**3. Potential targets for treatment to modulate oxidative stress status**

*Selenium (Se)* is a micronutrient and mineral. It is a structural component and a co-factor of the antioxidant enzyme glutathione peroxidase. For this reason, supplementation with selenium can modestly increase glutathione levels in persons who are selenium deficient. Selenium has an antagonistic action on mercury and other toxic metals. No association of autism with hair concentrations of selenium was found in a recent meta-analysis [43].

*Zinc (Zn)* Zinc is an important cofactor for metabolism relevant to neurotransmitters, prosta‐ glandins, and melatonin, and indirectly affects dopamine metabolism. It is necessary for 100 different metalloenzymes and metal–enzyme complexes [44], many of them in the central nervous system. It contributes to structure and function of the brain [45]. Zinc is considered to be an important mineral for children. It is suggested that infants need more zinc for growth and development than older children and that that lack of zinc early in life may be linked with the development of autism. Zinc deficiency has been implicated in hyperactivity and jitters [46]. Children with autism have been shown to have lower zinc/copper ratios than normally developing peers and disturbed zinc homeostasis is suggested as a risk factor for neurodege‐ nerative diseases [24, 25, 47].

*N-Acetyl-L-Cysteine (NAC)* NAC is sulphur-containing (sulfhydryl) amino acid which is present in many proteins, and is in the same class as the amino acid methionine. NAC is a naturally occurring amino sugar and is a form of cysteine which has been demonstrated to facilitate the short term cellular detoxification of alcohol, tobacco smoke, acetaminophen poisoning and environmental pollutants in several *in vitro* studies. NAC supplementation over long periods associates with modest increases in serum glutathione, but has not proven particularly useful in the treatment of chronic, long-term intracellular glutathione deficiencies. Furthermore, therapeutic levels of NAC are relatively toxic. At therapeutic doses, oral NAC supplementation can cause significant side effects. Cerebral symptoms, nausea, blurred vision, and vomiting are associated with NAC supplementation.

*Vitamin A and E* Retinol, the most useful form of vitamin A, (along with retinal and retinoic acid) and vitamin E are lipid-soluble. They can build up in the body and cause toxicity in excess amounts. Vitamin A is believed to improve sensory perception, language, and attention [48]. Autistic children could have a vitamin A deficiency because of gastrointestinal inflammation caused by leaky gut syndrome, allergies or viral infections. Lower levels of vitamin E are reported in ASD patients than in healthy controls [49, 50].

expression of transporters. The amino acids serve as precursors for neurotransmitters like dopamine and serotonin, which play a role in mood and pleasure-seeking, and whose activities

Recent studies have associated mitochondrial dysfunction with autism [39, 40]. Defects and malfunction observed in the mitochondria of autistic children suggest that oxidative stress in mitochondria could influence the onset of autism and explain the immunological anomalies present in autistic children. While many inherited genetic mitochondrial disorders occur in the mitochondria of all cells in the body, some are limited to specific cell sites, such as the brain

**3. Potential targets for treatment to modulate oxidative stress status**

*Selenium (Se)* is a micronutrient and mineral. It is a structural component and a co-factor of the antioxidant enzyme glutathione peroxidase. For this reason, supplementation with selenium can modestly increase glutathione levels in persons who are selenium deficient. Selenium has an antagonistic action on mercury and other toxic metals. No association of autism with hair

*Zinc (Zn)* Zinc is an important cofactor for metabolism relevant to neurotransmitters, prosta‐ glandins, and melatonin, and indirectly affects dopamine metabolism. It is necessary for 100 different metalloenzymes and metal–enzyme complexes [44], many of them in the central nervous system. It contributes to structure and function of the brain [45]. Zinc is considered to be an important mineral for children. It is suggested that infants need more zinc for growth and development than older children and that that lack of zinc early in life may be linked with the development of autism. Zinc deficiency has been implicated in hyperactivity and jitters [46]. Children with autism have been shown to have lower zinc/copper ratios than normally developing peers and disturbed zinc homeostasis is suggested as a risk factor for neurodege‐

*N-Acetyl-L-Cysteine (NAC)* NAC is sulphur-containing (sulfhydryl) amino acid which is present in many proteins, and is in the same class as the amino acid methionine. NAC is a naturally occurring amino sugar and is a form of cysteine which has been demonstrated to facilitate the short term cellular detoxification of alcohol, tobacco smoke, acetaminophen poisoning and environmental pollutants in several *in vitro* studies. NAC supplementation over long periods associates with modest increases in serum glutathione, but has not proven particularly useful in the treatment of chronic, long-term intracellular glutathione deficiencies. Furthermore, therapeutic levels of NAC are relatively toxic. At therapeutic doses, oral NAC supplementation can cause significant side effects. Cerebral symptoms, nausea, blurred vision,

*Vitamin A and E* Retinol, the most useful form of vitamin A, (along with retinal and retinoic acid) and vitamin E are lipid-soluble. They can build up in the body and cause toxicity in excess amounts. Vitamin A is believed to improve sensory perception, language, and attention [48].

are likely associated with autism.

nerative diseases [24, 25, 47].

cells which rely largely on mitochondria for energy [41, 42].

100 Pharmacology and Nutritional Intervention in the Treatment of Disease

concentrations of selenium was found in a recent meta-analysis [43].

and vomiting are associated with NAC supplementation.

*Vitamin B6and B12* Some forms of epilepsy are linked with deficiency of B vitamins. Lowered concentrations of B vitamins have been linked with cognitive decline and dementia in older adults. No statistically significant differences were found in plasma B12 levels between ASD cases and controls in meta-analysis [51]. Beneficial effects from high dose supplementation of vitamin B6 with magnesium are shown in a subgroup of ASD individuals. Magnesium is combined to the treatment to prevent hyperactivity that can be caused by vitamin B6 taken alone [52]. Peripheral neuropathy is a rare side effect of high dose vitamin B6 treatment which generally disappears when supplementation is finished.

*Folic acid* Folic acid, the synthetic form of folate or vitamin B9, during the first month of pregnancy may reduce child´s risk of autism [53]. Folate, vitamin B6 and vitamin B12 are important coenzymes of the homocysteine-degrading remethylation and transsulfuration pathways [54] and their deficiencies can lead to an elevated serum concentration of homocys‐ teine (hyperhomocysteinemia). In addition, B vitamins play a crucial role in the reduction of oxidative stress and in the methylation of different proteins [55]. Serum and plasma levels of folic acid are not affected in children with ASD when compared with control subjects and homocysteine show no association with ASD [51].

*Vitamin C* Higher or not abnormal plasma levels of vitamin C have been reported in individuals with ASD when compared with controls [49, 50]. There is evidence that vitamin C brings about significant improvement in people with autism [56]. Vitamin C softens stools and can help in constipation by making the stools easier to pass.

*Magnesium (Mg)* Plasma magnesium levels are shown to be lower in autistic than control children [57]. Magnesium is usually combined with vitamin B6 supplement in ASD [52]. The efficacy of this treatment in ASD remains to be verified. Magnesium has been helpful for many autistic children who suffer from constipation. Magnesium is a smooth muscle relaxant, and it helps to pass stools by promoting rhythmic contractions of the intestinal smooth muscle. High magnesium supplementation can cause diarrhea as a side effect and the dose of magne‐ sium should be increased gradually until the desired effects are achieved.

*Phenol sulfotransferase (PST)* Phenol sulfotransferase is an enzyme involved in liver detoxifica‐ tion. Researchers have proposed that PST is compromised in autistic children. A study [58] demonstrated that the PST enzyme system was functioning at sub-optimal levels in more than half of the autistic children tested. Since the deficiency of sulfur in the bloodstream and impairment of the PST system interferes with the body's ability to process and eliminate phenols, this may explain why many children with autism are so sensitive to phenols ingested via certain foods. Low levels of plasma sulphate are reported in autistic children when compared with age-matched control children [59].

*Coenzyme Q10* Classical mitochondrial diseases associate with a subset of autism cases. Both nuclear and mitochondrial genes can underlie mitochondrial dysfunction that is associated with autism [60]. Coenzyme Q10 administration in rats increases mitochondrial concentra‐ tions, extended survival times, and exhibited neuroprotective effects [61]. In human, coenzyme Q10 has also been shown to be beneficial in patients with mitochondrial disease [62].

and the occurrence of autism [72]. It was suggested that the cytokine interleukin-10 (IL-10) could play a key role in the mechanisms that lead to alterations in the adaptive immune response in individuals with autism [73]. Croonenberghs et al. [74] found elevated levels of interleukin-12 and gamma interferon in autistic patients. They reported that proinflammatory cytokines may induce some of the behavioral symptoms of autism, including social with‐

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103

Fragile X syndrome (FXS) is a common cause of inherited intellectual disability and the most common monogenic cause of autism [75]. Neurobehavioral symptoms of FXS include restrict‐ ed repetitive and stereotyped patterns of behavior, hyperactivity, defects in sensory integra‐ tion, and communication difficulties. About 30% of FXS males fulfill the standardized criteria of autism [76-78]. Improved understanding of the FXS etiology has facilitated clinical trials to identify targeted treatments with potential to reverse or improve behavioral and cognitive deficits in FXS. Studies of the animal models of FXS suggest that an imbalance in gammaaminobutyric acid (GABA)/glutamate transmission is involved in the pathogenesis of behav‐

Defects of antioxidant system is seen as altered levels of components of the glutathione system and higher levels of reactive oxygen species, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activation, lipid peroxidation and protein oxidation are found in the murine model of FXS [79]. These findings have led to research to treat FXS individuals with antioxidants. High doses of alpha-tocopherol have been shown to reduce hyperactivity, anxiety, and corticosterone levels in *Fmr1* knockout mice, the mouse model for FXS [80]. Similar beneficial effects have been shown experimentally with melatonin which is a sleep hormone in addition to its effects as an anti-oxidant [81]. GABA-B was identified as a drug target in treatment of FXS and autism. Recent studies show that arbaclofen and STX209, an oral selective GABA type B (GABA-B) receptor agonist, have the potential to normalize the deficient inhibitory neurotransmission in FXS and autism [82-84]. In addition, clinical trials with metabotrobic glutamate receptor antagonists and donepezil in FXS are on-going [82]. Further‐ more, beneficial effects of minocycline and lithium on the functional defects of FXS individuals

Several mechanisms can be involved in antioxidant function of zinc. First, zinc may protect protein sulfhydryl groups from oxidative modification by influencing the conformation and reducing potential of thiol groups. Since the sulfhydryl groups are required for the catalytic activities of several enzymes, zinc protects the enzyme's activity from oxidative inactivation. Second, zinc may antagonize the activity of transition metals such as iron and copper. Zago and Oteiza [85] showed that zinc may compete with copper and iron ions and prevent transition metal mediated oxidative modifications. Third mechanism for the antioxidant property of zinc is that zinc may reduce oxidative damage indirectly by modulating antioxi‐ dant defence including (a) enzymes which catalytically remove free radicals and reactive

**3.1. Defects of antioxidant system in fragile X syndrome, a variant of ASD**

drawal, resistance to novelty and sleep disturbances.

ioural defects.

remain to be properly evaluated.

**3.2. Antioxidant properties of zinc**

*Uric acid* Urate is the final oxidation product of purine metabolism. A subset of autistics display higher uric acid excretion to urine than controls [63].

*Amino acids* There is evidence that children with ASD are likely to have abnormalities in amino acid metabolism. Gastrointestinal problems and selective eating may contribute to these changes. In several studies, an increased plasma level of glutamate that is the major excitatory neurotransmitters in the central nervous system is a consistent finding [64]. The level of glutamine and proteins that are involved in transforming glutamate to GABA, an inhibitory neurotransmitter in brain, are shown to be decreased in children with autism [65]. In addition, the levels of essential amino acids are reduced in urine of ASD individuals. Raised glutamic acid levels and reduced plasma glutamine are also found in individuals with Asperger syndrome and their siblings and parents [66].

*L-Arginine* Arginine is a semi-essential amino acid because the human body does not always manufacture an adequate supply. Arginine deficiency may result from digestive [67]problems or selective eating in ASD. It has been found that autistic subjects have more essential amino acid deficiencies than controls [67] and high plasma levels of arginine were reported in a recent mass spectroscopy study of high-functioning ASD males [68].

*Lithium* Lithium appears to be an essential mineral in small quantities for good mental health. Low levels of lithium have been reported in the hair of children with autism and their mothers when compared with not autistic children and their mothers in Arizona.

*Prostaglandins (PGs)* Accumulation of the very long chain fatty acids (VLCFAs) in the mem‐ brane of red cells of autistic individuals [69] indicates involvement of peroxisomal beta oxidation in the pathophysiology of ASD. Peroxisomes are cellular organelles which are important in the biotransformation of endogenous compounds in lipid metabolism, including fatty acids, steroids, and prostaglandins They are pivotal for the formation of myelin, neuro‐ transmission, detoxification of exogenous compounds and xenobiotic [70]. Defects in peroxi‐ somal beta oxidation [71] may link disturbances in endocrine, gastrointestinal, and immune systems as well as cytochrome P450 enzyme nitric oxide synthase (NOS) and nitric oxide formation (NO) in ASD [69]. Plasma prostaglandin E2 (PGE2) and leukotriene levels have been shown to higher in ASD than control individuals [49].

*Cytokines* Inflammation and immune system dysfunction are implicated in neuropsychiatric disorders such as ASD, attention deficit hyperactivity disorder, and schizophrenia. In fact, there is a significant overlap in the pathogenic factors, structural and functional abnormalities of brain, and clinical manifestations. Altered immune responses in ASD children are seen as decreased responsiveness of peripheral blood mononuclear cells to mitogen stimulation, reduced number of T lymphocytes, elevated levels of interleukin 1 receptor antagonist and elevated production of tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β) by blood mononuclear cells. An emerging area of research in autism is the role of prenatal exposure to inflammatory mediators during critical developmental periods. Epidemiological data have revealed significant correlations between prenatal exposure to pathogens, including influenza, and the occurrence of autism [72]. It was suggested that the cytokine interleukin-10 (IL-10) could play a key role in the mechanisms that lead to alterations in the adaptive immune response in individuals with autism [73]. Croonenberghs et al. [74] found elevated levels of interleukin-12 and gamma interferon in autistic patients. They reported that proinflammatory cytokines may induce some of the behavioral symptoms of autism, including social with‐ drawal, resistance to novelty and sleep disturbances.

#### **3.1. Defects of antioxidant system in fragile X syndrome, a variant of ASD**

Fragile X syndrome (FXS) is a common cause of inherited intellectual disability and the most common monogenic cause of autism [75]. Neurobehavioral symptoms of FXS include restrict‐ ed repetitive and stereotyped patterns of behavior, hyperactivity, defects in sensory integra‐ tion, and communication difficulties. About 30% of FXS males fulfill the standardized criteria of autism [76-78]. Improved understanding of the FXS etiology has facilitated clinical trials to identify targeted treatments with potential to reverse or improve behavioral and cognitive deficits in FXS. Studies of the animal models of FXS suggest that an imbalance in gammaaminobutyric acid (GABA)/glutamate transmission is involved in the pathogenesis of behav‐ ioural defects.

Defects of antioxidant system is seen as altered levels of components of the glutathione system and higher levels of reactive oxygen species, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activation, lipid peroxidation and protein oxidation are found in the murine model of FXS [79]. These findings have led to research to treat FXS individuals with antioxidants. High doses of alpha-tocopherol have been shown to reduce hyperactivity, anxiety, and corticosterone levels in *Fmr1* knockout mice, the mouse model for FXS [80]. Similar beneficial effects have been shown experimentally with melatonin which is a sleep hormone in addition to its effects as an anti-oxidant [81]. GABA-B was identified as a drug target in treatment of FXS and autism. Recent studies show that arbaclofen and STX209, an oral selective GABA type B (GABA-B) receptor agonist, have the potential to normalize the deficient inhibitory neurotransmission in FXS and autism [82-84]. In addition, clinical trials with metabotrobic glutamate receptor antagonists and donepezil in FXS are on-going [82]. Further‐ more, beneficial effects of minocycline and lithium on the functional defects of FXS individuals remain to be properly evaluated.

#### **3.2. Antioxidant properties of zinc**

tions, extended survival times, and exhibited neuroprotective effects [61]. In human, coenzyme

*Uric acid* Urate is the final oxidation product of purine metabolism. A subset of autistics display

*Amino acids* There is evidence that children with ASD are likely to have abnormalities in amino acid metabolism. Gastrointestinal problems and selective eating may contribute to these changes. In several studies, an increased plasma level of glutamate that is the major excitatory neurotransmitters in the central nervous system is a consistent finding [64]. The level of glutamine and proteins that are involved in transforming glutamate to GABA, an inhibitory neurotransmitter in brain, are shown to be decreased in children with autism [65]. In addition, the levels of essential amino acids are reduced in urine of ASD individuals. Raised glutamic acid levels and reduced plasma glutamine are also found in individuals with Asperger

*L-Arginine* Arginine is a semi-essential amino acid because the human body does not always manufacture an adequate supply. Arginine deficiency may result from digestive [67]problems or selective eating in ASD. It has been found that autistic subjects have more essential amino acid deficiencies than controls [67] and high plasma levels of arginine were reported in a recent

*Lithium* Lithium appears to be an essential mineral in small quantities for good mental health. Low levels of lithium have been reported in the hair of children with autism and their mothers

*Prostaglandins (PGs)* Accumulation of the very long chain fatty acids (VLCFAs) in the mem‐ brane of red cells of autistic individuals [69] indicates involvement of peroxisomal beta oxidation in the pathophysiology of ASD. Peroxisomes are cellular organelles which are important in the biotransformation of endogenous compounds in lipid metabolism, including fatty acids, steroids, and prostaglandins They are pivotal for the formation of myelin, neuro‐ transmission, detoxification of exogenous compounds and xenobiotic [70]. Defects in peroxi‐ somal beta oxidation [71] may link disturbances in endocrine, gastrointestinal, and immune systems as well as cytochrome P450 enzyme nitric oxide synthase (NOS) and nitric oxide formation (NO) in ASD [69]. Plasma prostaglandin E2 (PGE2) and leukotriene levels have been

*Cytokines* Inflammation and immune system dysfunction are implicated in neuropsychiatric disorders such as ASD, attention deficit hyperactivity disorder, and schizophrenia. In fact, there is a significant overlap in the pathogenic factors, structural and functional abnormalities of brain, and clinical manifestations. Altered immune responses in ASD children are seen as decreased responsiveness of peripheral blood mononuclear cells to mitogen stimulation, reduced number of T lymphocytes, elevated levels of interleukin 1 receptor antagonist and elevated production of tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β) by blood mononuclear cells. An emerging area of research in autism is the role of prenatal exposure to inflammatory mediators during critical developmental periods. Epidemiological data have revealed significant correlations between prenatal exposure to pathogens, including influenza,

Q10 has also been shown to be beneficial in patients with mitochondrial disease [62].

higher uric acid excretion to urine than controls [63].

102 Pharmacology and Nutritional Intervention in the Treatment of Disease

syndrome and their siblings and parents [66].

mass spectroscopy study of high-functioning ASD males [68].

shown to higher in ASD than control individuals [49].

when compared with not autistic children and their mothers in Arizona.

Several mechanisms can be involved in antioxidant function of zinc. First, zinc may protect protein sulfhydryl groups from oxidative modification by influencing the conformation and reducing potential of thiol groups. Since the sulfhydryl groups are required for the catalytic activities of several enzymes, zinc protects the enzyme's activity from oxidative inactivation. Second, zinc may antagonize the activity of transition metals such as iron and copper. Zago and Oteiza [85] showed that zinc may compete with copper and iron ions and prevent transition metal mediated oxidative modifications. Third mechanism for the antioxidant property of zinc is that zinc may reduce oxidative damage indirectly by modulating antioxi‐ dant defence including (a) enzymes which catalytically remove free radicals and reactive species, like superoxide dismutase, catalase, and glutathione peroxidase; (b) proteins which minimize the availability of pro-oxidants, like transferrins, ceruloplasmin and metallothio‐ neins; (c) low-molecular-mass ROS and RNS scavengers, like glutathione, ascorbic acid, uric acid, and alpha-tocopherol.

display behavioural abnormalities such as over-responsivity and hyperactivity-like behaviour whereas prenatal zinc deficiency caused ASD-related behaviour such as deficits in vocalization and social behaviour. Furthermore, low zinc levels were shown to increase the incidence of seizures, hypotonia, attention deficits, and hyperactivity in patients with Phelan-McDermid syndrome, an ASD caused by haploinsufficiency of a member of ProSAP/Shank family.

Oxidative Stress and Dietary Interventions in Autism: Exploring the Role of Zinc, Antioxidant Enzymes and Other…

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105

In the brain, zinc is in its ionic form (Zn2+) within synaptic vesicles of glutamatergic nerve terminals or bound to metalloproteins and intracellulary mobilized by oxidative stress. Zn2+is thought to be released from the nerve terminals in an activity-dependent manner synaptically and it may be a key modulator of neuronal activity and survival. Extracellular Zn2+is involved in the regulation of the balance of excitation and inhibition, and exogenously applied Zn2+have

Zinc homeostasis in the brain is maintained by the blood-brain and blood-cerebrospinal fluid barriers. Researchers are beginning to understand zinc's role in maintaining the structural integrity of the endothelia, which line the blood vessels, and the epithelia, which line the gastrointestinal tract. In atherosclerosis, arterial endothelial cells are destroyed by oxidating fatty acids and inflammatory immune factors. Zinc deficiency magnifies the defect. Supple‐ mentation with zinc has recently been found to protect the integrity of the blood vessel cell lining which helps maintain immune function in the elderly [100]. Within two months of zinc supplementation, resistance improves, and the chances of surviving an infection increase [101]. The critical role of zinc to immune function is consistent with its beneficial effects against infection. Supplement of zinc, as well as vitamin C, may enhance the activity of natural killer cells. Deficiencies of zinc, as well as vitamins A and D, conversely reduce natural killer cell

Many metallic elements play an important role in the maintenance of human health and an imbalance in trace elements may be a significant factor in a wide variety of physical and psychiatric conditions. Copper and zinc are regarded as neurotransmitters and they are both found in high concentrations in the hippocampus of brain. Elevated copper and depressed zinc levels have been associated with hyperactivity, attention deficit disorders, depression, and ASD [103]. Also, many individuals with ASD or paranoid schizophrenia have elevated

The actions of selenium, zinc, and copper are all intermingled in the regulation of detoxify‐ ing and antioxidant enzymes. Zinc may induce a decrease in intracellular cadmium accumulation and the sequestration of cadmium by cadmium-induced metallothionine [104]. However, the actual activity of cadmium/zinc-metallothionine-whether protective or damaging-is believed to depend on various parameters governed by the extracellular and

blood copper levels combined with other biochemical imbalances [19,20].

effects on the activity of glutamate, GABAA, and glycine ionotropic receptors.

function [102].

**3.3. Copper-Zinc and Cadmium-Zinc Imbalance**

intracellular environment [105].

#### *3.2.1. Zinc deficiency*

Inadequate zinc intake has been implicated in many diseases; however, no laboratory test can clearly distinguish zinc deficiencies [86, 87]. Most at risk of zinc deficiency are young children, teenage girls, and people over age 71. Although zinc deficiency is largely uncommon in the developed world, it has been estimated to affect about 2 billion people worldwide. Elderly people, those with lower incomes, and those with less education may be apt to consume inadequate amounts of zinc. Zinc deficiency can be caused by insufficient dietary intake of the mineral and also by some underlying conditions including malabsorption syndrome, liver and renal disease, diabetes, sickle cell disease and other chronic illnesses [88, 89]. Appropriate absorption of nutrients depends on an intact intestine and intestinal injuries can lead to zinc deficiency. This may cause appetite loss and diarrhea which speeds the downward spiral of zinc loss and tissue damage [90]. Supplementation with 12.5-50 µM zinc has been shown to enhance epithelial cell restitution, the initial step of wound healing. Zinc has been found to help healing of intestinal lesions that associate with inflammatory bowel disease that is a serious intestinal disorder.

Improved neurophysiologic performance, positive growth response, and significantly reduced mortality and morbidity with zinc supplementation have been observed in Chinese children [91]. Zinc deficient animals display an increased susceptibility to exogenous oxidative stress such as endotoxin exposure and hyperoxia [92] and zinc is thought to protect macro‐ molecules such as proteins, lipids and DNA from oxidative damage. Mice defective in CuZnSOD develop neurological damage and cancer at an accelerated rate as they age [16]. Zinc depletion affects the expression of metallothioneins that are low-molecular-weight proteins with high cysteine content and high affinity for metal ions [93]. In addition, zinc depletion decreases α-tocopherol and ascorbate levels in liver and lung [94], but may not change α-tocopherol level in testes [95]. Moreover, plasma uric acid concentration has been shown to be elevated occasionally in zinc-deficient rats [96].

#### *3.2.2. The role of zinc in reversing synaptic deficits in autism*

There is evidence that zinc deficiency plays a role in autism [24] and the zinc-dependent mechanisms underlying the neurobiology of autism are under active investigation. Zinc plays important roles in nucleic acid/protein synthesis, cell replication, tissue growth and repair. Zinc finger proteins (ZNF81 and ZNF182) are the largest class of DNA binding proteins encoded in the human genome [97] and mutations involving genes encoding these proteins have been shown to associate with mental retardation [98]. Zinc is a regulator core component of the postsynaptic density (PSD), an active zone at the synapse. A low postsynaptic availability of zinc has been shown to affect the activity dependent increase of proteins of the ProSAP/ Shank family which are linked to ASD [99]. Mice with acute zinc deficiency were shown to display behavioural abnormalities such as over-responsivity and hyperactivity-like behaviour whereas prenatal zinc deficiency caused ASD-related behaviour such as deficits in vocalization and social behaviour. Furthermore, low zinc levels were shown to increase the incidence of seizures, hypotonia, attention deficits, and hyperactivity in patients with Phelan-McDermid syndrome, an ASD caused by haploinsufficiency of a member of ProSAP/Shank family.

In the brain, zinc is in its ionic form (Zn2+) within synaptic vesicles of glutamatergic nerve terminals or bound to metalloproteins and intracellulary mobilized by oxidative stress. Zn2+is thought to be released from the nerve terminals in an activity-dependent manner synaptically and it may be a key modulator of neuronal activity and survival. Extracellular Zn2+is involved in the regulation of the balance of excitation and inhibition, and exogenously applied Zn2+have effects on the activity of glutamate, GABAA, and glycine ionotropic receptors.

Zinc homeostasis in the brain is maintained by the blood-brain and blood-cerebrospinal fluid barriers. Researchers are beginning to understand zinc's role in maintaining the structural integrity of the endothelia, which line the blood vessels, and the epithelia, which line the gastrointestinal tract. In atherosclerosis, arterial endothelial cells are destroyed by oxidating fatty acids and inflammatory immune factors. Zinc deficiency magnifies the defect. Supple‐ mentation with zinc has recently been found to protect the integrity of the blood vessel cell lining which helps maintain immune function in the elderly [100]. Within two months of zinc supplementation, resistance improves, and the chances of surviving an infection increase [101]. The critical role of zinc to immune function is consistent with its beneficial effects against infection. Supplement of zinc, as well as vitamin C, may enhance the activity of natural killer cells. Deficiencies of zinc, as well as vitamins A and D, conversely reduce natural killer cell function [102].

#### **3.3. Copper-Zinc and Cadmium-Zinc Imbalance**

species, like superoxide dismutase, catalase, and glutathione peroxidase; (b) proteins which minimize the availability of pro-oxidants, like transferrins, ceruloplasmin and metallothio‐ neins; (c) low-molecular-mass ROS and RNS scavengers, like glutathione, ascorbic acid, uric

Inadequate zinc intake has been implicated in many diseases; however, no laboratory test can clearly distinguish zinc deficiencies [86, 87]. Most at risk of zinc deficiency are young children, teenage girls, and people over age 71. Although zinc deficiency is largely uncommon in the developed world, it has been estimated to affect about 2 billion people worldwide. Elderly people, those with lower incomes, and those with less education may be apt to consume inadequate amounts of zinc. Zinc deficiency can be caused by insufficient dietary intake of the mineral and also by some underlying conditions including malabsorption syndrome, liver and renal disease, diabetes, sickle cell disease and other chronic illnesses [88, 89]. Appropriate absorption of nutrients depends on an intact intestine and intestinal injuries can lead to zinc deficiency. This may cause appetite loss and diarrhea which speeds the downward spiral of zinc loss and tissue damage [90]. Supplementation with 12.5-50 µM zinc has been shown to enhance epithelial cell restitution, the initial step of wound healing. Zinc has been found to help healing of intestinal lesions that associate with inflammatory bowel disease that is a

Improved neurophysiologic performance, positive growth response, and significantly reduced mortality and morbidity with zinc supplementation have been observed in Chinese children [91]. Zinc deficient animals display an increased susceptibility to exogenous oxidative stress such as endotoxin exposure and hyperoxia [92] and zinc is thought to protect macro‐ molecules such as proteins, lipids and DNA from oxidative damage. Mice defective in CuZnSOD develop neurological damage and cancer at an accelerated rate as they age [16]. Zinc depletion affects the expression of metallothioneins that are low-molecular-weight proteins with high cysteine content and high affinity for metal ions [93]. In addition, zinc depletion decreases α-tocopherol and ascorbate levels in liver and lung [94], but may not change α-tocopherol level in testes [95]. Moreover, plasma uric acid concentration has been

There is evidence that zinc deficiency plays a role in autism [24] and the zinc-dependent mechanisms underlying the neurobiology of autism are under active investigation. Zinc plays important roles in nucleic acid/protein synthesis, cell replication, tissue growth and repair. Zinc finger proteins (ZNF81 and ZNF182) are the largest class of DNA binding proteins encoded in the human genome [97] and mutations involving genes encoding these proteins have been shown to associate with mental retardation [98]. Zinc is a regulator core component of the postsynaptic density (PSD), an active zone at the synapse. A low postsynaptic availability of zinc has been shown to affect the activity dependent increase of proteins of the ProSAP/ Shank family which are linked to ASD [99]. Mice with acute zinc deficiency were shown to

shown to be elevated occasionally in zinc-deficient rats [96].

*3.2.2. The role of zinc in reversing synaptic deficits in autism*

acid, and alpha-tocopherol.

104 Pharmacology and Nutritional Intervention in the Treatment of Disease

serious intestinal disorder.

*3.2.1. Zinc deficiency*

Many metallic elements play an important role in the maintenance of human health and an imbalance in trace elements may be a significant factor in a wide variety of physical and psychiatric conditions. Copper and zinc are regarded as neurotransmitters and they are both found in high concentrations in the hippocampus of brain. Elevated copper and depressed zinc levels have been associated with hyperactivity, attention deficit disorders, depression, and ASD [103]. Also, many individuals with ASD or paranoid schizophrenia have elevated blood copper levels combined with other biochemical imbalances [19,20].

The actions of selenium, zinc, and copper are all intermingled in the regulation of detoxify‐ ing and antioxidant enzymes. Zinc may induce a decrease in intracellular cadmium accumulation and the sequestration of cadmium by cadmium-induced metallothionine [104]. However, the actual activity of cadmium/zinc-metallothionine-whether protective or damaging-is believed to depend on various parameters governed by the extracellular and intracellular environment [105].
