Elevated levels of oxidative stress are a well-described feature of ASD ^{[1][2][3][4][5]}[61,62,63,64,65]. The actions in mitochondria that result in the synthesis of adenosine triphosphate (ATP) naturally produce free radicals, also known as reactive oxygen species (ROS). Reactive nitrogen species (RNS) and radicals with carbon and sulfur centers are also produced ^[6][66]. As a result, small amounts of ROS in brain tissue should be regarded as normal, but large amounts that cause oxidative stress are harmful. Excessive free radical generation or insufficient cellular mechanisms to alleviate oxidative stress can contribute to elevated oxidative stress levels. The brain is particularly vulnerable to oxidative stress because of its high energy (ATP) requirements.

The brain is a lipid-rich tissue. Lipid peroxidation thus poses a particular threat to brain cells ^[6][66]. However, excess ROS also causes protein oxidation in addition to lipid peroxidation. Together, the two processes might be responsible for aberrant neurogenesis, synaptic signaling, plasticity, brain cell death, and functional deficits ^{[7][8][9][10]}[67,68,69,70]. For example, oxidative stress may change brain-derived neurotrophic factor levels (BDNF) ^[11][71]. BDNF is a growth factor linked to the pathogenesis of ASD as a regulator of neurogenesis, neuron survival, and synaptic plasticity ^[12][72].

As underlying reasons for increased ROS in ASD, numerous studies implicate an increase in BBB permeability that results in neuroinflammation ^[13][14][73,74]. For example, it has been noted that exposure to mercury damages the BBB, exposing the CNS to undesired substances that might cause processes such as neuroinflammation ^[15][75]. Additionally, it has been suggested that excitotoxicity caused by aberrant glucocorticoid receptor signaling, and altered N-methyl-d-aspartate (NMDA) receptor signaling contribute to ROS ^[16][17][18][76,77,78].

One defense mechanism that can lower ROS levels is the antioxidant response system. It works by activating antioxidant enzymes such as catalase (CAT), glutathione peroxidase, glutathione reductase, glyoxalase, and superoxide dismutase (SOD) ^[19][79]. Interestingly, SOD enzymes require essential trace metals, for example, copper and zinc (Cu-Zn SOD). Low-molecular-weight antioxidants such as glutathione, uric acid, ascorbic acid, and melatonin are used in the second stage of the antioxidant response system. These primarily act through scavenging metals ^[20][80].

Elevated levels of oxidative stress are also a well-described feature of heavy metal exposure. Mercury is one of the toxic metals that raises oxidative stress levels and harms mitochondria ^[21][22][23][81,82,83]. Mercury can inhibit sulfhydryl-containing enzymes by forming a covalent bond with sulfhydryl (thiol) groups ^[24][84]. Similarly, other proteins and non-protein molecules, such as glutathione (GSH), may be impacted by the direct chemical interaction between MeHg and thiol ^[24][84]. Due to GSH’s function as an antioxidant, if GSH levels fall, oxidative stress rises ^[25][85]. However, it has also been demonstrated that MeHg directly interacts with nucleophilic protein groups to cause oxidative stress ^[19][79]. In addition, mercury exposure also results in glutamate excitotoxicity ^[26][86]. MeHg increases glutamate levels in the synaptic cleft by inhibiting glutamate re-uptake into synaptic vesicles and promoting spontaneous release, according to in vitro and animal studies ^{[26][27][28][29]}[86,87,88,89].

Abnormally high glutamate concentrations in the synaptic cleft can cause NMDA-type glutamate receptor (R) overactivation. Apart from direct consequences on synapse formation, stability, and function, this raises the amount of sodium and calcium that enters neurons through NMDARs. In response, excessive intracellular calcium will again boost oxidative stress (for example, by translocation into mitochondria) ^[30][90]. Because astrocytes help remove the glutamate from the synaptic cleft, mercury injury to astrocytes exacerbates excitotoxicity ^[31][91].

Excessive cadmium concentrations also result in oxidative stress. Due to its affinity for thiol groups, cadmium may accumulate inside cells as cadmium-thiol complexes ^[32][92]. As a result, GSH levels fall, impacting cellular defenses against ROS ^[33][93]. Additionally, cadmium interferes with cellular protein breakdown, which is demonstrated by an increase in the number of ubiquitinated proteins. As a result, cadmium disrupts many cellular functions, including the antioxidant response ^[34][35][94,95]. Besides, ROS production by lead has been identified as a key mechanism underpinning lead poisoning. For example, the δ-aminolevulinic acid (ALA) dehydrase (ALAD) is affected by the presence of lead, leading to elevated ALA levels, which induces the production of ROS. The oxidation product of ALA may, in turn, cause DNA damage ^[36][96]. In addition, lead impacts the antioxidant defense mechanisms ^[37][97]. By blocking functional sulfhydryl groups in various enzymes, including SOD, CAT, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, lead has been demonstrated to change antioxidant activity ^[37][97].

Oxidative stress and mitochondrial damage are intimately related. Thus, mitochondrial dysfunction may be linked to ASD ^{[38][39][40][41][42][43]}[98,99,100,101,102,103]. Mitochondria oxidize glucose and fatty acids to produce ATP. In addition, the mitochondria play critical roles in calcium homeostasis and programmed cell death (apoptosis). They impact CNS neuronal functions such as synaptic plasticity and neurotransmitter release ^[44][104]. Brain cells are cells with a high energy requirement. Therefore, they have more mitochondria and are particularly vulnerable.

Children with ASD may have mitochondrial (mt) damage, which manifests as mtDNA over-replication, mtDNA deletions, and mitochondrial malfunction (lower mitochondrial-dependent oxygen consumption and increased hydrogen peroxide (H₂O₂) generation) ^[38][45][98,105]. It was reported that symptoms discovered as a result of mitochondrial malfunction coincide with those of ASD, suggesting that mitochondrial damage may contribute to the phenotype of ASD ^[46][106]. A reduced synaptic neurotransmitter release and improper calcium signaling due to mitochondrial malfunction have been proposed as potential pathomechanisms. Notably, plasma membrane calcium channels are impacted by mitochondria, which serve as calcium reserves ^[47][107].

Numerous immunological disorders can arise from toxic metal exposure. In epidemiological research and mechanistic studies utilizing animal models, disorders of the immune system and inflammation have been linked to ASD. It has been demonstrated that lead, cadmium, and mercury directly influence gene expression. The altered genes included those that encode proteins key to regulating oxidative stress and inflammation, among others ^[48][49][50][108,109,110]. Furthermore, it was shown that lead and methylmercury could directly trigger glial reactivity ^[51][111], and a rise in ROS and BBB permeability may be a component of this. For example, mercury is concentrated in astrocytes and, to a lesser extent, in microglial cells inside the CNS ^[52][53][112,113], which causes these cells to become dysfunctional ^[54][114]. Notably, activated glial cells such as microglia and astrocytes release the proinflammatory cytokines Tumor Necrosis Factor (TNF), Interleukin (IL)-1, and IL-6 ^[55][115]. TNF, IL-1, and IL-6 are elevated in biosamples of individuals with ASD, and their levels positively correlate with ASD severity ^[56][57][58][116,117,118]. Animal studies have demonstrated that IL-6, in particular, may change synapse formation and plasticity ^[59][60][119,120].

In susceptible people, elevated mercury and lead levels result in increased production of proinflammatory cytokines and the development of autoantibodies even at low levels of chronic exposure ^[61][62][63][121,122,123]. For instance, one study discovered a correlation between increased blood levels of autoantibodies and increased mercury levels ^[64][124]. Neuronal cytoskeletal proteins, neurofilaments, and myelin basic protein (MBP) are among the proteins that lead and mercury-induced autoantibodies target ^[65][125].

Numerous studies have linked ASD to white matter abnormalities, including hypomyelination ^[66][67][68][126,127,128]. Neuronal axons in the CNS are myelinated when oligodendrocytes create myelin, a multi-lamellar membrane rich in lipids. Myelination is important in establishing connectivity in the developing brain. Toxic trace metals affect myelination. For example, lead exposure has been connected to disrupted connectivity ^[69][70][129,130]. Glial cells, including oligodendrocytes, take up lead ^[71][72][131,132]. A reduction in the activity of 2′,3′-Cyclic-nucleotide 3’-phosphodiesterase (CNPase) by lead, an enzyme that has been demonstrated to be essential for myelin formation, might be a factor explaining reduced myelinization ^[73][133]. The CNS white matter is also damaged by cadmium. It was shown that oligodendrocytes, and more so oligodendrocyte progenitors, are susceptible to cadmium. Cadmium toxicity was tied to excessive production of ROS, leading to oligodendrocyte death or dysfunction. In addition, MeHg toxicity is linked to the downregulation of MBP in pregnant and lactating rats ^[74][134]. MBP is essential for the construction and function of the myelin sheaths by oligodendrocytes and Schwann cells.

By competing with essential metals such as calcium and zinc for protein binding, toxic metals can affect activities mediated by these metals. In particular, calcium and zinc are critical intracellular signaling ions bound by many proteins. For example, around 10% of the human genome encodes for zinc-binding proteins ^[75][135]. Zinc and lead may compete for the same protein binding sites ^[76][77][136,137]. Zinc and mercury were also discovered to interact, and cadmium can inhibit the transport of zinc and calcium, including the transfer of zinc to the fetus ^[78][138]. In turn, this will influence the activity of enzymes and second messengers and interfere with cellular signaling, transport, and metabolism. Additionally, cadmium should compete somewhat with magnesium, copper, and iron ^[79][139]. The disturbance of physiological processes mediated by metals, such as calcium and zinc, may be a major pathomechanism of toxic metals ^[80][140] and contribute to the abovementioned processes.

For example, myelination is a zinc-dependent process. A study on rhesus monkeys showed that maternal zinc deficiency causes the offspring’s myelin protein profiles to change ^[81][141]. Additionally, it was discovered that MBP, a target of autoantibodies generated by lead and mercury, is a zinc-binding protein, indicating a function for zinc in myelin compaction ^[82][83][142,143]. Further, in addition to promoting the production of free radicals, toxic metals, such as cadmium, lead, and mercury, may also compromise the antioxidant system by displacing essential metals from antioxidant enzymes such as Cu-Zn SOD. Lead also disrupts mitochondrial calcium and substitutes calcium in calcium signaling systems, altering Calmodulin and numerous downstream protein kinases ^[84][144]. Thereby, lead impedes synaptic signaling and vital cellular processes, such as proliferation and differentiation, for example, by changing the activity of the protein kinase C (PKC) enzyme. Lead also reduces the calcium-dependent release of neurotransmitter vesicles ^[85][145]. This can be a way that lead affects glutamatergic neurotransmission ^[86][146], which is also dependent on critical zinc-regulated synaptic proteins ^[87][147].

Given that zinc can be replaced or the levels of zinc lowered by toxic metals, some of the consequences of zinc deficiency may be imitated by toxic metal overload. Thus, prenatal zinc deficiency and heavy metal overload may be two sides of the same coin.