The data from our experiment showed that METH exposure during pregnancy increased the level of apoptosis and autophagy in the PFC and AMY area of both male and female offspring’s brain. Also we found an elevation in the GSH content of the all three mentioned brain areas (ACC, PFC, and AMY) and catalase activity of PFC in the offspring's brain.
METH-induced neuropathological changes have been observed in various brain areas of users including the prefrontal cortex (PFC) and hippocampus (Proebstl et al., 2018). In addition, investigations revealed alteration in the structure and function of different brain regions of rats (Thanos et al., 2016). Administration of METH is associated with ROS production, and consequently apoptosis, autophagy, and DNA damage (Li and Trush, 1993, Nopparat et al., 2010). Our results revealed that METH exposure during pregnancy leads to substantial alterations in the different brain regions of offspring, and the mentioned alterations were detectable in adulthood. Protein assessment showed an increased level of Bax / Bcl2 and caspase-3 in the prefrontal cortex and AMY of offspring that were prenatally exposed to METH. This elevation was same as molecular changes reported in the hippocampal neurons of maternaly exposed animals to METH during pregnancy and lactation period (Bagheri et al., 2017).
An elevation in Bax / Bcl-2 ratio in the prefrontal cortex and AMY areas indicated the onset of apoptosis in these areas, and with an increase in the cleaved caspase-3 level as an important executive agent in apoptosis pathway, cell death is observed in offspring that exposed with METH during gestation. It is important to note that brain AMY and PFC are involved in cognitive processes such as anxiety, stress learning and memory. Consistent with our findings, some studies have shown that cognitive processes are disrupted following prenatal METH exposure (Acuff-Smith et al., 1996, Bubenikova-Valesova et al., 2009, Dong et al., 2018). Also, a brain imaging study in children with prenatal METH exposure indicated less connectivity in frontal and limbic hubs over time compared to healthy control children (Roos et al., 2020).
Furthermore, there is a complex interaction between autophagy and apoptosis molecular pathways. Autophagy acts as a double-edge sword and it has been shown to protect against apoptosis, as an anti-apoptotic pathway to reduce cell death, or, pro-apoptosis, as a combined or dependent mechanism for modulation of cell death. However, the precise tendency to death or controlling the fate of neurons in response to METH exposure is not well understood. Xu et al., Showed that METH abuse, at least in part, caused premature autophagy before apoptosis in a time-dependent manner, dominated the pathophysiological process earlier, and then gradually progressed to apoptosis. As we found that prenatal METH exposure influenced Beclin-1 level, Xu and colleagues observed an expression-regulated expression of Beclin-1 in METH used animals, indicating a representative of active autophagy (Xu et al., 2018). Subsequently, our result showed the accumulation of autophagosomes (LC3-I to LC3-II), which may cause cytotoxicity (Button et al., 2017) and induce apoptotic signals by increasing cleaved caspase expression. Beclin-1 as an essential mediator of autophagy interacts with Bcl-2 and can influence apoptosis level by activating proapoptotic proteins of the Bcl-2 family and increase the permeabilizing of the mitochondrial membrane. On the other hand, liberating Beclin-1 from its inhibition by Bcl-2 at the level of the endoplasmic reticulum modulates via these Bcl-2 family members and activate autophagy. Also, some studies have shown that binding of Beclin-1 to Bcl-2 leads to release of cytochrome c into the cytosol and activation of caspases-3/-9 by cleavage of them (Huang et al., 2014). Although the process of autophagy may initially activated to prevent apoptosis, it could not prevent cell death due to apoptosis, and finally the process of apoptosis recruit autophagy to proceed neuronal cell death (Xu et al., 2018). So, METH abuse not only can cause neuronal apoptosis and autophagy in several areas of the brain, including the striatum, cortex, hippocampus, and olfactory bulb (Deng et al., 2002, Deng et al., 2007, Krasnova and Cadet, 2009, Subu et al., 2020), but also could influence neuronal cell death in the brain PFC and AMY of next generation if used in pregnancy period.
One of the important endogenous defense mechanisms against oxidative stress that can defeat neuronal cell death is antioxidant system. Oxidative stress leads to direct or indirect damage by ROS to nucleic acids, proteins, and lipids (Ray et al., 2012). It has been shown that elevated oxidative damage by METH in embryonic and fetal brain causes long-term postnatal neurodevelopmental deficits. This impairments have no relation to dopaminergic neurotoxicity (Jeng et al., 2005). In addition, ROS activation by METH self-administration has been shown (Jang et al., 2017) The enzymatic–nonenzymatic antioxidant cellular defense system including catalase and GSH plays a key role in protecting cells from oxidative stress by regulating the production of free radicals and their metabolites (Patlevič et al., 2016). Our results indicated an increased activity of catalase in the PFC, as well as the amount of GSH in all three studied areas of offspring's brain. This compensatory effect may only eliminate oxidative stress in ACC area of brain after prenatal METH exposure. Because cell death eventually occurs in the other two areas of brain. No alteration in apoptosis and autophagy level in ACC may be consistent with Sowell et al., investigation that they found volume reductions in the striatum, thalamus, parietooccipital and anterior prefrontal cortices, and volume increases in the anterior and posterior cingulate, ventral and medial temporal, and perisylvian cortices in the children with prenatally METH exposure (Sowell et al., 2010).
After data analysis we found that METH exposure during pregnancy did not affect male and female offspring differently. Westbrook et al., showed that there was no significant difference between the sexes in the expression of D1 and NMDA receptors due to METH exposure (Westbrook et al., 2020). Perhaps the reason that we did not observe a significant difference between both sexes of offspring, was long-term changes that caused by prenatal METH exposure in the epigenome of the offspring (Itzhak et al., 2015). Moreover, our investigation revealed no difference between two doses of METH, and the dose of 2 mg/kg had a detrimental effect on different areas of the brain in male and female offspring. Smith et al., also observed that different doses of prenatal METH exposure could exert same effect on behavioral and eye development in rats (Acuff-Smith et al., 1996). So, METH abuse during pregnancy has destructive effects on the brain development of offspring even in lower doses.