The immature brains of fetuses or toddlers are more susceptible to environmental toxicants because the nerve cells are not fully developed until around the age of two years old. The rest of the developing nervous system is also sensitive to environmental toxicants because of temporal and regional developmental processes, i.e. proliferation, migration, differentiation, synaptogenesis, myelination, and apoptosis, during this period. Toxicants that interfere with one or more of these processes may be inhalation [69]. In the rat cortex, post-synaptic glutamate receptors increase rapidly from PND5 to PND20 and reach half the adulthood’s amount by PND40 followed by a continual increase to a plateau around PND50 [70]. Since glutamate receptors have been suggesting to play a role in the modulation of cell death after perinatal brain injury [71], age-specific vulnerability is somewhat dependent on the developmental expression of these neurotransmitter networks [72]. The exposure period of E0 to PND 42 covers the main neurodevelopmental events (including: the timing of neurogenesis, synaptogenesis, gliogenesis, and oligodendrocyte maturation) that occur in this developmental period.
On this subject, the immature rat’s vulnerability to toxins may be related not only to the neurodevelopmental stages, but also to the failure of other protective barriers, e.g. the placental, and the blood-brain barrier. The placental should be a protective barrier against the passage of the harmful substances like environmental toxins from the mother’s body. Howbeit, the placenta is not an effective protective barrier against environmental toxins during this time of fetal vulnerability [73]. In recent years, studies were conducted showing that only nano-sized particles can pass the placental barrier [74, 75]. Also, a recent study [76] found that black carbon particles were able to translocate from the mother’s lungs to the placenta. However, in this subject, various potential mechanism has been proposed, including both direct particle translocation, and/or through an indirect mechanism such as intrauterine inflammation [77, 78, 79]. An indirect mechanism may also be involved because of the exposure to particulate air pollution, and its constituents e.g., metals, and PAHs, can induce oxidative stress, and inflammation that leads to developmental toxicity [80, 81] by negatively affecting placental transportation.
This study design used ambient air pollution without modification to mimic real-world exposure scenarios. Since prior studies typically used high pollutant concentrations, it was not clear that exposure at commonly observed air pollution concentrations can induce ASD or not. Comparing the internal dose of PM2.5 per kg body weight indicates that the rat’s dose in our study were about 2–3 times higher than a human dose. Therefore, these results provide more realistic exposures compared to prior studies.
To the best of our knowledge, this is the first detailed, comprehensive assessment of air pollutant’s effect on the valproic acid-induced model of autism. Also, the hypothesis that air pollutants could play as an environmental trigger was investigated. PM2.5 is an air pollutant that elicits major concerns regarding public health, and contains toxic substances, such as PAHs, metals, and organic matter. In the present study, concentrations of PAHs, and metals in PM2.5, particularly lead, zinc, manganese, benzo (a)anthracene, benzo (k) fluoranthene, benzo (a) pyrene, and pyrene were considered potential neurotoxic risks. Some of these toxicants have been associated with the triggering of inflammation, generation of ROS, and lipid peroxidation [14, 15, 82, 83]. Mostafa, et al. [84] hypothesized that lead as a one of the main neurotoxicants acting as environmental triggers for ASD through neuroinflammation, and autoimmunity. While the PM2.5, and components in it are likely candidates for inducing autism-like phenotype, it is possible that gaseous pollutants mainly nitrogen oxides, also contribute to induced ASD [26, 85].
Since prenatal exposure to VPA has become a reliable tool to model autism, more brain alternations were investigated in rodents exposed to this teratogen [37]. Nonetheless, different doses of this toxin have different functions in causing autism i.e. dose 350 mg/kg. BW can affect the brain by reducing the number of motor neurons from the hypoglossal, and oculomotor nuclei [30]. A dose of 500 mg/kg.BW can altered the distribution of 5-HT neurons in the dorsal raphe nucleus [86]. Therefore, we investigated the effect of air pollutants on rates subjected to these two VPA doses.
Currently, two main clinical features are enumerated to diagnose ASD: impaired social interaction, and communication, and repetitive behaviors. Therefore, since diagnosis of autism are mainly based on clinical symptoms [87], we used clinical type behavioral tests to examine the autism characteristics in these rat groups.
In the open field arena, a hypokinetic condition that is consistent with a reduction in exploratory activity was observed, as has been previously reported by others in VPA-treated rats using the same test. All VPA-injected subjects indicated poorer locomotor activity vs negative controls (CAE). In the sociability phase of the three-chamber social test, all VPA-injected rats showed a sociability defect, a major feature of autism. Our findings in this phase were also in agreement with Dai et al..[88] In the social novelty phase of that test, all VPA-injected rats demonstrated an unwillingness to establish new social relationships. Increased marble burial activity in all VPA-injected rats indicated enhanced repetitive behavior. This finding agress with the results of Schneider et al. [35]. Also when repetitive digging behavior in this test was examined, the PGE- high rats showed a significant increase in digging behavior vs two control group of rats, CAE-low, and CAE-high. Therefore, air pollutants can have an additive effect on repetitive behavior. Also, the reduction of mean spontaneous alternation indicates restricted behavior that is another core of the autism. In this test, PGE-low, PGE-high, and GE-high (that were in contact with gases) vs control group of rat (CAE) showed a significant decrease of mean spontaneous alternation. Therefore, air pollutants can have additive effect on restricted pattern of behavior.
The activity of CAT in the cerebellum, hippocampus, and prefrontal cortex. decreased significantly in all VPA-injected groups of rats compared to the negative controls (CAE), This finding is in agreement with Khongrum, et al.[89]. Moreover, the CAT activity in several groups of animals exposed to both air pollution, and VPA decreased dramatically, compared to control animal groups that were exposed to only low, and high doses of valproic acid. Air pollution might provide toxins that induce oxidative stress.
The GSH levels in cerebellum, hippocampus, and prefrontal cortex decreased significantly in all VPA-injected groups of rats relative to the negative controls (CAE), This finding is in agreement with Simon [90], who reported that valproic acid reduced the intracellular level of glutathione (GSH) in the human body.Our results agrees with Seckin et al. [91]. GSH levels in several groups of animals exposed to both air pollution, and VPA decreased dramatically, compared to control groups exposed only to low or high doses of VPA. Again suggesting that air pollution may trigger oxidative stress.
The level of the OXTR (as a biomarker of ASD) in the cerebellum, hippocampus, and prefrontal cortex decreased significantly in all VPA-injected rats compared to the negative controls (CAE), Our results are in agreement with Bertelsen et al. [92] who reported that VPA-induced– rat models of autism demonstrated a decreased level of OXTR compared to controls. The OXTR levels in several group of animals exposed to both air pollution, and VPA decreased dramatically, compared to control animals exposed only to low or high doses of VPA. This result suggests the possible role of air pollution on valproic acid in inducing ASD by the OXTR mechanism.