**11. Environmental factors**

#### **11.1. Toxicity during neurodevelopment**

Although the involvement of genetic alterations in ASD is clearly accepted, new studies point to a similar or even greater contribution by environmental factors, particularly environmental toxicants. Toxic chemicals cause dysregulation of the developing human brain by interacting with the genome or through direct toxicity.

The developing human brain is exceptionally sensitive to injury caused by toxic chemical exposure with several developmental processes being highly vulnerable. The dissemination of chemical industrial agents in the environment is important contributor to the call "global silent pandemic of neurodevelopmental toxicity" [63]. An estimative by U.S. National Academy of Sciences (NAS) shows that 3% of all neurobehavioral disorders including ASD, attention-deficit hyperactivity disorder and dyslexia are caused directly by exposure to environmental toxics and that another 25% are caused by interactions between environmental factors and inherited susceptibility [103].

About 80,000 new synthetic chemicals have been developed in the past 50 years, but only about 20% was screened for potential toxicity in early neurodevelopment. The human fetus is susceptible and not well protected against industrial chemicals, because the placenta does not block the passage of all toxicants from the maternal to fetal circulation. The individual's brain is unique and individual variability in genetic susceptibility can influence responses to environmental toxicants [62, 79, 106].

Exposure to many environmental toxicants has been correlated to autism including pesticides, polychlorinated biphenyls, solvents, toluene, toxic waste, and heavy metals such as mercury, lead and arsenic, besides exposure to automotive air pollution [115, 132, 152]. On the other hand, there was a false indicative association between these disorders and vaccines such as the measles, mumps, rubella vaccine and thimerosal-containing vaccines against diphtheria, tetanus and pertussis vaccine. One meta-analysis, combining the results of five previous studies involving 1,256,407 children with five case-controlled studies with a further 9,920 children show that there was no relationship between vaccination and autism [144].

There is thus strong evidence that the relation between environmental and genetic factors may converge to neurotoxic mechanisms that could lead to behavior disorders. But what kind of biological mechanisms would be involved in the causation of autism due to toxic agents? Epigenetic mechanisms are known to affect the subsequent gene expression in the brain as has already been described in this chapter. Toxics can lead to a modification in epigenetic gene expression, resulting in methylation, histone modification or changes in non-protein-coding RNA [60]. However, possible individual susceptibilities to toxicants implicated in ASD, including altered detoxification, genetic factors, oxidative stress, altered neuronal develop‐ ment, epigenetic mechanisms, synaptic function, hormonal factors, etc., may amplify the effects of toxicants during the prenatal and early postnatal periods [123]. The susceptibility to effects of environmental toxicants needs more attention and studies.

#### **11.2. Oxidative stress**

**11. Environmental factors**

328 Autism Spectrum Disorder - Recent Advances

**11.1. Toxicity during neurodevelopment**

with the genome or through direct toxicity.

factors and inherited susceptibility [103].

environmental toxicants [62, 79, 106].

Although the involvement of genetic alterations in ASD is clearly accepted, new studies point to a similar or even greater contribution by environmental factors, particularly environmental toxicants. Toxic chemicals cause dysregulation of the developing human brain by interacting

The developing human brain is exceptionally sensitive to injury caused by toxic chemical exposure with several developmental processes being highly vulnerable. The dissemination of chemical industrial agents in the environment is important contributor to the call "global silent pandemic of neurodevelopmental toxicity" [63]. An estimative by U.S. National Academy of Sciences (NAS) shows that 3% of all neurobehavioral disorders including ASD, attention-deficit hyperactivity disorder and dyslexia are caused directly by exposure to environmental toxics and that another 25% are caused by interactions between environmental

About 80,000 new synthetic chemicals have been developed in the past 50 years, but only about 20% was screened for potential toxicity in early neurodevelopment. The human fetus is susceptible and not well protected against industrial chemicals, because the placenta does not block the passage of all toxicants from the maternal to fetal circulation. The individual's brain is unique and individual variability in genetic susceptibility can influence responses to

Exposure to many environmental toxicants has been correlated to autism including pesticides, polychlorinated biphenyls, solvents, toluene, toxic waste, and heavy metals such as mercury, lead and arsenic, besides exposure to automotive air pollution [115, 132, 152]. On the other hand, there was a false indicative association between these disorders and vaccines such as the measles, mumps, rubella vaccine and thimerosal-containing vaccines against diphtheria, tetanus and pertussis vaccine. One meta-analysis, combining the results of five previous studies involving 1,256,407 children with five case-controlled studies with a further 9,920

There is thus strong evidence that the relation between environmental and genetic factors may converge to neurotoxic mechanisms that could lead to behavior disorders. But what kind of biological mechanisms would be involved in the causation of autism due to toxic agents? Epigenetic mechanisms are known to affect the subsequent gene expression in the brain as has already been described in this chapter. Toxics can lead to a modification in epigenetic gene expression, resulting in methylation, histone modification or changes in non-protein-coding RNA [60]. However, possible individual susceptibilities to toxicants implicated in ASD, including altered detoxification, genetic factors, oxidative stress, altered neuronal develop‐ ment, epigenetic mechanisms, synaptic function, hormonal factors, etc., may amplify the effects of toxicants during the prenatal and early postnatal periods [123]. The susceptibility to

children show that there was no relationship between vaccination and autism [144].

effects of environmental toxicants needs more attention and studies.

ASD also can be characterized by some physiological abnormalities, such as oxidative stress and immune dysregulation/inflammation. Many of the behavioral and cognitive features of ASD appear to arise from dysfunctions of the brain but studies have documented physiological abnormalities in organs other than the brain [122].

Many studies have reported genetic variations in glutathione-related pathways associated with autism, such as lower concentrations of reduced glutathione (GSH), higher levels of oxidized glutathione (GSSG) and a decrease in the GSH/GSSG redox ratio, along with a lower mitochondrial GSH reserve in individuals with ASD compared to controls [19, 2952, 65, 75 , 121]. Moreover, ASD severity has been correlated with lower GSH levels and markers of increased oxidative stress [2, 56, 123]. Oxidative stress studied in postmortem brain samples from individuals with ASD demonstrated low levels of GSH, the major cellular antioxidant, oxidative damage to proteins, lipids and deoxyribonucleic acid (DNA) as well as alterations in the activity of enzymes important for redox metabolism [123, 142].

Adaptive responses to oxidative stress were shown by [102], when significantly lower methionine synthase mRNA levels and lower homocysteine and cystathionine concentrations were observed in the frontal cortex of the brains of ten individuals with autism compared to ten controls.

Also it is possible that the reduced transportation of folate into the brain as a consequence of the folate receptor alpha autoantibody or mitochondrial dysfunction could reduce the methylation process and glutathione metabolism. Many findings reported for the brain (oxidative damage to lipids, protein and DNA, glutathione abnormalities and reduced function of enzymes essential in oxidative stress regulation) have been found in the blood, immune cells and cell lines from individuals with ASD, raising the question of whether these findings are specific to the brain or whether they represent a more general process [53, 123].

#### **11.3. Immune system**

Dysfunctional immune activity, both innate and adaptive, can impact neurodevelopment, cognitive function and behavior, suggesting a profound effect on neurodevelopment. Some studies in individuals with ASD have reported evidence of immune dysregulation and inflammation related to ASD, including disruption of genes of the immune system with elevations of tumor necrosis factor-alpha (TNF-α) in lymphocytes and amniotic fluid, altera‐ tions of immune proteins, and increased levels of cytokines, expression in genes related to immunity and microglial cell activation in brain tissue [113]. The expression of inflammatory genes in brain tissue was studied by [163], who reported that nuclear factor-kappa β expression in the orbitofrontal cortex was increased in individuals with autism compared to controls.

Microglias, a type of glial cell, are the resident macrophages of the brain and spinal cord. They therefore act as the first and main line of active immune defense of the CNS and participate in immune surveillance of the CNS and synaptic pruning in normal neurodevelopment. They are activated to eliminate some agents and damaged cells via phagocytosis, but, they may increase inflammation if chronically activated by releasing proinflammatory cytokines and free radicals [41]. Changes in microglial morphology and gene expression are seen in many psychiatric disorders and neurodegenerative diseases, such as Alzheimer's, Parkinson's, Multiple Sclerosis and autism. [100] and [165] reported that microglias in the dorsolateral prefrontal cortex were frequently located closer to neurons in ASD individuals compared to controls. Another study with autism showed microglial activation in the cerebellum, brain‐ stem, corpus callosum, fusiform gyri, superior temporal gyri, anterior cingulate, orbitofrontal, and parietal lobes [139].

Proteomic analysis indicates that the levels of many immune proteins in plasma, such as cytokines, chemokines, complement proteins, adhesion molecules and growth factors are altered in ASD [113]. Increases in plasma levels of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, IL-8 and IL-12p40] as well as macrophage migration inhibitory factor (MIF) and platelet-derived growth factor (PDGF), have been reported in ASD [11, 64].

Alterations in immune mediators can occur at an early stage of development and might alter NMDA and NMD receptor-mediated excitatory synaptic transmission and plasticity, which is relevant to ASD (Escobar et al., 2011). These immune mediators may be related in LTP which is involved in synaptic plasticity in learning and memory processes. IL-1 influences many molecular components of LTP, such as NMDA and AMPA receptor signaling and glutamate release. IL-6 is involved in the generation of LTP and an increase in IL-6 leads to a reduction of insulin-like growth factor (IGF)-binding protein 3 and IGF1 [43, 117]. Furthermore, other cytokines, such as IL-18, IL-1b, and TNF-α, act on AMPA and NMDA receptors in different ways [25].

Increases of pro-inflammatory cytokines (including TNF-α, IL-6 and granulocyte-macrophage colony-stimulating factor), a T helper cell type 1 cytokine (interferon gamma) and a chemokine (IL-8) were reported in postmortem frontal cortex brain samples of autistic individuals, but no significant differences in T helper cell type 2 cytokines were identified (IL-4, IL-5, and IL-10) [86]. The cytokines IL-2 and IL-4 have been shown to influence repetitive and cognitive behaviors. Mice treated with IL-2 showed an increased "climbing behavior" that is thought to denote repetitive behavior, a pattern of behavior that is characteristic of ASD.

There is evidence suggesting a role of immune dysfunction in ASD symptoms, but further investigations should be carried out to clarify these findings because it is still unknown whether these changes are a primary cause or a secondary consequence of neuronal deficits.

#### **11.4. Microbiota and autism**

Gastrointestinal (GI) disorders are a comorbidity of autism and interestingly have a strong correlation with disease severity [7, 16]. Thus, a growing number of papers point to the importance of the so-called "brain-gut axis", revealing the central role of the intestinal micro‐ biota (the new name of "microflora") in postnatal development and maturation of the immune and endocrine systems for control signaling in the CNS, brain function, and behavior [51; 158].

Individuals with ASD often suffer from GI illnesses, e.g., diarrhea, constipation, bloating, and gastroesophageal reflux [22, 3]. The composition of the microbiota is influenced primarily by

genetic factors, age and diet [80, 168], although the interactions between these are multifactorial and not well defined yet [21].

free radicals [41]. Changes in microglial morphology and gene expression are seen in many psychiatric disorders and neurodegenerative diseases, such as Alzheimer's, Parkinson's, Multiple Sclerosis and autism. [100] and [165] reported that microglias in the dorsolateral prefrontal cortex were frequently located closer to neurons in ASD individuals compared to controls. Another study with autism showed microglial activation in the cerebellum, brain‐ stem, corpus callosum, fusiform gyri, superior temporal gyri, anterior cingulate, orbitofrontal,

Proteomic analysis indicates that the levels of many immune proteins in plasma, such as cytokines, chemokines, complement proteins, adhesion molecules and growth factors are altered in ASD [113]. Increases in plasma levels of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, IL-8 and IL-12p40] as well as macrophage migration inhibitory factor (MIF) and

Alterations in immune mediators can occur at an early stage of development and might alter NMDA and NMD receptor-mediated excitatory synaptic transmission and plasticity, which is relevant to ASD (Escobar et al., 2011). These immune mediators may be related in LTP which is involved in synaptic plasticity in learning and memory processes. IL-1 influences many molecular components of LTP, such as NMDA and AMPA receptor signaling and glutamate release. IL-6 is involved in the generation of LTP and an increase in IL-6 leads to a reduction of insulin-like growth factor (IGF)-binding protein 3 and IGF1 [43, 117]. Furthermore, other cytokines, such as IL-18, IL-1b, and TNF-α, act on AMPA and NMDA receptors in different

Increases of pro-inflammatory cytokines (including TNF-α, IL-6 and granulocyte-macrophage colony-stimulating factor), a T helper cell type 1 cytokine (interferon gamma) and a chemokine (IL-8) were reported in postmortem frontal cortex brain samples of autistic individuals, but no significant differences in T helper cell type 2 cytokines were identified (IL-4, IL-5, and IL-10) [86]. The cytokines IL-2 and IL-4 have been shown to influence repetitive and cognitive behaviors. Mice treated with IL-2 showed an increased "climbing behavior" that is thought to

There is evidence suggesting a role of immune dysfunction in ASD symptoms, but further investigations should be carried out to clarify these findings because it is still unknown whether these changes are a primary cause or a secondary consequence of neuronal deficits.

Gastrointestinal (GI) disorders are a comorbidity of autism and interestingly have a strong correlation with disease severity [7, 16]. Thus, a growing number of papers point to the importance of the so-called "brain-gut axis", revealing the central role of the intestinal micro‐ biota (the new name of "microflora") in postnatal development and maturation of the immune and endocrine systems for control signaling in the CNS, brain function, and behavior [51; 158].

Individuals with ASD often suffer from GI illnesses, e.g., diarrhea, constipation, bloating, and gastroesophageal reflux [22, 3]. The composition of the microbiota is influenced primarily by

denote repetitive behavior, a pattern of behavior that is characteristic of ASD.

platelet-derived growth factor (PDGF), have been reported in ASD [11, 64].

and parietal lobes [139].

330 Autism Spectrum Disorder - Recent Advances

ways [25].

**11.4. Microbiota and autism**

Thus, several studies have been conducted in an attempt to profile the microbiota in ASD compared with healthy siblings and controls [4, 51, 59]. While some authors have reported little or no differences in the composition of the intestinal flora between children with ASD and their unaffected siblings, the imbalance in the intestinal microbial composition in samples of ASD has been identified, especially when compared to controls [49, 50].

Analyses of the feces of patients with ASD have revealed that the microbiome of ASD is significantly different to controls and consists in over 1,000 different species compared to unaffected children. Of the phyla, patients with autism have underrepresented *Firmicutes* and actinobacteria, especially bifidobacteria, and over-represented *Bacteroidetes* and proteobacteria compared to control individuals [49, 51, 119].

Interestingly, non-autistic siblings often presented intermediate microbiota profiles between ASD and controls [49, 155], possibly due to genetic factors, but also as a reflection of shared environmental conditions. These studies are complicated by the fact that autistic individuals often receive medications such as antibiotics and are often on special diets or have repetitive eating habits, both of which can alter the composition of the microbiota [89].

Interestingly, although *Bacteroidetes* are over-represented in ASD feces, the presence of the genus *Prevotella* and other fermenters, albeit in lesser amounts, has been described in the gut of children with ASD. *Prevotella* do not only have the ability to synthesize vitamin B1, which attenuates the ASD symptoms, but it is also considered a central niche to maintain the structure of the intestinal microbial community of healthy humans [10, 13, 162]. Moreover, *Propionibac‐ terium* and *Clostridium* that are over-represented in the intestine of ASD, produce propionic acid, a short-chain fatty acid capable of passing the gut-blood-brain barrier thereby changing neurophysiological processes by binding to acetyl-CoA and acetyl-carnitine which are involved in mitochondrial lipid transport. Recent experiments have shown that administration of propionic acid in young mice causes mental retardation with cognitive disabilities, innate neuroinflammatory response and restricted, repetitive behavioral, symptoms consistent with autism [53, 92]. Propionic acid induces behavioral and functional alterations in the brain of animals. Most likely, the presence of this acid alters mitochondrial function. Taken together, these reports support a view that the metabolism of proteins and the host/pathogen relation‐ ship is altered in patients with autism [99].

Few studies have considered a deregulated metabolism of fecal levels free of amino acids in autism. Certain amino acids, in particular glutamic acid (Glu), act as neurotransmitters in the center of the CNS. An excess of Glu leads to neuronal cell death and plays an important role in the pathophysiology of several neuropsychiatric disorders [127]. High levels of Glu were found in fecal samples of autistic children [37]. As it has a role in brain development, the findings support the hypothesis that glutamatergic neurotransmission is involved in ASD [130].

The microbiota might also be involved in the etiology of the disease via interactions with the immune system. Some of the possible mechanisms described above are likely to involve changes in the global balance of the entire microbial community, while others may be exercised by certain bacteria. More studies are needed to clarify whether the gut microbio‐ ta in fact plays a role in ASD. These include prospective studies to address the issue of cause and consequence, and intervention studies aimed at modulating the microbiota with probiotics or dietary interventions. The right profile, the categorization of patients and control groups, with the application of molecular techniques to verify the profile of fecal microbiota and urinary metabolome will corroborate the underlying relationship between gut microbes and the host [34].

Some authors believe that the action of microbiota may have much wider effects on the physiology of the host than originally thought, and emerging evidence shows that it may include modulation of brain activity and behavior. Differences in the composition of the microbiota between individuals with ASD and healthy controls were identified in several studies, both based on bacterial cultures and on molecular methods [18]. However, changes in bacterial diversity that were reported in one study [51] were not confirmed by another [158]. The direct comparison between studies is complicated because different methodologies are employed and study groups may not be directly comparable due to the heterogeneous nature of ASD [89]. However, if the differences in gut microbiota between autistic children and controls are one of the causes of the disorder or the result, could have implications on the diagnosis, treatment and prevention [37].

Nevertheless, despite the substantial amount of data, it is not possible to clarify whether the results represent the presence of a causative agent, or reflect a consequence of treatment, or whether they are nothing more than confounders. This has to be considered and caution is needed before recommending "miracle diets" to the child and family that may only increase the anxiety in respect to symptom improvement.
