**Acknowledgements**

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

Epigenetics, a gene expression regulatory process that involves heritable and reversible biochemical modifications of DNA or histones, independent of the DNA sequence, acts at the interface between genes and the environment. These processes include DNA methylation, histone methylation and acetylation events, and post-transcriptional regulation by non-coding RNAs, which are known to be involved in brain development [138]. Environmental factors can modulate genetics through epigenetic mechanisms and xenobiotics implicated ASD are known to alter epigenetic patterns. For instance, valproic acid inhibits histone deacetylases up-regulating the expression of various genes [139]. 5-MethylTHF, a metabolite of folic acid produced by MTHFR enzymatic activity, is a donor of the carbon group used to methylate DNA [140]. Consequently, *MTHFR* gene polymorphisms that result in a diminished activity of the enzyme (i.e. *MTHFR* 677C > T polymorphism) might affect methyl donation and lead to impaired epigenetic regulation [141]. Epigenetic effects of air pollutants [142], BPA [143] and PCBs [144] have

Neuropathological mechanisms that putatively lead to ASD, such as oxidative stress, neuro-inflammation, hypoxic damage, abnormal signaling pathways and endocrine disruption, can be induced by exposure to xenobiotics. Reduced brain levels of glutathione, the major endogenous cellular antioxidant responsible for the detoxification of xenobiotics, and other oxidative stress biomarkers have been observed in ASD subjects [145]. Evidence for increased levels of neuroinflammation biomarkers in ASD, including brain levels of pro-inflammatory cytokines and microglia activation, which may be stimulated by allergens such as pesticides, has been reported [146]. Proxies for fetal and newborn hypoxia, indicating a deprivation of oxygen supply, have been reported in neonates that later develop ASD [26] and may be elicited by early-life events. Xenobiotics also interact directly with intracellular neurotransmitter pathways [108] leading to signaling impairments. For example, acetylcholinesterase, the enzyme that catalyzes the acetylcholine neurotransmitter breakdown, is the primary target of inhibition by organophosphate pesticides [147] Most of the identified xenobiotics are endocrine disruptors and a role for hormonal imbalances in the disorder is plausible, particularly given the male skewness in ASD diagnoses. Atypical steroidogenic activity, namely increased androgen [148] and estrogen [149] levels in the amniotic fluid, has been reported in affected males. Gender-specific effects of environmental toxins [110] and consequent hormonal imbalances may also be implicated in the female protective effect, a hypothesis proposed to explain the ASD male bias. A novel area of interest in ASD is the role of gut-brain axis, which refers to biochemical signaling connections between the gastrointestinal tract and the central nervous system. Dysbiosis of the gut microbiome likely accounts for a high comorbidity of gastrointestinal symptoms in ASD patients [150]. While the liver is the predominant site of xenobiotic metabolism, the gastrointestinal tract is the first line of defense against ingested compounds, and is rich in both host and microbial enzymes. As the gut microbiota metabolize hundreds of dietary, pharmaceutical and industrial chemicals, dysbiosis could lead to impairments in the gut-brain axis

This review highlights the accumulating evidence for a role of exposure to xenobiotics in ASD risk, and reinforces the need of developing strategies that consider genetics and the environment as interacting components in ASD etiology. This is

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resulting in neurological insults.

**5. Conclusion**

also been described.

This work was supported by Foundation for Science and Technology (FCT), through funding of the project "Gene-environment interactions in Autism Spectrum Disorder" [Grant PTDC/MED-OUT/28937/2017]. JXS was supported by a BioSys PhD programme fellowship from FCT (Portugal) with reference PD/BD/114386/2016. CR was supported by a grant from FCT (Ref: POCI- 01-0145-FEDER-016428).
