Transgenerational Impacts of Oxidative Damage Induced by Prenatal Ethanol Exposure on Spatial Learning/Memory and BDNF in the Hippocampus of Male Rats

doi:10.21203/rs.3.rs-3633347/v1

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Transgenerational Impacts of Oxidative Damage Induced by Prenatal Ethanol Exposure on Spatial Learning/Memory and BDNF in the Hippocampus of Male Rats

https://doi.org/10.21203/rs.3.rs-3633347/v1

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Background and Aim:

Alcohol use during pregnancy causes damage to normal fetal development and induces oxidative stress (OS), and intervenes in the expression of neurotrophic factors (NTFs).

Materials and Methods

A rat model of fetal alcohol spectrum disorder (FASD) was initially generated to reflect on the deficits in the first generation, and then those transmitted via the male germline to the unexposed male ones. The pregnant rats were thus divided into four groups, namely, the control group (CTRL) receiving only distilled water, and three groups being exposed to ethanol (20%, 4.5 g/kg) by oral gavage, during the first 10-days gestation (FG), the second 10-days gestation (SG), and the entire gestation (EG) periods. Half of the male offspring were then subjected to the Morris water maze (MWM) test on the 30 postnatal days. The other half was further recruited to evaluate the activity of the antioxidant enzymes, including glutathione peroxidase (GPx), superoxide dismutase (SOD), and malondialdehyde (MDA), in the hippocampus. Ultimately, the qRT-PCR was practiced to measure the BDNF gene expression.

Results

Prenatal ethanol exposure caused spatial learning/memory deficits during the SG and EG in both generations. The elevated MDA levels, along with the decreased GPx and SOD activity and BDNF gene expression occurred all through the SG and EG of the first filial generation (F1), and the reduction in the GPx and SOD activity together with the BDNF gene expression was only observed in the EG of the second filial generation (F2) of the male rats.

Conclusion

OS could play a leading role in the diminished BDNF gene expression as well as the spatial learning/memory decline induced by prenatal ethanol exposure.

Ethanol

Oxidative Stress

Brain-Derived Growth Factor

Spatial Learning/Memory

Transgenerational Impact

Rat

Following the increased use of alcohol in various societies, its harmful effects are gradually being revealed. Extensive research is thus being conducted on the negative impacts of this drink. To date, fetal alcohol spectrum disorder (FASD) has been detected in children with prenatal ethanol exposure, presenting with the primary symptoms of delayed growth, facial birth deformities, and mental retardation (Abel and Hannigan 1995). Strong experimental evidence further suggests that even low amounts of ethanol may have negative effects on the brain during normal fetal development (Everett et al. 2012). In this respect, the hippocampus is a critical target for the toxic effects of ethanol in the course of brain development. Furthermore, spatial learning and memory impairments are prevalent symptoms observed in animals exposed to ethanol during fetal development, as substantiated by both experimental and clinical research (Streissguth et al. 1994; Sulik et al. 1981). The effects of alcohol on the hippocampus vary depending on the developmental stages, potentially inducing numerous changes during gestation, adolescence, and adulthood (Alfonso-Loeches and Guerri 2011). Abundant studies have accordingly revealed the time-dependent impacts of ethanol exposure (Gianoulakis 1990; Hamilton et al. 2010; Patten et al. 2014) whose intensity might show a discrepancy at different stages of brain development. In spite of this, some investigations on various ethanol exposure times have established no safe time to drink while being pregnant (May et al. 2013). Many mechanisms have been additionally proposed so far to explain the neurotoxicity caused by ethanol consumption. For instance, Alcohol metabolism can either induce OS inside cells by increasing the amount of reactive oxygen species (ROS) (Guerri et al. 1994) and also has the capacity to modify the activity of endogenous antioxidant enzymes through oxidation (Brocardo et al. 2011). Once the levels of ROS within cells reach an excessively high threshold, the endogenous protective mechanisms are unable to effectively mitigate oxidative damage over an extended duration (Tran et al. 2005). Free radicals exert detrimental effects on mitochondria and other subcellular organelles by facilitating the oxidation of nucleic acids, proteins, inducing DNA fragmentation, and promoting lipid peroxidation (Cabral-Costa and Kowaltowski 2020; Singh et al. 2022). Oxidative stress causes apoptosis via the mitochondria-mediated endogenous pathway, which the proapoptotic protein, Bax, causes breakdown of mitochondrial membranes and cytochrome c release (Mo et al. 2017). Cytochrome c could interact with pro-caspase 9 to separate and stimulate caspase-3 in the brain (Young et al. 2003) (Fig. 8). These processes expedite the apoptosis of neurons, ultimately culminating in neuronal death, particularly in the more vulnerable regions of the brain like hippocampus, cerebellum and prefrontal cortex. Indeed, the neuronal defects and cognitive impairments arising out of various ethanol doses can be attributed to the disruption of the neurotrophic factor (NTF) support, which results in some changes in neuronal survival and their protective mechanisms. According to in vivo studies, alcohol exposure during brain development decreases the BDNF gene expression, and then makes changes in its receptor, tropomyosin receptor kinase B (TrkB) (Climent et al. 2002). Some investigations have correspondingly found a link between OS and the BDNF gene expression as well as their involvement in multiple spatial learning/memory deficits (Beheshti et al. 2019; Molteni et al. 2004). In a study on ethanol exposure during the gestational days 5–20, the reduced BDNF gene expression and mRNA levels had been thus detected in the cortex and hippocampus of rat models (Feng et al. 2005), while other investigations had reported an upward trend or no changes in the BDNF mRNA expression in this respect (Caldwell et al. 2008). Accordingly, the effects of ethanol exposure on the hippocampal BDNF gene expression seem to be unpredictable. Some inconsistencies in the study results can be further related to the dose, administration time, exposure time, as well as method of ethanol administration, such as inhalation, intraperitoneal (IP) injection, or gavage. Remarkably, the alterations induced by ethanol exposure can exhibit heritable traits, being transmitted across generations and affecting gene expression in individuals who have never been directly exposed to ethanol (Govorko et al. 2012). However, little research has looked into the way prenatal alcohol exposure affects future generations. Data from multiple sources have additionally identified an augmented susceptibility to ethanol and heightened alcohol consumption in subsequent generations (Mead and Sarkar 2014; Nizhnikov et al. 2016).

In line with this perspective, Koabel et al. (2021) reported a higher probability of anxious behavior and increased alcohol consumption in the second generation, whose grandparents had been previously exposed to ethanol (Koabel et al. 2021). In the present study, the main objective was to evaluate the transgenerational effects of different prenatal ethanol exposure times (namely, the first and second 10-day as well as entire pregnancy periods) in male rats through the male germline.

Adult male and female Wistar rats, weighing 200 − 180 g, were purchased from the Razi Vaccine and Serum Research Institute, Karaj, Iran. Then, they were transferred to the animal house, kept in a cage with 12 hours of light and 12 hours of darkness, with a controlled environmental temperature (20–22 ^oC) and free access to food and water. All the experiments were carried out and authorized by the Research Ethics Committee affiliated to Islamic Azad University (IAU), Damghan Branch, Damghan, Iran, in accordance with the international laws of ethics and protection of laboratory animals, with the code no. IR.IAU.DAMGHAN.REC.1401.016.

The pregnant rats (F0) were accordingly divided into four groups. the control group (CTRL) receiving only distilled water (DW) and three other groups being exposed to ethanol (20% v/v, 4.5 g/kg) by oral gavage, during the first 10-day gestation (FG), the second 10-day gestation (SG), and the entire gestation (EG) periods. Upon exposing one generation to ethanol, further generations were arisen through the male germline. To create the second filial generation (F2) subset, the first filial generation (F1) males were mated with the females in the CTRL group. The litter size was also planned to have approximately 12 offspring (namely, 7–8 males and 4–5 females). On the postnatal day 21, a total of 14 male offspring in the F1 and the F2 were separated. Some offspring were included in the analysis set as 1 per litter for the biochemical tests and 1–2 per litter for the behavioral ones. In total, 5 male rats from each group were anesthetized with ketamine and xylazine, afterward the hippocampus was removed and stored in a -70 ^oC until the biochemical and molecular studies were performed. The other nine male offspring were further subjected to behavioral tests 24 hours later to measure spatial learning/memory, using the Morris water maze (MWM) test (Fig. 1).

2.1. MWM test

The MWM task consist of two parts: four days of training, to evaluate learning ability and probe test, to evaluate spatial memory 24h after training days. It was performed as described before (Toosi et al. 2019), 24 days following birth to investigate spatial learning and memory. The water maze was made up of a black pool containing water. The tank was divided into four quadrants. The detachable platform was located below the water's surface. During the probe test, the rats had to remember the location of the platform in a variety of external cues. Each rat was allowed to swim to discover the platform during trial days. The time it took animals to move up on the hidden platform was recorded as escape latency, and the distance traveled to reach the platform was recorded as distance moved. In order to determine the memory of the animals, probe test was performed. The platform was eliminated 24 hours after the last training sessions and the rat was placed into the water. Time spent in the "target quadrant" (the quadrant that previously contained the platform) was measured, and time to reach in the zone that initially includes the platform was noted as escape location latency. A video camera placed on the ceiling above the core of the pool recorded all procedures, and an automated monitoring device tracked the animal's following behavior (latency, distance, and swim speed) (Ethovision; Noldus IT, the Netherlands).

2.2. Malondialdehyde (MDA) assessment

The amount of MDA produced is dependent on the amount of unsaturated fatty acids broken down and separated. As a consequence, measuring MDA is an appropriate index for lipid peroxidation. In this method, MDA reacts with thiobarbituric acid (TBA) in an acidic pH to produce a pink color. The spectrophotometric was used to measure the absorbance of the reaction mixture at 535 nm (Shafahi et al. 2018).

2.3. Assessment of the anti-oxidative enzyme’s activity

2.3.1. SOD activity measurement

SOD activity was measured based on inhibition of photochemical reduction of nitroblue terazolium chloride (NBT) in the samples. The mixed solution was consisting of the following ingredients: K-phosphate buffer (210 M at pH = 7.2), methionine (2.2 M), Riboflavin (2.2 M) and NBT (82.5 µM). A spectrophotometer was used to measure the absorbance of different groups at 560 nm. One unit of SOD (U) was defined as the amount of enzyme that prevents NBT reduction by 50%. The activity was measured in terms of units per mg of protein (Bagheri et al. 2022).

2.3.2. GPx Activity measurement

GPx activity was measured using tert-butyl hydroperoxide as a substrate and NADPH usage by glutathione reductase for 2 minutes at 37°C and 340 nm.Reduced glutathione (2 mM), glutathione reductase enzyme (0.15 u/ml), sodium azide (0.4 mM), tert-butyl hydroperoxide (0.5 mM), NADPH (1 mM), and K-phosphate buffer (10 mM at pH = 7.2) were all prepared for the reaction (Bagheri et al. 2015).

2.4. Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

Extracted RNA was prepared from hippocampus using TriZol reagent. Using spectrophotometer, the optical density at A260/280 ratio (1.8-2.0 was considered pure) was measured and gel electrophoresis were used to determine RNA quality. cDNA synthesis was performed in compliance with the manufacturer's instructions for the PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara, Japan). A thermalcycler was applied to perform PCR (Eppendorf, Germany). PCR products were electrophoresed using agarose gel. The BDNF gene was relative quantified using image J software, and the PCR results were normalized with the GAPDH mRNA expression. Table 1 provides the forward and reverse primer sequences used in the study. The RTq-PCR products were 405 bp for BDNF, and 203 bp for GAPDH.

Primer	Sequence	Primer size (bp)	Product size (bp)
GAPDH-F	5'-TGACATCAAGAAGGTGGTGAA-3'	21	203
GAPDH-R	5'-CCCTGTTGCTGTAGGCGTATT-3'	21	203
BDNF-F (Soltanian et al. 2013)	5'-GCCCAACGAAGAAAACCATA-3'	20	405
BDNF-R	5'-GATTGGGTAGTTCGGCATTG-3'	20	405

Graph Pad prism 9 software was used to analyze the data, which included two-way and one-way ANOVA. Tukey's post hoc test was applied to the data in groups. The results are presented as mean standard error of the mean (SEM) and are considered significant at P ≤ 0.05.

Additionally, Shapiro-Wilk test was performed to test normality of data set.

4.1. Impacts of Prenatal Ethanol Exposure on Spatial Learning/Memory in F1 Male Rat Offspring

The MWM test results were assessed to learn about the spatial learning/memory ability of the animals (n = 9 in each group). The repeated measures two-way analysis of variance (ANOVA) results of the distance traveled during this test showed the main effects of days (F_3,24=41.63, P < 0.001), groups (F_3,24=21.09, P < 0.001), and group×day (F_9,27=2.144, P < 0.03). As evidenced by Tukey’s post-hoc test outcomes, the total distance traveled significantly augmented in the SG and EG groups compared with that in the CTRL group on days 2–4 (P < 0.01, P < 0.001, respectively) in the FG period (Fig. 2a). The comparison between the study groups also demonstrated a significant difference between the FG group with two other ones exposed on days 3 and 4 (P < 0.05, P < 0.01, respectively).

The data analysis of the escape latency parameter in the F1 male offspring (Fig. 2b) further confirmed the significant effect of days (F_3,24=45.56, P < 0.001), groups (F_3,24=10.89, P < 0.001), and no interaction in group×day (F_9,27=0.929, P < 0.505). Moreover, the Tukey’s post-hoc test outcomes confirmed a significant rise in the escape latency in the SG and EG groups on days 3 and 4 (P < 0.05, P < 0.01respectively) than in the CTRL group. The within-group comparison additionally evidenced a significant difference between the FG and EG groups on days 3 (P < 0.05) and 4 (P < 0.01).

The probe test was correspondingly performed to evaluate spatial memory retention, and then record the time spent in the target quadrant (Fig. 2c). The one-way ANOVA results accordingly demonstrated a significant difference between the study groups (F_3,32=10.49, P < 0.001). In this line, the SG (P < 0.05) and EG (P < 0.001) groups spent less time in the target zone compared with the CTRL group, and a significant decrease was detected in the EG group as compared with the FG group (P < 0.01).

4.2. Impacts of Prenatal Ethanol Exposure on OS Markers in F1 Male Rat Offspring

As per the one-way ANOVA outputs, a significant difference was observed between the study groups in terms of the SOD (F_3,16=32.33, P < 0.001) and GPx (F_3,16=13.86, P < 0.001) levels (n = 5 male rats in each group). The SOD and GPx activity in the SG and EG groups were notably lower than those in the CTRL group (P < 0.001), but the SOD activity showed a significant decrease in the FG group compared with the CTRL group (Fig. 3a, b). Comparing the study groups exposed to ethanol also established a significant difference in the SOD activity between the FG and the SG and EG groups (P < 0.05, P < 0.001, respectively). Furthermore, the GPx activity significantly reduced in the EG compared with the FG group (P < 0.01). Moreover, there was a significant change in the MDA level (F_3,16=17.84, P < 0.001). The post-hoc comparison additionally showed that the hippocampal MDA level (Fig. 3c) significantly elevated in the SG and EG groups than in the CTRL one (P < 0.01, P < 0.001, respectively). Of note, the MDA level was significantly different in the FG and EG groups (P < 0.001).

4.3. Impacts of Prenatal Ethanol Exposure on BDNF mRNA Expression in F1 Male Rat Offspring

The one-way ANOVA outcomes of the BDNF mRNA expression showed a significant difference between all study groups (F_3,16=32.9, P < 0.001). Compared with the CTRL group, the gene expression in the SG and EG animals significantly downregulated (P < 0.01, P < 0.001, respectively), and the reduction of the BDNF gene expression was significant in both SG (P < 0.05) and EG (P < 0.001) groups compared with the FG group. On the other hand, the BDNF mRNA expression significantly declined in the EG group than in the SG group (P < 0.01) (Fig. 4).

4.4. Impacts of Prenatal Ethanol Exposure on Spatial Learning/Memory in F2 Male Rat Offspring

According to the repeated measures two-way ANOVA results of the distance traveled, the main effects of days (F_3,21=129.4, P < 0.001), groups (F_3,21=20.4, P < 0.001), and group×day (F_9,63=3.18, P < 0.003) were established (n = 8). On day 2, the total distance traveled significantly increased in the FG (P < 0.05), SG, and EG (P < 0.001) groups compared with that in the CTRL group. As well, a significant upward trend was observed in the SG (P < 0.001, P < 0.01, respectively) and EG (P < 0.001) groups during days 3 and 4 than that in the CTRL group (Fig. 5a). The comparison between the groups exposed to ethanol also established a significant change on days 3 and 4 between the FG and SG (P < 0.01, P < 0.05, respectively) and FG and EG (P < 0.01, P < 0.001, respectively) groups.

Moreover, the escape latency parameter in the F2 male offspring disclosed significant effects in days (F_3,21=90.65, P < 0.001), groups (F_3,21=20.49, P < 0.001), and no interaction in group×day (F_9,63=1.33, P < 0.240). In the SG (P < 0.05, P < 0.01, respectively) and EG (P < 0.01, P < 0.001, respectively) groups, there was also a significant rise in the escape latency parameter on days 2 and 3 as compared with that in the CTRL group. On day 4, only the EG group showed a significant increase in comparison with the CTRL group (P < 0.01) (Fig. 5b). Moreover, the escape latency indicated a significant difference between the FG and EG groups on days 3 and 4 (P < 0.05, P < 0.01, respectively).

According to the probe test, the time spent in the target quadrant was recorded. The one-way ANOVA outcomes accordingly proved a significant difference between the study groups (F_3,32=16.11, P < 0.001). The EG group also spent less time in the target quadrant compared with the CTRL group (P < 0.001) (Fig. 5c). Likewise, the within-group comparison showed that the EG group spent notably less time than the FG (P < 0.001) and SG (P < 0.01) groups.

4.5. Impacts of Prenatal Ethanol Exposure on OS Markers in F2 Male Rat Offspring

The one-way ANOVA outputs revealed a significant change in the SOD (F_3,16=6.049, P < 0.006) and GPx (F_3,16=3.70, P < 0.03) activity between the study groups (n = 5 in each group). As established by Tukey’s post-hoc test results, the SOD activity significantly decreased in the EG (P < 0.01) compared with that in the CTRL group (Fig. 6a). Moreover, the GPx activity remarkably declined in comparison with that in the CTRL group (P < 0.05) (Fig. 6b). In general, no significant change was found between the study groups in terms of the MDA level of the F2 generation (F_3,16=2.38, P = 0.107) (Fig. 6c).

4.6. Impacts of Prenatal Ethanol Exposure on BDNF mRNA Expression in F2 Male Rat Offspring

A significant difference was observed between the study groups (F_3,16=14.7, P < 0.001) according to the one-way ANOVA results of the BDNF mRNA expression. The Tukey’s post-hoc test outcomes further indicated the gene expression only decreased significantly in the EG (P < 0.001) than in the CTRL group (Fig. 7). Furthermore, the BDNF mRNA expression suggested a significant reduction in the EG compared with the FG (P < 0.001) and SG (P < 0.05) groups in the F2.

The study findings revealed that ethanol exposure and toxicity at various stages of pregnancy could induce OS in the brain, resulting in spatial learning/memory deficits in the later life of the F1. The severity of the impairments might thus differ according to the time and duration of the exposure. However, limited knowledge has been acquired thus far regarding the implications experienced by subsequent generations that were not exposed to adverse intrauterine conditions. Therefore, the current study aimed to explore the effects of prenatal ethanol exposure on the F2 male rat offspring, specifically focusing on spatial learning and memory deficits, as well as potential associations with oxidative damage in the hippocampus.

To unravel the significant role of OS as a mediator in the cognitive abnormalities associated with ethanol exposure, the OS markers were thus measured in the hippocampal tissues of offspring. Furthermore, the MWM test along with the BDNF gene expression was assessed as the crucial components in the hippocampal development and neuronal survival, as well as the basis of spatial learning/memory.

Based on previous research, ethanol exposure during normal fetal development could raise OS markers in the brains of the newborn or adult rats (Boschen and Klintsova 2017; Virgolini et al. 2019). The oxidative damage observed in different animal models of FASD accordingly depended on the brain region, the time and pattern of ethanol consumption, alcohol dosage, the blood alcohol concentration, and the age at the time of the analysis (Bagheri et al. 2015; Brocardo et al. 2011; Mahdinia et al. 2021). Even so, there is no strong evidence that the effects of ethanol exposure during the trimesters might be different. The pattern of drinking and the developmental period of ethanol intake can thus result in a wide variety of effects on offspring, ranging from moderate to severe cognitive and behavioral disorders (Cuzon Carlson et al. 2020; Guerri et al. 2009; Maier and West 2001). In this study, the induction of oxidative damage was confirmed by the evaluation of the OS markers in the F1 all through three time periods of gestation. The results correspondingly indicated that the MDA levels significantly elevated in the rats in the SG and EG groups exposed to ethanol. A significant decrease was further observed in the GPx and SOD activity in these groups as well. In this regard, many studies focusing on the role of oxidative stress in neurodegenerative disorders such as depression, Alzheimer’s disease, Parkinson disease and ischemic stroke (Bhatt et al. 2020; Huang et al. 2016; Orellana-Urzúa et al. 2020). As the central nervous system (CNS) also contained far fewer antioxidants than other tissues, the elevated levels of unsaturated fatty acids within the CNS render it more susceptible to the detrimental effects of free radicals. OS plays a detrimental role in neurodegeneration by causing damage to neural cells through free radical attacks. The toxic effects of ROS contribute to various harmful processes, including protein misfolding, activation of glial cells, disruption of mitochondrial function, and ultimately, cellular apoptosis (Fulda et al. 2010). The amount of LP could accordingly increase in the SG and EG groups, implying that the hippocampus might face greater damage during the SG than the FG. Additionally, augmenting the prenatal ethanol exposure length (namely, the total gestational days) could be efficient in enhancing toxicity induced by ethanol metabolism.

To evaluate the association between OS in the hippocampus and memory impairment, the qRT-PCR technique was exploited to examine the BDNF gene expression in the hippocampal tissue of male offspring over two generations. Of note, it is well known that the expression of neurotrophins and their receptors is typically regulated during the CNS development, and the accurate regulation seems critical for this purpose. Furthermore, neurotrophins and their receptors are mostly expressed in the hippocampus and cortex, which are the vital areas of neuronal plasticity related to spatial learning/memory (Chao 2003). The interaction between BDNF and the TrkB receptor triggers a series of intracellular signaling pathways that lead to the phosphorylation and activation of the transcription factor known as cAMP response element binding protein (CREB). Once phosphorylated, CREB is transported into the nucleus, where it facilitates the transcription of BDNF by binding to specific regions called BDNF promoters. The binding of CREB to BDNF promoters promotes the expression of BDNF, which in turn regulates critical processes such as neuronal survival, differentiation, and synaptic plasticity (Pradhan et al. 2019; Song et al. 2013) (Fig. 8). Indeed, the BDNF is a potent synaptic facilitator, whose deficiency leads to long-term potentiation of abnormalities as well as spatial learning/memory deficits, which can be then alleviated by administering exogenous BDNF (Boschen and Klintsova 2017). In the present study, the BDNF mRNA expression significantly declined in the SG and EG groups of the F1. In this context, Kown (2013) had indicated that repeated treadmill exercise could improve cognitive impairments caused by restraint stress-induced OS and reduced BDNF levels (Kwon et al. 2013). According to previous research, this descending trend might play a key role in spatial learning/memory deficits in offspring (Alzoubi et al. 2013; Wang et al. 2020). The evaluations of the spatial learning/memory have further shown that alcohol intake in the SG as well EG might cause learning difficulties and poor spatial and cognitive memory in rat offspring. When comparing the CTRL group to the SG and EG groups, there was a noticeable increase in the time it took to find the platform and the distance traveled. Additionally, the time spent in the target quadrant significantly decreased in the SG and EG groups, as opposed to the CTRL group. These findings strongly suggest that the SG and EG groups exhibited deficits in spatial learning and memory. In agreement with the present report, many research has discovered that prenatal ethanol exposure can impair cognition and lead to lasting neurobehavioral impacts (Kodituwakku and Kodituwakku 2014; Mahdinia et al. 2021; Marquardt and Brigman 2016).

Most studies in the field of prenatal ethanol exposure have primarily focused on the negative effects on the F1 generation. However, research on alcohol exposure has also revealed the potential for multigenerational ethanol impacts that might be mediated by epigenetic changes found in the male germline of rats (Govorko et al. 2012). In this study, alcohol was given to pregnant rats to produce the F1, and then male F1 rats were mated with the CTRL females to produce the F2. All F1 male offspring studies were also conducted on the F2 ones. Based on the most recent investigations, maternal ethanol exposure might promote tissue-specific epigenetic changes that are transferred to the subsequent generations, thereby resulting in malfunction in non-treated offspring (Chen et al. 2013; Gangisetty et al. 2020). In the second part of the study, there were no significant modifications of the OS markers in any group except the EG group, and there were no significant differences in LP across all groups. In this context, the qRT-PCR results revealed a significant reduction in BDNF specifically in the EG group, which coincided with the spatial learning/memory deficits observed in this group. However, the present study was not specifically designed to evaluate the factors related to the epigenetic changes. A possible explanation for the outcomes might be that prenatal ethanol exposure could cause epigenetic changes in the regulatory genes of brain development, such as the DNA methylation, histone acetylation, etc., which play an important role in the abnormal neural development of FASD. These epigenetic changes can then be transmitted to the F2 via the germline, altering the gene expression of their cells to exhibit the same phenotype in the absence of external impacts. Since the epigenetic changes generated by ethanol depend on the dose and duration of exposure, it was realized that only exposure during the EG caused a decrease in the BDNF gene expression in the offspring. In this vein, Govorko (2012) had found that some epigenetic changes had happened in the pituitary proopiomelanocortin (POMC) regulatory gene region during in utero ethanol exposure, including the DNA methylation and histone marks. As a consequence, at least three generations of the POMC neurons could be affected (Govorko et al. 2012).

Another study had similarly established the poor performance of spatial working memory in the F2 gestational diabetes female offspring, probably related to the changes in the OS markers (Huerta-Cervantes et al. 2021). An issue not addressed in this study was whether prenatal ethanol exposure could lead to epigenetic changes in the genes in the germline as well as its relationship with alcohol-induced cognitive impairments in the subsequent generations. Therefore, further experimental investigation is needed to address this possibility.

The study findings suggested the involvement of OS in producing developmental and behavioral abnormalities in a rat model of FASD. In addition, the F2 studies indicated that the problems associated with prenatal ethanol exposure could be inherited in offspring. However, further investigation is still required to demonstrate the epigenetic changes in the genes involved in the hippocampal development and memory formation to evaluate this hypothesis.

Ethical approval

All applicable international and national guidelines for the care and use of animals were followed.

Conflict of interests

The authors declare no competing interests.

Author contributions M.Sh performed all experiments and wrote the manuscript. V.H designed the experiment and review the manuscript. All authors verified the final manuscript.

Funding

This study was supported by Islamic azad university of Damghan branch.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Acknowledgment

The authors would like to thank Islamic Azad University, Damghan, Iran

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Transgenerational Impacts of Oxidative Damage Induced by Prenatal Ethanol Exposure on Spatial Learning/Memory and BDNF in the Hippocampus of Male Rats

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Abstract

Background and Aim:

Materials and Methods

Results

Conclusion

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1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. MWM test

2.2. Malondialdehyde (MDA) assessment

2.3. Assessment of the anti-oxidative enzyme’s activity

2.3.1. SOD activity measurement

2.3.2. GPx Activity measurement

2.4. Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

3. Statistical analysis

4. RESULTS

4.1. Impacts of Prenatal Ethanol Exposure on Spatial Learning/Memory in F1 Male Rat Offspring

4.2. Impacts of Prenatal Ethanol Exposure on OS Markers in F1 Male Rat Offspring

4.3. Impacts of Prenatal Ethanol Exposure on BDNF mRNA Expression in F1 Male Rat Offspring

4.4. Impacts of Prenatal Ethanol Exposure on Spatial Learning/Memory in F2 Male Rat Offspring

4.5. Impacts of Prenatal Ethanol Exposure on OS Markers in F2 Male Rat Offspring

4.6. Impacts of Prenatal Ethanol Exposure on BDNF mRNA Expression in F2 Male Rat Offspring

5. DISSCUSION

6. CONCLUSION

Declarations

References

Additional Declarations

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