encodes full-length membrane-type rat *Atrn*, was cleaved from pCAGGS-neo-*Atrn* (kind gift from Dr. Kenzo Sato, Tottori University) and inserted into the pAcGFP1-N1 vector as a ~4.3-kb *Eco* R1-*Bam* HI fragment. After cloning, insertion of the fusion sequence into the plasmid was confirmed. HEK293 cells (1.5 × 104 cells/10-mm glass bottom dish) were transfected with 0.1 μg *Atrn*-GFP or GFP (as a control) plasmid using Lipofectamine Plus Reagent. At 18 h after transfection, cells were incubated with 0.5 μM MTR for 30 min at 37°C. Fluorescence images of living cells were scanned with a confocal laser-scanning microscope (LSM 510 META; Carl Zeiss, Jena, Germany) using a 60× 1.4-NA oil-immersion objective. Under the microscope, cells were exposed to 1 mM H2O2 in Opti-MEM and observed for 60 min.

## **3. Results**

**2.3.** *Ex vivo* **study**

142 Free Radicals and Diseases

ty of 2 × 105 cells/cm2

shown).

Cont.

content.

**2.4.** *In vitro* **study**

MEM and observed for 60 min.

Ventral mesencephalic neurons were prepared from fetal *zi* and SD rats (Charles River Laboratories, Tsukuba, Japan) at gestation day 14. Cells were seeded in 24-well plates at a densi-

immunofluorescence, 15-mm diameter) were pre-treated with poly-l-lysine (0.1 mg/ml). After 2 h of initial plating, the medium was changed to neurobasal medium (containing penicillin/streptomycin and l-glutamine) supplemented with B27. Cultures were maintained at 37°C in a humidified 5% CO2 incubator (Revco Ultima RCO5000T, Thermo Fisher Scientific; Asheville, NC). To obtain neuron-enriched cultures, cytosine-arabinofuranoside (7.5 μM) was added beginning at 2 days *in vitro* (DIV) to 6 DIV. At 6 DIV, neurons were divided into two groups and cultured in neurobasal medium supplemented with B27 (Cont) or B27 without antioxidants (AO (−)), consisting of B27 supplement without vitamin E, vitamin E acetate, superoxide dismutase (SOD), catalase, or glutathione (GSH). Cultures were used for experiments at 11 DIV. Over 95% of the cells in the cultures were neurons as determined by immunostaining for the neuron-specific marker microtubule-associated protein-2 (data not

The cell viability assay was performed by using the cell proliferation reagent WST-1 according to the manufacturer's protocol. Cell viability was expressed as a percent of the values of SD-

ROS accumulation was monitored with a plate reader using MitoTracker Red CM-H2Xros (MTR). Neurons were co-stained with 0.5 μM MTR and 0.2 μM MitoTracker green (MTG: mitochondria marker) in Opti-MEM for 30 min at 37°C and then assessed (MTR, excitation 535 nm, barrier filter 595 nm; MTG, excitation 485 nm, barrier filter 535 nm). The MTR/MTG ratio of each group was normalized to that of SD-Cont, which was used as an index of ROS

*Atrn* cDNA, which encodes full-length membrane-type rat *Atrn*, was cleaved from pCAGGS-neo-*Atrn* (kind gift from Dr. Kenzo Sato, Tottori University) and inserted into the pAcGFP1-N1 vector as a ~4.3-kb *Eco* R1-*Bam* HI fragment. After cloning, insertion of the fusion sequence into the plasmid was confirmed. HEK293 cells (1.5 × 104 cells/10-mm glass bottom dish) were transfected with 0.1 μg *Atrn*-GFP or GFP (as a control) plasmid using Lipofectamine Plus Reagent. At 18 h after transfection, cells were incubated with 0.5 μM MTR for 30 min at 37°C. Fluorescence images of living cells were scanned with a confocal laser-scanning microscope (LSM 510 META; Carl Zeiss, Jena, Germany) using a 60× 1.4-NA oil-immersion objective. Under the microscope, cells were exposed to 1 mM H2O2 in Opti-

Immunofluorescence procedure was described previously [16].

. Wells (for the cell viability assay and ROS assay) or cover glasses (for

#### **3.1. The effect of antioxidants on DA neurodegeneration in** *zi* **rats**

We treated *zi* rats with the antioxidants vitamin E and melatonin. Between 1 and 10 months of age, we fed *zi* rats a diet supplied with vitamin E, which is a lipid-soluble peroxyl radical scavenger. Chronic vitamin E feeding suppressed DA neuron death in the SNc (**Figure 2B**). The number of DA neurons in *zi* rats treated with vitamin E was 1.5 times higher than that in control *zi* rats [15]. However, vitamin E protected few DA fibers and terminals in the CPu of *zi* rats (data not shown).

Melatonin, which is a synthetic product of the pineal gland, is a direct free radical scavenger and an indirect antioxidant. Melatonin readily crosses the blood-brain barrier and plays a role in protecting against ROS in the brain. Chronic melatonin administration suppressed DA neuron death in the SNc of *zi* rats (**Figure 2C**). The number of DA neurons in *zi* rats treated with melatonin was two times higher than that in control *zi* rats [13]. In the CPu, DA fibers and terminals were protected by melatonin, and *zi* rats administered chronic melatonin showed twice as much dopamine as control *zi* rats [8, 13]. Melatonin protected DA neuronal somas, fibers, and terminals in both the SNc and the CPu of *zi* rats.

**Figure 2.** Antioxidants suppressed dopaminergic neurodegeneration in *zi* rats. Tyrosine hydroxylase-immunoreactive cells in the substantia nigra at 10 months of age in *zi* rats (A) or *zi* rats treated with vitamin E (B) or melatonin (C) for 9 months. Scale bar: 500 μm.

Antioxidants were effective for preventing DA neurodegeneration in *zi* rats, indicating that the DA neurodegeneration in the mutant rats is caused by oxidative stress.

#### **3.2. Accumulation of ROS in** *zi* **mesencephalic neurons** *ex vivo*

To investigate whether ROS accumulation in mesencephalic neurons is caused by a genetic factor of the *Atrn* mutation, we established primary mesencephalic neuronal cultures from *zi* rats and compared them with cultures from wild-type SD rats. Neurons were cultured under an individual condition with (Cont) or without antioxidants (AO (−)), consisting of B27 supplement without vitamin E, vitamin E acetate, SOD, catalase, or GSH. We analyzed the cell viability and ROS content according to two factors: genotype (*zi* or wild type) and supplement (AO (−) or Cont).

Cell viability was shown in **Figure 3A**. Two-way analysis of variance (ANOVA) revealed significant effects of the supplement (*P* = 1.98 × 10–8) and an interaction of genotype × supplement (*P* = 8.73 × 10–6), but no effect of genotype (*P* = 0.493). Mesencephalic neurons from *zi* rats showed significantly lower cell viability in AO (−) supplement compared to Cont supplement (*P* < 1 × 10–8). However, the cell viability of neurons from wild-type SD rats was significantly lower in AO (−) supplement (*P* = 0.0085), the decreasing rate was considerably less than that of *zi* rats.

Next, ROS accumulation in mitochondria was monitored using MTR, which produces intense fluorescence depending on ROS accumulation in mitochondria (**Figure 3B**). Two-way ANOVA revealed significant effects of genotype (*P* = 6.21 × 10–4), supplement (*P* = 0.0016), and an interaction of genotype × supplement (*P* = 0.0026). Mesencephalic neurons from *zi* rats showed significantly higher ROS content in AO (−) (*P* = 0.0011 compared with Cont supplement in *zi* rats), whereas that of the wild type showed low ROS content and no difference between AO (−) and Cont supplements (*P* = 0.994). With Cont supplement, the ROS content was not different according to genotype (*P* = 0.875 *zi* neurons vs. SD neurons).

Because the neurons from *zi* rats showed excessive ROS accumulation, we examined the morphology of DA neurons using immunofluorescence with anti-TH antibody (**Figure 3C**). With Cont supplement, TH-immunoreactive (TH-ir) neurons from *zi* rats had long processes similar to those from wild-type rats (**Figure 3C**, upper column). However, in AO (−), TH-ir neurons had no long processes and formed cell clusters (**Figure 3C**, lower column). These culture experiments revealed that mesencephalic neurons from *zi* rats accumulate excess ROS followed by DA neurodegeneration and death in the absence of exogenous antioxidants.

**Figure 3.** Effects of genotype and supplement factors in primary neuronal cultures from the ventral mesencephalon. Neurons from *zi* rats and wild-type rats (SD rats) were supplemented with B27 (Cont: open bars) or B27 without antioxidants (AO (−): closed bars) for 5 days. (A) Cell viability was measured with a plate reader using WST-1 assay. Data are the percent absorbance at 450 nm versus SD-Cont (*n* = 4). (B) ROS production by mitochondria was detected with a plate reader using MitoTracker Red CM-H2Xros (MTR). Data are normalized to MitoTracker Green and represent the percent absorbance at 535 nm/485 nm versus SD-Cont (*n* = 3–4). All data are expressed as means ± SEM. Statistical analysis was performed with two-way ANOVA with Turkey post-hoc adjustment for multiple comparisons with a significant level set at #*P*< 0.01 and ##*P*< 0.0001. (C) Immunofluorescence demonstrating the morphological alteration of TH-immunoreactive neurons from *zi* rats in medium with B27 (Cont: upper column) or B27 without antioxidants (AO (−): lower column). Scale bar: 10 μm.

#### **3.3. Suppression of ROS accumulation in** *Atrn***-overexpressing cells** *in vitro*

Cell viability was shown in **Figure 3A**. Two-way analysis of variance (ANOVA) revealed significant effects of the supplement (*P* = 1.98 × 10–8) and an interaction of genotype × supplement (*P* = 8.73 × 10–6), but no effect of genotype (*P* = 0.493). Mesencephalic neurons from *zi* rats showed significantly lower cell viability in AO (−) supplement compared to Cont supplement (*P* < 1 × 10–8). However, the cell viability of neurons from wild-type SD rats was significantly lower in AO (−) supplement (*P* = 0.0085), the decreasing rate was considerably less than that

Next, ROS accumulation in mitochondria was monitored using MTR, which produces intense fluorescence depending on ROS accumulation in mitochondria (**Figure 3B**). Two-way ANOVA revealed significant effects of genotype (*P* = 6.21 × 10–4), supplement (*P* = 0.0016), and an interaction of genotype × supplement (*P* = 0.0026). Mesencephalic neurons from *zi* rats showed significantly higher ROS content in AO (−) (*P* = 0.0011 compared with Cont supplement in *zi* rats), whereas that of the wild type showed low ROS content and no difference between AO (−) and Cont supplements (*P* = 0.994). With Cont supplement, the ROS content was not different according to genotype (*P* = 0.875 *zi* neurons vs. SD neurons).

Because the neurons from *zi* rats showed excessive ROS accumulation, we examined the morphology of DA neurons using immunofluorescence with anti-TH antibody (**Figure 3C**). With Cont supplement, TH-immunoreactive (TH-ir) neurons from *zi* rats had long processes similar to those from wild-type rats (**Figure 3C**, upper column). However, in AO (−), TH-ir neurons had no long processes and formed cell clusters (**Figure 3C**, lower column). These culture experiments revealed that mesencephalic neurons from *zi* rats accumulate excess ROS followed by DA neurodegeneration and death in the absence of exogenous antioxidants.

**Figure 3.** Effects of genotype and supplement factors in primary neuronal cultures from the ventral mesencephalon. Neurons from *zi* rats and wild-type rats (SD rats) were supplemented with B27 (Cont: open bars) or B27 without antioxidants (AO (−): closed bars) for 5 days. (A) Cell viability was measured with a plate reader using WST-1 assay. Data are the percent absorbance at 450 nm versus SD-Cont (*n* = 4). (B) ROS production by mitochondria was detected with a plate reader using MitoTracker Red CM-H2Xros (MTR). Data are normalized to MitoTracker Green and represent the percent absorbance at 535 nm/485 nm versus SD-Cont (*n* = 3–4). All data are expressed as means ± SEM. Statistical analysis was performed with two-way ANOVA with Turkey post-hoc adjustment for multiple comparisons with a significant level set at #*P*< 0.01 and ##*P*< 0.0001. (C) Immunofluorescence demonstrating the morphological alteration of TH-immunoreactive neurons from *zi* rats in medium with B27 (Cont: upper column) or B27 without antioxidants (AO

of *zi* rats.

144 Free Radicals and Diseases

(−): lower column). Scale bar: 10 μm.

To investigate the function of *Atrn* during oxidative stress, ROS accumulation in mitochondria was observed in HEK293 cells that overexpressed *Atrn*-GFP.

ROS accumulation in *Atrn*-GFP-transfected cells was detected 30 min after MTR incubation (**Figure 4**). *Atrn*-GFP-transfected cells (**Figure 4A**) showed lower MTR intensity than the surrounding untransfected cells (**Figure 4C** and **D**).

**Figure 4.** ROS accumulation in *Atrn*-GFP-transfected HEK293 cells. *Atrn*-GFP (A), the corresponding phase-contrast image (B), and MitoTracker Red CM-H2Xros (C) were merged as shown in (D). White dotted lines (C) show the outline of *Atrn*-GFP-positive cells (A). Scale bar: 50 μm.

Under the microscope, the time of addition of H2O2 as a form of oxidative stress was set at 0 min, and live images were scanned at 0 and 60 min (**Figure 5**). *Atrn*-GFP-transfected cells showed little MTR fluorescent intensity at 0 min (**Figure 5C 2**–**3**) and faint intensity at 60 min (**Figure 5D 2**–**3**), whereas control transfected cells showed high MTR intensity at 0 min (**Figure 5A 2**–**3**) and markedly higher intensity at 60 min (**Figure 5B 2**–**3**). Compared with adjacent cells, *Atrn*-overexpressing cells maintained low MTR intensity under oxidative stress (**Figure 5D 2**–**3**).

**Figure 5.** *Attractin* suppressed the accumulation of mitochondrial ROS under oxidative stress. HEK293 cells transfected with Cont-GFP (A, B) or *Atrn*-GFP (C, D) were incubated in medium containing 1 mM H2O2 for 60 min. Time-lapse images at 0 min (A, C) and 60 min (B, D) are shown. GFP (1), MitoTracker Red CM-H2Xros: MTR (2), and the corresponding phase-contrast image were merged into (3). White dotted line (2) shows the outline of GFP-positive cells (1). Scale bar: 50 μm.

#### **4. Discussion**

This study revealed that the loss of *Atrn* led to ROS accumulation in mitochondria and resulted in DA neurodegeneration in *zi* rats.

Z*i* rats accumulate ROS and are vulnerable to oxidative stress, as observed with fibroblast cultures from the *zi/zi* kidney [11]. Moreover, DA neurons are particularly vulnerable to oxidative stress [1, 2]. Based on these observations, our previous studies revealed that the antioxidants vitamin E and melatonin suppress DA neurodegeneration in *zi* rats [5, 13, 15]. Therefore, we hypothesized that the DA neurodegeneration in *zi* rats was caused by oxidative stress. However, whether oxidative stress is directly caused by the genetic factor, *Atrn* gene mutation, was not clear. In the present study, we used *ex vivo* neuronal cultures and showed that this mutation directly led to ROS accumulation in mesencephalic neurons and caused DA neurodegeneration. B27 without the antioxidants vitamin E, vitamin E acetate, SOD, catalase, and GSH was used in this study. *In vivo*, SOD and catalase are produced by neurons, whereas vitamin E and vitamin E acetate are not synthesized in animal cells; GSH is mainly produced by astrocytes [17]. Thus, our result showing that wild-type neurons accumulate little ROS in medium without these five antioxidants indicates that the generated ROS is removed by scavenging agents including SOD and/or catalase in neurons. However, *zi* neurons accumulated significantly excessive ROS in the same medium, indicating that *zi* neurons have little or no endogenous scavenging agents and produce ROS levels that exceed their scavenging ability. This idea is consistent with previous assays using homogenates of *zi* brain tissues, which show abnormal H2O2 metabolism [10] and mitochondrial function [14]. In addition, morphological analysis using primary neuronal cultures from *zi* cerebral cortex shows protection from neurodegeneration by the antioxidants, vitamin E and catalase [11]. Additionally, *in vitro* study using DA neuroblastoma cell line shows that overexpressing *Atrn* protects against cell death induced by the neurotoxin 1-methyl-4-phenylpyridinium, which enhances ROS production [12]. Thus, *Atrn* may play a role in the activation of antioxidant enzymes in mitochondria. The observation that overexpression of *Atrn* suppressed ROS accumulation supports this hypothesis. We suggest that a deficiency in *Atrn* directly results in an abnormal antioxidant system and excess ROS accumulation in DA neurons. The findings in the present study will contribute to our understanding of the mechanism of not only DA neurodegeneration but also neurodegeneration of other neurons caused by oxidative stress. Further studies are needed to elucidate these possibilities.

## **Acknowledgements**

This work was supported by research grant from Dokkyo Medical University (Investigator-Initiated Research Grant No.2014-01).

## **Author details**

**Figure 5.** *Attractin* suppressed the accumulation of mitochondrial ROS under oxidative stress. HEK293 cells transfected with Cont-GFP (A, B) or *Atrn*-GFP (C, D) were incubated in medium containing 1 mM H2O2 for 60 min. Time-lapse images at 0 min (A, C) and 60 min (B, D) are shown. GFP (1), MitoTracker Red CM-H2Xros: MTR (2), and the corresponding phase-contrast image were merged into (3). White dotted line (2) shows the outline of GFP-positive cells (1).

This study revealed that the loss of *Atrn* led to ROS accumulation in mitochondria and

Z*i* rats accumulate ROS and are vulnerable to oxidative stress, as observed with fibroblast cultures from the *zi/zi* kidney [11]. Moreover, DA neurons are particularly vulnerable to oxidative stress [1, 2]. Based on these observations, our previous studies revealed that the antioxidants vitamin E and melatonin suppress DA neurodegeneration in *zi* rats [5, 13, 15]. Therefore, we hypothesized that the DA neurodegeneration in *zi* rats was caused by oxidative stress. However, whether oxidative stress is directly caused by the genetic factor, *Atrn*

Scale bar: 50 μm.

146 Free Radicals and Diseases

**4. Discussion**

resulted in DA neurodegeneration in *zi* rats.

Ayuka Ehara1\*, Shin-ichi Sakakibara2 and Shuichi Ueda1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Departments of Histology and Neurobiology, Dokkyo Medical University School of Medicine, Mibu, Tochigi, Japan

2 Laboratory for Molecular Neurobiology, Graduate School of Human Sciences, Waseda University, Tokorozawa, Saitama, Japan

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#### **Oxidative Stress and Parkinson's Disease: Effects on Environmental Toxicology Oxidative Stress and Parkinson's Disease: Effects on Environmental Toxicology**

Genaro Gabriel Ortiz, Fermín P. Pacheco-Moisés, Mario A. Mireles-Ramírez, L. Javier Flores-Alvarado, Héctor González-Usigli, Angélica L. Sánchez-López, Lorenzo Sánchez-Romero, Irma E. Velázquez-Brizuela, Erika Daniela González-Renovato and Erandis Dheni Torres-Sánchez Genaro Gabriel Ortiz, Fermín P. Pacheco-Moisés, Mario A. Mireles-Ramírez, L. Javier Flores-Alvarado, Héctor González-Usigli, Angélica L. Sánchez-López, Lorenzo Sánchez-Romero, Irma E. Velázquez-Brizuela, Erika Daniela González-Renovato and Erandis Dheni Torres-Sánchez Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63794

#### **Abstract**

Epidemiological studies have found an increased risk of Parkinson's disease (PD) with environmental factors such as exposure to substances derived from industrial processes, use of agrochemicals, or living in a rural environment. The hypothesis that certain environmental toxins could be the source of the EP is supported by the discovery that chemicals such as herbicides paraquat, diquat, and the fungicide maneb are selectively toxic in nigrostriatal dopaminergic neurons. Also, one of the insecticides produced by plants, such as rotenone, and by-product of the synthesis of synthetic heroin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) can be reproduced in animal models where neurochemicals, histopathological, and clinical characteristic of PD can be found. Interestingly, there are similarities in the chemical structure of paraquat and MPTP. Recent evidence exhibited that inflammation and oxidative stress play an essential role in the development of PD. So, in our laboratory we found that in an animal model melatonin decreases the products of lipid oxidation, nitric oxide metabolites, and the activity of cyclooxygenase 2, which are induced by an intraperitoneal injection of MPTP. This suggests that the neuroprotective effects of melatonin are partially attributed to its antioxidant scavenging and anti-inflammatory action.

**Keywords:** dopaminergic neurons, melatonin, MPTP, paraquat, Parkinson, Parkinsonism

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Parkinson's disease (PD) is a neurodegenerative disorder characterized by tremor and disruptions to voluntary movement. The main neuropathology in PD involves the death of dopaminergic cells in the pars compacta of the substantia nigra with intracytoplasmic inclusions (Lewy bodies) in the remaining intact nigral neurons [1]. Neural loss in the substantia nigra increases with age in PD, consistent with a worsening prognosis and increased symptom severity in older patients. The substantia nigra is an anatomical region of the brain implicated in dopamine synthesis and voluntary motor control and is a part of the basal ganglia. Neural circuits in the basal ganglia, particularly the nigrostriatal pathway, appear to be crucial to the successful execution of both innate and learned motor behaviors [2].

PD primarily affects people of ages 50 and older, and the prevalence and risk of developing sporadic PD increases substantially with age and has an incidence rate of 18 per 100,000 per year [3]. As the disease progresses, significant motor disability is seen in PD patients even when treated with symptomatic medications. Symptoms like dysphagia, sialorrhoea, microphagia, and dystonia are also well-known.  Interestingly, increased mortality risk has been linked with both motor and nonmotor features in newly diagnosed PD patients, especially with features like postural instability, hallucinations, and cognitive impairment [7].

No definitive diagnostic test such as magnetic resonance imaging or computed topography scans or genetic tests can confirm PD. Its diagnosis is typically based on the presence of a combination of key motor features, such as associated and exclusionary symptoms, and response to levodopa [8].

The molecular mechanisms underlying the loss of these neurons still remain elusive. Different modes of cell death, apoptotic, necrotic, and autophagic, have been described to contribute to the neuronal loss occurring in PD [9]. Oxidative stress plays an important role in dopaminergic neurotoxicity. Mitochondrial complex I deficiencies of the respiratory chain account for the neuronal degeneration in PD. Neurotoxins and other environmental factors, such as pesticides, insecticides, dopamine metabolites, heavy metals, microbial toxins, and genetic mutations, in PD-associated proteins contribute to mitochondrial dysfunction [10, 11]. A byproduct of an illicit narcotic drug, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as well as its metabolite MPP+, has been shown to cause the same signs and symptoms as PD. In fact, inhibitors of mitochondrial complex 1 (MPTP, rotenone, and paraquat) are able to reproduce parkinsonism with selective dopaminergic neuronal loss in mice and primate models [12]. Furthermore, a chronic infusion of either rotenone [13] or MPTP [14] in rodents induces the formation of α-synuclein positive aggregates. These data support the suggestion that sporadic PD may be caused by a combination of genetic predisposition and environmental toxins, which act via inhibition of the mitochondrial complex I to produce selective dopaminergic cell loss. Human epidemiological studies have implicated a higher incidence of PD in residents of rural environments with exposure to herbicides and pesticides [15].

## **2. Oxidative stress**

**1. Introduction**

152 Free Radicals and Diseases

impairment [7].

response to levodopa [8].

Parkinson's disease (PD) is a neurodegenerative disorder characterized by tremor and disruptions to voluntary movement. The main neuropathology in PD involves the death of dopaminergic cells in the pars compacta of the substantia nigra with intracytoplasmic inclusions (Lewy bodies) in the remaining intact nigral neurons [1]. Neural loss in the substantia nigra increases with age in PD, consistent with a worsening prognosis and increased symptom severity in older patients. The substantia nigra is an anatomical region of the brain implicated in dopamine synthesis and voluntary motor control and is a part of the basal ganglia. Neural circuits in the basal ganglia, particularly the nigrostriatal pathway, appear to

be crucial to the successful execution of both innate and learned motor behaviors [2].

PD primarily affects people of ages 50 and older, and the prevalence and risk of developing sporadic PD increases substantially with age and has an incidence rate of 18 per 100,000 per year [3]. As the disease progresses, significant motor disability is seen in PD patients even when treated with symptomatic medications. Symptoms like dysphagia, sialorrhoea, microphagia, and dystonia are also well-known.  Interestingly, increased mortality risk has been linked with both motor and nonmotor features in newly diagnosed PD patients, especially with features like postural instability, hallucinations, and cognitive

No definitive diagnostic test such as magnetic resonance imaging or computed topography scans or genetic tests can confirm PD. Its diagnosis is typically based on the presence of a combination of key motor features, such as associated and exclusionary symptoms, and

The molecular mechanisms underlying the loss of these neurons still remain elusive. Different modes of cell death, apoptotic, necrotic, and autophagic, have been described to contribute to the neuronal loss occurring in PD [9]. Oxidative stress plays an important role in dopaminergic neurotoxicity. Mitochondrial complex I deficiencies of the respiratory chain account for the neuronal degeneration in PD. Neurotoxins and other environmental factors, such as pesticides, insecticides, dopamine metabolites, heavy metals, microbial toxins, and genetic mutations, in PD-associated proteins contribute to mitochondrial dysfunction [10, 11]. A byproduct of an illicit narcotic drug, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as well as its metabolite MPP+, has been shown to cause the same signs and symptoms as PD. In fact, inhibitors of mitochondrial complex 1 (MPTP, rotenone, and paraquat) are able to reproduce parkinsonism with selective dopaminergic neuronal loss in mice and primate models [12]. Furthermore, a chronic infusion of either rotenone [13] or MPTP [14] in rodents induces the formation of α-synuclein positive aggregates. These data support the suggestion that sporadic PD may be caused by a combination of genetic predisposition and environmental toxins, which The body is constantly exposed to the influence and attack of free radicals, which have been associated with various disorders of the nervous system such as Parkinson disease, the motor neuron disease, and other disorders of the central nervous system (CNS). A free radical is considered any molecule containing one or more unpaired electrons. It is produced by biochemical redox reactions occurring as a result of normal cellular metabolism (biochemical reactions with oxygen or produced as a result of oxidative stress), as well as by phagocytes in inflammatory reactions controlled in response to exposure to different environmental factors, including ionizing radiation, ultraviolet rays, smoking, air pollution, gamma radiation, hyperoxia, excessive exercise, ischemia, and toxic compounds such as cancer drugs, some anesthetics, and painkillers [16].

The main free radicals are superoxide anion (O2•–), hydroxyl (OH•), nitric oxide (NO•), and peroxyl (ROO•) [17]. Some of them are considered highly reactive molecules that can cause cell damage and even death. Usually, the most damaged cellular components are unsaturated fatty acids in cell membranes and proteins such as enzymes conveying ions across membranes.

It is estimated that mitochondria are the main source of oxygen radicals (**Figure 1**) [18] in which anion superoxide (O2•) is generated during electron transport. Superoxide dismutase (SOD) converts O2•– to hydrogen peroxide (H2O2) by the Fenton reaction; the latter in the presence of Fe2+ produces hydroxyl radical (OH•) by reacting Fe2+ and Cu+. Then, the Fenton reaction is expressed as

O2•– + Fe3+ \_\_\_\_\_\_\_\_\_\_\_\_ Fe2++O2 Fe2+ + H2O2 \_\_\_\_\_\_\_\_\_\_\_\_ Fe3+ + OH– + OH• O2• + H2O2 \_\_\_\_\_\_\_\_\_\_\_\_ OH– + OH• + O2

According to Burdon and Mattson 1995 and 1998, the O2•– may also interact with nitric oxide (NO•) to form peroxynitrite (ONOO–). Of all free radicals, OH– is the most damaging free radical, since its presence–though is only a fraction of a second–is able to destroy proteolytic enzymes causing rupture of polysaccharide and lipid membrane peroxidation (LMP) altering its permeability and associated features [19] (**Figure 2**). Peroxynitrite (ONOO–) can cause direct damage to proteins and DNA, and is also a potent inducer of LMP that can destroy neurons, which are especially sensitive to this process. Oxidative stress can occur in different acute degenerative conditions such as cerebral ischemia, traumatic brain injury, and chronic degenerative processes such as Parkinson's disease [20]; it is observed, to a lesser extent, in neural circuits during normal physiological activity.

**Figure 1.** Mitochondria are considered the main source of free radicals, which come from the electron transport chain. Within mitochondria, 2O2+ is produced by the one-electron reduction of O2. Therefore, it is the kinetic and thermodynamic factors underlying the interaction of potential one-electron donors with O2 that control mitochondrial ROS production.

**Figure 2.** Damage generated by free radicals (ONOO-, OH-) directly affects proteins, membrane phospholipids, and DNA molecules. The resulting free radicals, such as superoxide anion (O2-) and hydroxyl radical (OH•), as well as the nonradical hydrogen peroxide, can damage macromolecules, including DNA, proteins, and oxidized lipids have all been related in such damages. The superoxide radical, although it is unreactive in comparison with many other radicals, biological systems can convert it into other more reactive species, such as peroxyl (ROO•), alkoxyl (RO•), and hydroxyl (HO•) radicals.

On the other side, oxidative stress can cause the onset of a disturbance in cellular calcium homeostasis. This action is usually related to an effect on the receptor mobilizing Ca2+, but the decrease of Ca2+ ATPase activity is also evident. Reactive oxygen species (ROS) also interfere with other transduction signal systems through the action of nitric oxide (NO) [21] (**Figure 3**). Reactive oxygen metabolites affect ligand binding to membrane receptors such as β-adrenergic, cholinergic, muscarinic, histamine, and serotonin. Some reactive oxygen species can affect enzymatic pathways such as the phospholipase A [22].

**Figure 3.** ROS can disrupt calcium homeostasis through its effect on receptors on the cell gate ionic membrane, calcium ATPase, and interfere with signal transduction. Interactions between Ca2+ and reactive oxygen species signaling coordinate signaling, which can be either beneficial or detrimental. In neurodegenerative disorders, cellular Ca2+-regulating systems are compromised. Oxidative stress, perturbed energy metabolism, and alterations of disease-related proteins result in Ca2+-dependent synaptic dysfunction.

Free radicals formed in the organism can initiate a series of chain reactions which continue, after several reactions, until removed with other free radicals or by the antioxidant system, which protects tissues from their deleterious effects. According to the mode of action, three main types of antioxidants are known: (a) those preventing the formation of new free radicals, (b) those making them less harmful before they can react, and (c) those preventing the formation of free radicals from other molecules.

The enzymes involved in the antioxidant system are superoxide dismutase glutathione peroxidase (GPx), catalase, glutathione reductase, glutathione S transferase, and other proteins that bind metals (ferritin, transferrin, and ceruloplasmin) limiting the availability of iron necessary to form the •OH radical.

**Figure 2.** Damage generated by free radicals (ONOO-, OH-) directly affects proteins, membrane phospholipids, and DNA molecules. The resulting free radicals, such as superoxide anion (O2-) and hydroxyl radical (OH•), as well as the nonradical hydrogen peroxide, can damage macromolecules, including DNA, proteins, and oxidized lipids have all been related in such damages. The superoxide radical, although it is unreactive in comparison with many other radicals, biological systems can convert it into other more reactive species, such as peroxyl (ROO•), alkoxyl (RO•), and

**Figure 1.** Mitochondria are considered the main source of free radicals, which come from the electron transport chain. Within mitochondria, 2O2+ is produced by the one-electron reduction of O2. Therefore, it is the kinetic and thermodynamic factors underlying the interaction of potential one-electron donors with O2 that control mitochondrial ROS pro-

On the other side, oxidative stress can cause the onset of a disturbance in cellular calcium homeostasis. This action is usually related to an effect on the receptor mobilizing Ca2+, but the decrease of Ca2+ ATPase activity is also evident. Reactive oxygen species (ROS) also interfere with other transduction signal systems through the action of nitric oxide (NO) [21] (**Figure 3**). Reactive oxygen metabolites affect ligand binding to membrane receptors such as β-adrenergic, cholinergic, muscarinic, histamine, and serotonin. Some reactive oxygen species can affect

hydroxyl (HO•) radicals.

duction.

154 Free Radicals and Diseases

enzymatic pathways such as the phospholipase A [22].

Excessive ROS reactive nitrogen species (RNS) generation may contribute to cell injury and death. In particular, accumulation of nitrosative stress due to excessive generation of nitric oxide (NO) appears to be a potential factor contributing to neuronal cell damage and apoptosis. In this process, overstimulation of N-methyl-D-aspartate-type of glutamate receptors permit calcium influx Ca2+ to cell, increasing NO and promoting ROS formation through a process named S-nitrosylation which consists in a reaction between NO and cysteine thiol to form Snitrosothiols (SNOS). In addition, NO can react with superoxide to generate peroxynitrite (ONOO–), which is highly toxic to cell, as well [23].

## **3. Clinical manifestations of Parkinson's disease**

PD is a common neurodegenerative disorder characterized by progressive loss of *substantia nigra* dopaminergic neurons, and the concomitant loss of dopaminergic nerve terminals in the *caudo-putamen* nuclei, which is the main neuron projection area of the *substantia nigra*. PD affects about 1% of the population over 65 years, which increases to up to 4% after the age of 80 [24]. Its main symptoms were described in 1817 by James Parkinson in an essay called the "shaking palsy." From a clinical point of view, PD is characterized by tremor at rest, slow movements (bradykinesia), rigidity, postural instability, stiffness of the muscles, serious inability to initiate movement (akinesia), and mask-like face expression. Other symptoms may be a flexed posture, freezing (motor blocks), loss of arm swing on one side, loss of smell, or a persisting glabellar tap reflex. However, the above mentioned symptoms may not all be present in one patient [25]. Bradykinesia, slowness of movement, is the most characteristic symptom. PD patients may therefore show slowness in daily activities and slow reaction times and may have difficulties in fine motor control [26].

A number of nonmotor features can precede the motor symptoms of PD. These symptoms probably arise from extra-nigral structures. For instance, olfactory deficits [27] and cardiac sympathetic denervation [28, 29] are present in a very high proportion of patients presenting with the earliest motor signs, suggesting that such features probably precede the motor signs and may be more useful in characterizing early disease status. Other nonmotor symptoms include autonomic, mood, cognitive, and sensory dysfunctions, as well as sleep disturbances [30, 31]. Depression is the most frequent psychiatric complication in PD. Although depression often precedes motor symptoms in PD [32], it may still reflect impairments of the nigrostriatal dopaminergic circuit [33]. Anxiety is also comorbid with PD [34]. These nonmotor symptoms significantly contribute to the reduced quality of life in PD patients, but are frequently undiagnosed and left untreated [35, 36].

## **4. Physiopathology of Parkinson disease**

One of the most surprising aspects of PD is the selective vulnerability of neuronal population to damage. PD can occur when an external or an internal toxin selectively destroys dopaminergic neurons. When the neurons which connect two specific brain regions, the compact part of the substantia nigra (SNPC) and the striatum, essential to maintain the motor function die, the dopaminergic pathway progressively degenerates, and the level of dopamine in the striatum decreases causing changes in brain circuitry and motor features of PD deficiencies appear.

The main pathological findings of PD are the loss of dopaminergic neurons and formation of fibril aggregates composed of α-synuclein, called Lewy bodies, in the remaining dopaminergic neurons located in substantia nigra pars compacta [37]. DA neurons of SNPC also have a tendency to degenerate with aging at a rate of approximately 5% per decade. In contrast, the rate of neurodegeneration in PD patients is about 10-fold faster and occurs mainly in the ventro-lateral part of SNPC [38].

**3. Clinical manifestations of Parkinson's disease**

have difficulties in fine motor control [26].

156 Free Radicals and Diseases

undiagnosed and left untreated [35, 36].

appear.

**4. Physiopathology of Parkinson disease**

PD is a common neurodegenerative disorder characterized by progressive loss of *substantia nigra* dopaminergic neurons, and the concomitant loss of dopaminergic nerve terminals in the *caudo-putamen* nuclei, which is the main neuron projection area of the *substantia nigra*. PD affects about 1% of the population over 65 years, which increases to up to 4% after the age of 80 [24]. Its main symptoms were described in 1817 by James Parkinson in an essay called the "shaking palsy." From a clinical point of view, PD is characterized by tremor at rest, slow movements (bradykinesia), rigidity, postural instability, stiffness of the muscles, serious inability to initiate movement (akinesia), and mask-like face expression. Other symptoms may be a flexed posture, freezing (motor blocks), loss of arm swing on one side, loss of smell, or a persisting glabellar tap reflex. However, the above mentioned symptoms may not all be present in one patient [25]. Bradykinesia, slowness of movement, is the most characteristic symptom. PD patients may therefore show slowness in daily activities and slow reaction times and may

A number of nonmotor features can precede the motor symptoms of PD. These symptoms probably arise from extra-nigral structures. For instance, olfactory deficits [27] and cardiac sympathetic denervation [28, 29] are present in a very high proportion of patients presenting with the earliest motor signs, suggesting that such features probably precede the motor signs and may be more useful in characterizing early disease status. Other nonmotor symptoms include autonomic, mood, cognitive, and sensory dysfunctions, as well as sleep disturbances [30, 31]. Depression is the most frequent psychiatric complication in PD. Although depression often precedes motor symptoms in PD [32], it may still reflect impairments of the nigrostriatal dopaminergic circuit [33]. Anxiety is also comorbid with PD [34]. These nonmotor symptoms significantly contribute to the reduced quality of life in PD patients, but are frequently

One of the most surprising aspects of PD is the selective vulnerability of neuronal population to damage. PD can occur when an external or an internal toxin selectively destroys dopaminergic neurons. When the neurons which connect two specific brain regions, the compact part of the substantia nigra (SNPC) and the striatum, essential to maintain the motor function die, the dopaminergic pathway progressively degenerates, and the level of dopamine in the striatum decreases causing changes in brain circuitry and motor features of PD deficiencies

The main pathological findings of PD are the loss of dopaminergic neurons and formation of fibril aggregates composed of α-synuclein, called Lewy bodies, in the remaining dopaminergic neurons located in substantia nigra pars compacta [37]. DA neurons of SNPC also have a tendency to degenerate with aging at a rate of approximately 5% per decade. In contrast, the The degeneration of neuronal cells may be the consequence of many pathogenic factors (toxic, infectious, genetic, metabolic, vascular, etc.) and the final consequence is apoptosis and cell death in which caspases (particularly caspase 3) and Bcl-2 protein families are central components with up and down regulatory mechanism that promotes apoptosis. Two main caspasemediated pathways of cell death have been described in mammals: (a) the extrinsic or death receptor-mediated pathway, which has a critical role in the maintenance of tissue homeostasis, and (b) the intrinsic, mitochondria-dependent pathway that is mainly activated in response to extracellular cues and internal insults such as DNA damage, growth factor deprivation, cytoskeletal disruption, accumulation of unfolded proteins, hypoxia, and many others [12, 19– 21, 39–42].

*In vivo*, the dysfunction of the proteasomal system in order to cleave misfolded α-syn due to lack of energy associated to mitochondrial damage, and problems with the autophagylysosomal degradation pathway, which disrupts damaged mitochondrial clearance, leads to the formation of protein inclusions and accumulation of damaged mitochondria, a major source of ROS, which activate the apoptotic cascade as has been mentioned. ROS accumulation may result in detrimental effects such as lipid peroxidation, protein oxidation, and further DNA damage. The maintenance of a pool of healthy mitochondria that can meet the bioenergetic demands of a neuron is therefore of critical importance; this is achieved by maintaining a careful balance between mitochondrial biogenesis, mitochondrial trafficking, mitochondrial dynamics, and mitophagy. Removal of damaged mitochondria through mitophagy can lead to the release of compartmentalized mitochondrial molecules. Once in the cytosol, some of these molecules, such as cyt-C, Smac/DIABLO, and HtrA2/OMI, are capable of activating apoptotic routines that lead to cell death. It is then reported that the failure of mitophagy results in the release of mtDNA into the cell cytosol contributing to mechanisms of cell death [43–47].

The central events in the mitochondrial-dependent cell death pathway are the activation of the mitochondrial permeability transition pore (mPTP) and the disruption of mitochondrial membrane potential, which cause the release of apoptogenic molecules and finally lead to cell death [48]. The protein α-syn is a small acidic protein containing 140 amino acids. This protein is able to undergo self-aggregation in a nucleation-dependent process to form nonfibrillar oligomers, protofibrils, and fibrillar aggregates with amyloid-like properties that are potentially cytotoxic to the neurons and it has been shown to be directly degraded *in vitro* by the 20S proteasome. Studies of the degradation of aggregated α-syn led us to suspect that oxidation of Met may play a role in α-syn degradation by the proteasome [46].

It is important to take into account that neuronal loss in PD is also associated to chronic neuroinflammation through microglial activation by some different mechanism including overexpression of inducible NOS (iNOS), COX-2, the cytokines tumor necrosis factor-alpha (TNF-α), and IL-1β and NO or accumulation of heat shock protein 60 (Hsp60) participating in DA cell death in PD via a mechanism unrelated to cytokine release and could serve as a signal of CNS injury through activation of microglial cells. Neuromelanin is released from dying dopaminergic neurons in the SNpc and activates microglia, provoking increase in the sensitivity of DA neurons to oxidative stress-mediated cell death. Parkinson's disease-associated proteins such as α-syn, parkin, LRRK2, and DJ-1 have also been reported to activate microglia, as well [11, 49].

One of the most surprising aspects of neurodegenerative diseases is the selective vulnerability of neuronal population to damage. Parkinson's disease can occur when an external or an internal toxin selectively destroys dopaminergic neurons. When the neurons which connect to two brain regions, the compact part of the substantia nigra (SNPC) and the striatum, essential to maintain the motor function die, the dopaminergic pathway progressively degenerates and the level of dopamine in the striatum decreases causing changes in brain circuitry, and motor features of PD deficiencies appear. The high vulnerability of the SNPC neurons to oxidative agents, compared to neurons in other cerebral regions, may be explained by a combination of different factors such as reduced antioxidant activity, increased iron concentration, increased susceptible to DA oxidation, and reduced activity of the mitochondrial complex I (NADH oxidoreductase) (**Figure 4**).

**Figure 4.** SNPC neurons and oxidative agents. External factors, such as neurotoxins, pesticides, insecticides, and endogenous factors such as dopamine (DA), and genetic mutations in PD-associated proteins contribute to oxidative damage and disruptions in the maintenance of the redox potential leading to destruction of dopaminergic neurons and cell death. Different pathways contribute to the substantia nigra pars compacta (SNPC) neurons vulnerability to oxidative damage including (a) high susceptibility of DA auto-oxidation, (b) reduced antioxidant activity such as glutathione, and (c) increased iron concentration d) deficits in mitochondrial complex I of the respiratory chain.

Biochemical abnormalities in the brain with PD show deficits in mitochondrial complex I, decreased extracellular thiols, increased oxidative iron in the substantia nigra, and oxidative damage, including DNA oxidation, nitration, and increased carbonyl groups of proteins. Lewy bodies inclusions contain phosphorylated neurofilaments and a protein called α-synuclein [50]. Currently, there is evidence of properties of α-synuclein and its possible association with oxidative stress state present in PD. One such evidence is the notion that one type of amyloid aggregates of α-synuclein, similar to those observed *in vivo*, by coincubation with copper (II), Fe/hydrogen peroxide, or are induced cytochrome c/ hydrogen peroxide. Many motor characteristics defining the PD result primarily from the loss of substantia nigra neurons [28]. Deficiencies in mitochondrial function, increased oxidative stress, apoptosis, excitotoxicity, and inflammation, all of them are part of the processes that eventually result in neurodegeneration [51].

#### **4.1. Genetic and Parkinson's disease**

tivity of DA neurons to oxidative stress-mediated cell death. Parkinson's disease-associated proteins such as α-syn, parkin, LRRK2, and DJ-1 have also been reported to activate microglia,

One of the most surprising aspects of neurodegenerative diseases is the selective vulnerability of neuronal population to damage. Parkinson's disease can occur when an external or an internal toxin selectively destroys dopaminergic neurons. When the neurons which connect to two brain regions, the compact part of the substantia nigra (SNPC) and the striatum, essential to maintain the motor function die, the dopaminergic pathway progressively degenerates and the level of dopamine in the striatum decreases causing changes in brain circuitry, and motor features of PD deficiencies appear. The high vulnerability of the SNPC neurons to oxidative agents, compared to neurons in other cerebral regions, may be explained by a combination of different factors such as reduced antioxidant activity, increased iron concentration, increased susceptible to DA oxidation, and reduced activity of the mitochondrial complex I (NADH

**Figure 4.** SNPC neurons and oxidative agents. External factors, such as neurotoxins, pesticides, insecticides, and endogenous factors such as dopamine (DA), and genetic mutations in PD-associated proteins contribute to oxidative damage and disruptions in the maintenance of the redox potential leading to destruction of dopaminergic neurons and cell death. Different pathways contribute to the substantia nigra pars compacta (SNPC) neurons vulnerability to oxidative damage including (a) high susceptibility of DA auto-oxidation, (b) reduced antioxidant activity such as gluta-

Biochemical abnormalities in the brain with PD show deficits in mitochondrial complex I, decreased extracellular thiols, increased oxidative iron in the substantia nigra, and oxidative damage, including DNA oxidation, nitration, and increased carbonyl groups of proteins. Lewy bodies inclusions contain phosphorylated neurofilaments and a protein called α-synuclein

thione, and (c) increased iron concentration d) deficits in mitochondrial complex I of the respiratory chain.

as well [11, 49].

158 Free Radicals and Diseases

oxidoreductase) (**Figure 4**).

A relatively new theory explores the role of genetic factors in the development of Parkinson's disease. A total of 15–25% of Parkinson's patients have a close relative who had experienced parkinsonian symptoms (such as tremor).

There are many genes linked to familial forms of PD, which have produced advances in PD basic research, increasing our understanding of possible mechanism of dopaminergic cells damage in patients with this condition. These genes include those associated with α-syn, parkin, DJ-1, PINK-1, LRRK-2, ATP13A2, mitochondrial phosphatase, and phosphatase and tensin homolog (PTEN)-induced kinase gene, and they have been demonstrated to be involved in apoptosis regulation. Parkin is associated with the outer mitochondrial membrane (OMM) and prevents cell death by inhibiting mitochondrial swelling, cyt-C release, and caspase activation. Another finding in pathogenesis has been centered in the ATP13A2 gene, which regulates intracellular Mn2+ homeostasis, playing an important role in preventing damage induced by Mn2+ cytotoxicity. Overexposure of cells to Mn2+ may determine cell death by induction of DNA damage, oxidative stress, disruption of Ca2+, iron homeostasis, and mitochondrial dysfunction. In an experimental model, monomeric α-Syn-expressing dopaminergic cells significantly attenuated Mn-induced neurotoxicity for initial exposures. However, overexposure to Mn produces precipitation of α-syn, which at the same time impairs antioxidative defense mechanism in this experimental model [39, 44, 52].

One important finding of PD is the presence of cytoplasmic inclusions containing α-synuclein and ubiquitin, known as Lewy bodies, in the SNPC and other brain regions. Some cases of familial PD are clearly attributed to mutations in the genes for α-synuclein and parkin.

#### **4.2. Dopamine as a source of ROS in the CNS**

Since the discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydrodropyridine infusion causes parkinsonism by selective inhibition of mitochondrial complex-1, raised the possibility that mitochondrial dysfunction is at the heart of PD [53].

In patients with PD, there is an excess amount of cytosolic DA outside of the synaptic vesicles due to damaged neurons with impaired reuptake, and a possible increased damage when an overload is related to levodopa treatment. Dopamine is easily metabolized via monoamine oxidase (MAO). Also DA suffers auto-oxidation to cytotoxic ROS producing mitochondrial impairment either by activation of the intrinsic apoptotic pathway or by inhibiting the respiratory chain. Also, the auto-oxidation of DA produces electron-deficient DA quinones or DA semiquinones, which, at the same time, modify some PD-related proteins, such as α-syn, parkin, DJ-1, superoxide dismutase-2 (SOD2), and UCH-L1. Additionally, DA quinones can be oxidized to aminochrome, whose redox-cycling leads to the generation of the superoxide radical and the depletion of cellular nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), which ultimately forms the neuromelanin, contributing to this degenerative mechanism [11, 47] (**Figure 5**).

**Figure 5.** Dopamine as a source of ROS in the CNS. In patients with PD there is an excess amount of cytosolic dopamine (DA), this molecule is unstable and undergoes auto-oxidation to form dopamine quinones and free radicals that leads to modification of PD-related proteins such as α-syn and mitochondrial impairment. The products of dopamine oxidation, dopamine quinones, can also contribute to neurodegeneration. Dopamine quinones can cyclize to form dopaminocromo who is the precursor of neuromelanin, a brain pigment that may contribute to neurodegeneration.

The microtubule (MT) system may play an important role in PD pathogenesis, as well. It is crucial for many aspects of neuronal function, including motility, differentiation, and protein and organelle trafficking. In experimental models, both acute and chronic sub-lethal settings, 6OHDA-induced oxidative stress elicited significant alterations in microtubule (MT) dynamics; very important for axonal transportation; these includes reductions in MT growth rate, increased frequency of MT pauses/retractions, and increased levels of tubulin acetylation [54].

DA metabolism act as proneurotoxins in the development of PD. Certain components of snuff smoke can react with these proneurotoxins preventing its activation. This may explain the possible beneficial effect of smoking on the incidence of PD. The ROS generated by the autooxidation of dopamine have been implicated in the neuron loss; related to age and other neurodegenerative disorders such as PD. To date, there have been proposed two mechanisms by which the DA stimulates the production of ROS. These depend on the presence or absence of enzymatic mediators [55]. The DA in the substantia nigra and striatum is spread by the enzyme monoamine oxidase (MAO), located in the outer membrane of mitochondria [56], from this reaction results the superoxide and hydroxyl radicals plus hydrogen-peroxide production. Another derivative compound is 1,2,3,4 tetrahydropapaverolin DA (THP) obtained from enzymatic catabolism. TTP itself is capable of inducing necrosis in neuroblastoma cells and is related to the pathogenesis of PD [57].

respiratory chain. Also, the auto-oxidation of DA produces electron-deficient DA quinones or DA semiquinones, which, at the same time, modify some PD-related proteins, such as α-syn, parkin, DJ-1, superoxide dismutase-2 (SOD2), and UCH-L1. Additionally, DA quinones can be oxidized to aminochrome, whose redox-cycling leads to the generation of the superoxide radical and the depletion of cellular nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), which ultimately forms the neuromelanin, contributing to this degenerative

**Figure 5.** Dopamine as a source of ROS in the CNS. In patients with PD there is an excess amount of cytosolic dopamine (DA), this molecule is unstable and undergoes auto-oxidation to form dopamine quinones and free radicals that leads to modification of PD-related proteins such as α-syn and mitochondrial impairment. The products of dopamine oxidation, dopamine quinones, can also contribute to neurodegeneration. Dopamine quinones can cyclize to form dopaminocromo who is the precursor of neuromelanin, a brain pigment that may contribute to neurodegeneration.

The microtubule (MT) system may play an important role in PD pathogenesis, as well. It is crucial for many aspects of neuronal function, including motility, differentiation, and protein and organelle trafficking. In experimental models, both acute and chronic sub-lethal settings, 6OHDA-induced oxidative stress elicited significant alterations in microtubule (MT) dynamics; very important for axonal transportation; these includes reductions in MT growth rate, increased frequency of MT pauses/retractions, and increased levels of tubulin acetylation [54]. DA metabolism act as proneurotoxins in the development of PD. Certain components of snuff smoke can react with these proneurotoxins preventing its activation. This may explain the possible beneficial effect of smoking on the incidence of PD. The ROS generated by the autooxidation of dopamine have been implicated in the neuron loss; related to age and other neurodegenerative disorders such as PD. To date, there have been proposed two mechanisms

mechanism [11, 47] (**Figure 5**).

160 Free Radicals and Diseases

DA is a molecule with a catechol group which cannot be easily enzymatically oxidized to form an array of electrochemical species (quinone type). First, the initial step in the oxidation of DA involves a reaction with molecular oxygen to form DA quinone and two molecules of superoxide anion. Second, the superoxide anions formation during autoxidation of DA leads to the production of hydrogen peroxide by the dismutation of superoxide. Third, in this way, iron mediated in substantia nigra showed high amounts of oxidative stress. Last, the total iron increased but does not necessarily mean an oxidative stress state as excess iron always store into the proteins, such as ferritin, which leaves the iron inert and harmless [58].

However, that state of iron stability can change by continuing entry and release of iron from ferritin to more active form entering the Fenton reaction generating the hydroxyl radical. The iron accumulates in astrocytes in the substantia nigra (experimental data), increasing the rate of Fe(III)/Fe(II) and reduced glutathione. Aging may be a factor which predisposes the brain to the PD, due to, according to our interpretation, the kidnapping by the mitochondria of Fe(II) in astroglia in the substantia nigra. Furthermore, there is evidence that the intracellular loss of redox balance results in aberrant dopamine oxidation in 6-hydroxydopamine, which in turn can undergo auto-oxidation to quinones and simultaneously generate superoxide. This cascade reaction, either by itself, or amplified by the generation of ROS, may explain the neuronal loss as an end result. The DA-o-quinone then undergoes intramolecular cyclization to form 5,6-dihydroxyquinoline, which is subsequently oxidized by the DA-o-quinone to form dopaminocromo; this compound undergoes a rearrangement to form 5,6-dihydroxyindole, which in turn is oxidized into a quinone indole. The following polymerization process finally leads to the generation of a dark pigment called neuromelanin. The dark appearance of the black substance is due to the presence of this pigment containing oxidation products of the cysteinyl-DA.

Dopaminergic neurons are particularly exposed to oxidative stress due to the metabolism of dopamine that causes a series of molecules that are potentially toxic if they are not adequately removed. Dopamine acts as a free radical generating compound, and can oxidize itself at physiological pH, generating dopamine-forming toxic quinones, superoxide radicals, and hydrogen peroxide [40]. It can also be enzymatically deaminated by monoamino oxidase (MAO) to 3,4-dihydroxyphenylacetic, a nontoxic metabolite acid (DOPAC), hydrogen peroxide [41], and by other oxidative processes. Thus, the metabolism of dopamine generates high concentrations of ROS, which can activate and induce apoptotic cell death cascades [19, 20]. ROS accumulation is toxic *per se*, and generates oxidative stress as a result of depletion of cellular antioxidants (e.g., vitamin E and reduced glutathione), increase the membrane lipid peroxidation, DNA damage, and oxidation alteration of protein folding [20]. Besides the general oxidative damage, there is evidence that the interaction between α-synuclein and dopamine metabolites determines the preferential neurodegeneration of dopaminergic neurons. Along with a number of possible changes, abnormal protein aggregates could also act as irritants, causing a chronic inflammatory reaction which can induce synaptic changes and neuronal death. Findings which suggest the existence of a chronic inflammation process include the presence of microglial activation and astrocytosis in the brain of these patients, particularly in the vicinity of protein aggregates. In PD, along with several toxic and genetic mechanisms producing neuronal damage, the compounds released from damaged neurons can induce microglial release of neurotoxic factors aggravating neurodegeneration [36]. Among those released is neuromelanin compound which is a strong iron chelator. The neuromelanin-ferrum complex activates microglia *in vitro*, causing the release of neurotoxic compounds such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO). Increased total iron concentration had been described in the substantia nigra of PD severe cases, although the underlying mechanism is not known [59]. Iron also contributes to increasing the generation of oxygen radicals (ROS), oxidative stress, and increased protein aggregation, including α-synuclein aggregation. The rapid aggregation of α-synuclein protein in turn can induce the formation of ROS. Lastly, dopamine stabilizes the protofibril form of α-synuclein, which would be toxic. Thus, in the oxidative atmosphere of a dopaminergic neuron, α-synuclein is involved in generating a vicious circle, which leads to neuronal death [59] (**Figure 6**).

**Figure 6.** Interaction between α-synuclein and dopamine metabolites. Dopamine metabolites such as neuromelanin-Ferrum complex produce microglial activation that leads to chronic inflammation process, causing the release of interleukins, and free radicals. The reactive oxygen radicals interact with total iron concentration in the substantia nigra and increase the propensity of the α-synuclein to aggregate. This protein is present in Lewy bodies and the formation of aggregates is associated with increased oxidative stress, neurodegeneration, and cell death.

Finally, till date there have been at least five different mechanisms of death in neurons with dopaminergic dysfunction associated to oxidation of the dopamine (DA): (1) proteasome dysfunction, (2) mitochondrial dysfunction, (3) oxidative stress, (4) α-syn oligomers precipitation, and (5) lysosomal autophagy dysfunction.

Another possible mechanism implicated in neuronal destruction and death is associated with diminished neuroprotection due to the dysfunction of neuromelanin and diaphorase enzyme [45, 56].

#### **4.3. MPTP and Parkinson's disease**

general oxidative damage, there is evidence that the interaction between α-synuclein and dopamine metabolites determines the preferential neurodegeneration of dopaminergic neurons. Along with a number of possible changes, abnormal protein aggregates could also act as irritants, causing a chronic inflammatory reaction which can induce synaptic changes and neuronal death. Findings which suggest the existence of a chronic inflammation process include the presence of microglial activation and astrocytosis in the brain of these patients, particularly in the vicinity of protein aggregates. In PD, along with several toxic and genetic mechanisms producing neuronal damage, the compounds released from damaged neurons can induce microglial release of neurotoxic factors aggravating neurodegeneration [36]. Among those released is neuromelanin compound which is a strong iron chelator. The neuromelanin-ferrum complex activates microglia *in vitro*, causing the release of neurotoxic compounds such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO). Increased total iron concentration had been described in the substantia nigra of PD severe cases, although the underlying mechanism is not known [59]. Iron also contributes to increasing the generation of oxygen radicals (ROS), oxidative stress, and increased protein aggregation, including α-synuclein aggregation. The rapid aggregation of α-synuclein protein in turn can induce the formation of ROS. Lastly, dopamine stabilizes the protofibril form of α-synuclein, which would be toxic. Thus, in the oxidative atmosphere of a dopaminergic neuron, α-synuclein is involved in generating a vicious circle, which leads to neuronal death

**Figure 6.** Interaction between α-synuclein and dopamine metabolites. Dopamine metabolites such as neuromelanin-Ferrum complex produce microglial activation that leads to chronic inflammation process, causing the release of interleukins, and free radicals. The reactive oxygen radicals interact with total iron concentration in the substantia nigra and increase the propensity of the α-synuclein to aggregate. This protein is present in Lewy bodies and the formation

of aggregates is associated with increased oxidative stress, neurodegeneration, and cell death.

[59] (**Figure 6**).

162 Free Radicals and Diseases

In 1982 (California, USA), a group of recreational drugs were developed which severely causes parkinsonian syndrome days after injection; 1-methyl-4-phenyl-4-propionoxipiperidina (MPPP), a synthetic analog of meperidine, was used for that purpose. This analog-product, according to the analysis of samples provided by the seller, was contaminated by 1-methyl-4 phenyl-1,2,3,6 tetrahydropyridine, which was discovered to cause parkinsonism at a concentration of 2.5–2.9% by weight. Initial clinical symptoms shown by these patients were treated with carbidopa/L-dopa. In the following years, the clinical treatment was insufficient to halt the progression of the disease and died 12 and 16 years, respectively, after injection. Pathological analysis of their brains showed moderate to severe decrease of neurons in the substantia nigra without Lewy bodies. Besides, gliosis and clumping of microglia around nerve cells were found [60].

In 1991, an individual, 39 years old, tried to produce the MPPP following the instructions of a chemistry textbook and obtained, without knowing, the by-product MPTP. Approximately, 45 g of the drug in the course of a week were injected. At the end of this period, he had language problems, remained lethargic, and developed rigid posture. These symptoms worsened over the next week, so he had to be admitted to a hospital. In the next two weeks, he was treated with selegiline and carbidopa/L-dopa, which greatly improved their symptoms. Over the next 3.5 years, the patient responded transiently to treatment with carbidopa less/L-dopa and bromocriptine; however, his condition progressed to severe parkinsonism immobility and a significant hypophonia and died. Neuropathological analysis of this patient showed similar findings to those found in the brains of those patients studied from California. Besides, large amounts of extraneuronal melanin were found indicating a progressive death of nerve cells in response to a brief temporary aggression the nigrostriatal system. The above data show that the acute phase of parkinsonian syndrome is completed within a few days after administration of MPTP; however, neurodegeneration caused by MPTP silently continue for several years or even decades [61, 62].

From a neurochemical point of view there is a great similarity between the MPTP-induced parkinsonism and PD. The neurotoxic action of MPTP involving dopaminergic transmission produces a variety of neurochemical changes: (1) decrease in the concentration of dopamine and its metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid), (2) decrease activity of the enzyme tyrosine hydroxylase, and (3) alteration in the density of dopamine receptors.

Since the discovery of the effect of MPTP in human, extensive research in animal models, cell cultures, etc. was initiated to characterize in more detail the effects of the toxin.

MPTP produces selective death of dopaminergic neurons and parkinsonian syndrome in several species, including Rhesus monkeys, squirrel monkeys, and beagle dogs. The effects of MPTP, presented in a variety of nonhuman primates, consist in a very significant reduction in spontaneous activity, rigidity, tremor, and bradykinesia. Very few human subjects develop resting tremor characteristic of PD. MPTP initially produces temporary parkinsonian symptoms but become permanent with repeated administration of the toxin. The mechanisms involved in spontaneous recovery experienced by these animals are not known, but may be related to a transient alteration of other neurotransmitter systems other than nigrostriatal. On the other hand, rats and guinea pigs do not show permanent impairments of DA in the striatum nor they present movement disorders such as those observed in primates. In mice of the C57BL/ 6 strain MPTP induces toxic changes, but at higher concentrations.

There is no evidence that MPTP induces alterations in the cholinergic, GABAergic, and glutamatergic systems in primates. However, the levels of various neuropeptides, substance P, dynorphin, and enkephalin in striatum, substantia nigra, and globus pallidus are reduced in animals chronically treated with MPTP. These same abnormalities have been described in patients with PD. However, it is not known whether they are due to the degeneration of neurons containing these peptides or represent adaptive nigrostriatal denervation to changes.

The susceptibility of different animal species to MPTP may be related to differences in the metabolism of MPTP, cerebral distribution, and retention of the final metabolite [63].

#### **4.4. Metabolism of MPTP**

MPTP metabolism is a complex process, after systemic administration it readily crosses the blood brain barrier due to their lipophilicity. Once in the brain, it is transformed extraneuronally in astrocytes, a monoamine oxidase B rich cells, into the intermediate, 1-methyl-4 phenyl-2,3-dihydropyridinium (MPDP+). This is a very reactive ion undergoing autoxidation to the radical 1-methyl-4-phenyl pyridinium (MPP+) with the formation of superoxide anion (O2•). Besides, the MPDP+ readily crosses the cell membrane and into the extracellular space to form MPP+ and O2•. Extracellular MPP+ is leaking from astrocytes and used by the presynaptic DA system uptake, resulting in an energy-dependent concentration in dopaminergic neurons [64].

Intraneuronal concentration of MPP+ increases by binding to neuromelanin and then brought into the mitochondrial matrix through active transport system, which acts as a potent inhibitor of complex I of oxidative phosphorylation system. This effect is due MPP+ binding to complex I at a distal sulfur iron site core and near the binding site of ubiquinone Q10. This leads to the cessation of oxidative phosphorylation, ATP level is depleted and decreases the concentration of the major cellular antioxidant, reduced glutathione. This also leads to changes in cellular calcium, altered transmembrane potential, and ultimately neuronal death is manifested [65].

The inhibition of mitochondrial complex I can increase oxidative stress induced by MPP+, since the electrons inside the mitochondria may contribute to the toxic effects of MPTP. For example, it has been demonstrated that MPP+ induced lipid peroxidation in cultured cells and the DA is blocked by specific inhibitors of lipid peroxidation [66] (see **Figure 2**).

#### **4.5. Toxicology of paraquat**

Since the discovery of the effect of MPTP in human, extensive research in animal models, cell

MPTP produces selective death of dopaminergic neurons and parkinsonian syndrome in several species, including Rhesus monkeys, squirrel monkeys, and beagle dogs. The effects of MPTP, presented in a variety of nonhuman primates, consist in a very significant reduction in spontaneous activity, rigidity, tremor, and bradykinesia. Very few human subjects develop resting tremor characteristic of PD. MPTP initially produces temporary parkinsonian symptoms but become permanent with repeated administration of the toxin. The mechanisms involved in spontaneous recovery experienced by these animals are not known, but may be related to a transient alteration of other neurotransmitter systems other than nigrostriatal. On the other hand, rats and guinea pigs do not show permanent impairments of DA in the striatum nor they present movement disorders such as those observed in primates. In mice of the C57BL/

There is no evidence that MPTP induces alterations in the cholinergic, GABAergic, and glutamatergic systems in primates. However, the levels of various neuropeptides, substance P, dynorphin, and enkephalin in striatum, substantia nigra, and globus pallidus are reduced in animals chronically treated with MPTP. These same abnormalities have been described in patients with PD. However, it is not known whether they are due to the degeneration of neurons containing these peptides or represent adaptive nigrostriatal denervation to changes. The susceptibility of different animal species to MPTP may be related to differences in the

metabolism of MPTP, cerebral distribution, and retention of the final metabolite [63].