Substance abuse during pregnancy is a pandemic proportion worldwide. Primarily the effects of prenatal cigarette use on the fetus were first identified in the 60's decade, and then effects of alcohol, opiate, and the effects of several illicit drugs have been revealed. Recently studies have focused on the effects of prenatal exposure to CNS stimulant drugs (14, 17). Although there are several reports about the molecular, cognitive and brain structure of methamphetamine abuse, there is a great lack of information about PMEs. Few animal model reports studied the PME effects on molecular pathways in the brain (18,19,20). In addition, some psychological studies evaluated the cognitive effects of PME in children at the age of six or seven or adolescence (21,22). To our knowledge, the present study is the first gene expression analysis of PME children and also is the first follow-up study of PMEs for a long term of a period and in a cohort big sample size. Especially the long term follow-up may help to separate the acute and temporary alterations of meth toxicity such as hepatotoxicity from chronic and constant changes such as neurodevelopment or immunodeficiency.
Gene ontology assessments
Genome expression profiling on methamphetamine treated animal model studies showed changes in critical genes for brain functions, including neuronal plasticity, mitochondrial energy metabolism and immune response (23). METH treatment induction on mitochondrial energy metabolism and associated glutamate receptor alterations as a neurotoxic response in the brain (including the amygdala, prefrontal cortex, hippocampus and striatum) of rats accompanied with a significant behavioral sensitization had reported (24,25). The present study may suggest that PME may induce chronic bioenergetic deregulations in children that, in turn, could increase the possibility of severe psychiatric disorders caused by mitochondrial dysfunction. Mitochondrial energy metabolism provides energy for brain functions and mitochondrial dysfunction are biomarkers for severe psychiatric disorders such as schizophrenia, bipolar disorder, and major depression disorders (26,27) especially complex I of the respiratory chain that is highly affected by PME are known to be associated with psychosis and number of cognitive dysfunctions in schizophrenia (28). Neuron projection, which is the most affected cellular component during the follow up is any process extending from a neural cell, such as axons or dendrites and is related to addiction tendency and stress problems and changes in brain circuitry (29,30). Synaptic transmission and transmission of nerve impulse are two biological processes that are affected in all samplings during the 36 months in PMEs. Synaptic transmission is essential for normal cognitive functions such as learning and memory (31). Dysfunction in synaptic transmission and transmission of nerve impulses had reported in a number of psychiatric disorders such as schizophrenia and autism(32,33). Findings may explain the learning and cognitive problem (34,35) and even anatomical changes in the brain of PMEs, such as reduced caudate and thalamus volume (36).
Probably the major and sever Primary immunodeficiency is the most worrying effect of PME observed in present study. METH treatment may cause an inflammatory response that plays a potential role in METH-induced neuronal injury and d regulation of cellular immune responses (37,38,39). METH also could increase blood-brain barrier (BBB) permeability that may explain similarities between PMEs and methamphetamine abusers(8), but taking together with primary immunodeficiency could increase the risk of several CNS infections in PMEs. There is comprehensive literature about methamphetamine role in immune deficiency and pathogenesis of Human immunodeficiency virus (HIV) (40,41). Immunodeficiency caused by methamphetamine can get escalated when accompanied by mitochondrial damage and increased levels of oxidative stress (42). By the way, primary immunodeficiency diseases (PIDs) are a group of more than 300 different heterogeneous disorders, and many PIDs patients are diagnosed late. Due to poor prognosis, many cases suffer from complications by the burden of chronic infections, irretrievable end-organ damage, or even death before the definitive diagnosis (43,44). Results revealed that PMEs are born at risk of lethal immunodeficiency disorders. But the bright side is that the targeted prognosis of PMEs may lead to timely diagnosis and appropriate treatment of those PMEs who may affect by PIDs. Recent epidemiological cohort studies showed that methamphetamine abuse increases the risk for developing Parkinson's disease (PD) and there are similar neurodegenerative processes in PDs and Meth abusers that target the nigrostriatal system (45,46.). In addition, Parkinson's disease pathway is mostly involved with dopaminergic and GABAergic pathways(47). Addiction tendency, number of psychiatric disorders including schizophrenia and attention deficit hyperactivity disorder (ADHD) and behavioral abnormalities such as anxiety and low-stress resilience are associated with overexpression of dopamine and GABA receptors, dopamine synthesis genes and downexpression of genes involved in the degradation of dopamine (48). Constant and severe deregulation of Parkinson related genes in PMEs provide evidence that prenatal methamphetamine exposure can cause long-lasting disabilities or degeneration of dopaminergic cell bodies, and it may increase the risk of PD in PMEs as well as Meth abusers. Also, these potential risks for PD can be detected in neonates.
The number of deregulations caused by methamphetamine, including reactive oxygen species activation and mitochondrial dysfunctions may lead to death receptor and ER pathways of apoptosis (49,50). Methamphetamine-induced apoptosis pathways are mostly mitochondria-mediated (51), but also the footprint of the mTOR signaling pathway were detected (52,53). Several genes from both mitochondrial and mTOR were differentially expressed in PMEs. mTOR pathway is also associated with autistic-like behaviors and locomotor activity problems (54).
Nine genes have met the criteria of biomarker selection in all three samplings. These genes were related to dopamine signaling, transcription factors, and immune system regulations. Also, the deregulation of four genes was getting worse during PME life and had met the criteria in twelve months and twenty-four months’ samplings and remained deregulated in thirty-six months’ samplings. These genes were related to synaptogenesis, memory formation, and dopamine degradations.
Dopaminergic pathway dysfunction
Methamphetamine (METH) is an illicit toxic psychostimulant that is widely abused. Its toxic effects depend on the release of excessive levels of dopamine (DA) that activates striatal DA receptors (55). Psychostimulant can produce robust rewarding effects through modulating the dopaminergic synapse, dopamine re-uptake and dopamine- regulated downstream signals. Five genes of the biomarker list are involved in the dopamine pathway, dopamine receptors (D1, D2, D3), brain-derived neurotrophic factor (BDNF) were overexpressed and MAOA down expressed. BDNF is involved in regulating the expression of dopamine receptors, especially in methamphetamine abuse and triggers behavioral sensitization, and MAOA are involved in the degradation of dopamine and its downregulation are related to violent behavior (56,57).
The similarity of expression pattern in PME and methamphetamine abuse in adults may support the evidence that methamphetamine can pass through the blood-brain barrier of infants. The up-regulation of dopamine receptors was reduced after twelve months but still significantly high during the next 24 months. About BDNF up-regulation was reduced after 12 months but chronically increased again. These patterns of dopamine pathway over function are well known in several psychiatric disorders such as schizophrenia and bipolar disorder (58). MAOA down-regulation started from first sampling but met criteria of significancy at the twelve months sampling and remains almost constantly during the next 24 months. Findings may suggest that PME children could be at high risk for psychiatric disorders, also may explain the stress and violence problem of PMEs (4,5,59,60).
Immune system dergulation
Immune system abnormalities in PMEs are the most significant findings of the present study. By the way, the exact genes related to the immune system that meet the criteria of biomarkers are all involved in neurodevelopmental disorders, especially autism spectrum disorder(ASD). (Imbalances in cytokines level such as IL-1Β, IL-6, IL-4, IFN-γ, and TGF-Β had reported in ASD. Cytokines are mediators of immunological activity and an increase in infection and toxic exposures. Cytokines participate in normal neural development and function, and abnormal cytokine activity caused by meth neurotoxic effects may lead to several neurological dysfunctions. It may explain some of the similar symptoms of ASDs and PMEs, such as language impairments and lack of communication (4,5,61).
Another shared biomarkers between PMEs and ASDs were SHANK2 and DLG4 genes that are related to synaptogenesis. Lack of function in SHANK2 which is detected in late of follow up (at 24 and 36 months sampling) may cause ASD-like behaviours such as reduced social interaction and social communication and repetitive behaviors (62,63,64).
Transcription factors alterations
Four transcription factors that are well known in the development of the central nervous system were revealed in the PMEs biomarker list, CREB1, DLG4, MECP2, EGR2, and FOXP2. These genes are essential in memory formation, language function, and cognition. Dysfunction of these genes is involved in several psychiatric disorders such as schizophrenia, bipolar disorder, and depression. Findings may suggest that PMEs are vulnerable to affect by psychiatric disorders as well as syndromic and heterogeneous disabilities in executive functions such as memory abnormalities and language impairments (65,66,67).
CYP2E1 was the only gene that appeared in the list of biomarkers just in the first time of samplings. CYP2E1 is an essential protein for cytochrome P450 mixed-unction oxidase system that is involved in the metabolism and degradation of drugs, toxic environmental chemicals, and carcinogens along with several basic metabolic reactions such as fatty acid oxidations, ethanol, and glucose metabolism(68). It is reported that inflammatory cytokines such as (IL)-1beta inhibit the CYP2E1 expression that may cause metabolic abnormalities (69). It seems that abnormal activity of the immune system in PMEs could cause acute but not constant effects on xenobiotic metabolism and several digestive pathways related to glucose and lipids in PME infants. But it may be reregulated during the years.