1 INDUCED OXIDATIVE STRESS BY DEFEROXAMINE ( DESFERAL ) DURING THE PREGNANCY : PRE AND POST-NATAL DEVELOPMENTAL STUDY

The aim of the study is to evaluate the effect of induced oxidative stress by the deferoxamine (desferal) during the pregnancy on pre and post-natal developmen. One hundred twenty adult female albino rats, 3 months old, with an average weight of 175-200 g, were randomly distributed into two main groups (60 rats/group). The 1st (control group) was injected with 0.3 ml physiological salt solution / animal, and the 2nd group was injected with 100 mg deferral/kg B.W. which was given intraperitoneal once daily for three consecutive weeks (during the pregnancy period). At the end of the treatment, each group in turn was divided into two parts, each section comprising 10 animals, blood samples were collected for biochemical analysis, then uterine and fetal tissues collected to observation the studied parameters. Results showed decrease in the numbers of implantation and blastocysts. The number of alive embryos were significantly reduced with an increasing number of dead and reabsorbed embryos, malformation of fetus increase in uterine of the stressed female group compared with control group. Postnatal observations showed significant variations in postnatal development parameters. Our study suggests that the deferral injection has a pro-oxidant capacity on rats' model at dose of 100 mg/kg, and the oxidative stress plays an important role during pregnancy especially on prenatal development as well as persist up to postnatal life.


INTRODUCTION
Oxidative stress occurs naturally in the body because of an imbalance between oxidants and antioxidants, and it has a vital and effective role in many metabolic processes necessary for the growth and development of various body systems as well as its physiological role in many body functions (Agarwal et al., 2005). Although reactive oxygen species (ROS) are necessary to maintain fetal development, overproduction of the ROS during embryonic life has a detrimental effect due to their interaction with different important cellular processes (Amin et al., 2014). The ROS attack large cellular molecules, and cell structures such as the mitochondria membrane and the endoplasmic reticulum causing oxidative damage leading to changes in the cellular activity through their effect on gene regulatory action on many cellular proteins (He et al., 2017).Most of the biological processes in the uterine The ROS regulate many of the transcription pathway by direct interaction with proteins tissue and ovarian are naturally involved free radicals that act as secondary messengers to perform their cellular response (Torres-Osorio et al., 2019).

‫الــرافـديــن‬ ‫زراعـــة‬ ‫مـجـلـــة‬
( ‫المجلد‬ 49 ( ‫العدد‬ ) 1 ) 2021 and modification of transcription factors and gene expression (Allen and Tresini, 2000). During pregnancy, an increase of stress severity may lead to unwanted Pregnancy outcome for instance spontaneous abortion, recurrent pregnancy loss, birth defects, pre-eclampsia, intrauterine growth restriction, minor congenital anomalies, and future diseases in adulthood (Pereira and Martel, 2014;Duhig et al., 2016). It was found that there is a strong link between increased ROS and early pregnancy loss (Bertout et al., 2004). In another study conducted on mice, defects in the mitochondrial respiratory chain resulted in impaired fertility and increased miscarriage rate due to increased oxidative stress (Takamasa et al., 2014). Hubel (1999) found that oxidative stress causes impairment the blood vessels in the placenta leading to decreased oxygen supply, causing abnormal differentiation of the Trophoblast. In addition to that, Min et al. (2009) recorded decreased blood flow in the placenta and damage to fetal tissue due to intrauterine stress. The placenta considered as a rich source of oxidants and antioxidants due to an increase in metabolic efficiency in the placental mitochondria (Agarwal et al., 2012). The formation of an abnormal placenta causes damage to the syncytiotrophoblast that is sensitive to stress during the first trimester of pregnancy, this contributes to recurrent pregnancy loss (Agarwal et al., 2012).
Desferal (deferoxamine mesylate) is an iron-chelating agent used to treat iron overload caused by blood transfusions in adults and children at least 3 years old (Brittenham, 2011). Desferal can lead to increase oxidative stress via activation of free radical production signaling pathway (Francisco et al., 2010). In this research work, our aims are to prove that desferal has pro-oxidant activity during pregnancy and to figure out its effect on pre and postnatal life in rats' model.

MATERIALS AND METHODS Animals
The study was conducted in the Laboratory of the Physiology, Biochemistry and Pharmacology department / Faculty of Veterinary Medicine, University of Mosul. One hundred twenty adult females albino rats at 3 months of age were used, with an average weight of 175-200 g. They were distributed randomly into two main groups, which are control and treatment by 60 rats / group according to the design of the experiment. The laboratory animals were placed in plastic cages for breeding for a period of two weeks, and for environment acclimatization and observing the health status of the rats. The diseased animals were excluded and replaced with healthy rats. The laboratory animals were placed under appropriate environmental conditions in terms of temperature (24 ± 1°C), humidity (60 ± 10%), ventilation and adequate lighting, and water and feed were provided to them freely throughout the experiment period (3 weeks).

Experiment of induced oxidative stress
For inducing oxidative stress, 10 adult females were used in this experiment, were injected with desferal a dose of 100 mg /ml /kg BW, i.p once daily for three consecutive weeks. Blood samples were collected one day before the injection of desferal, and after the completion of the injection period for measuring the level of each of MDA and glutathione as evidence of induced oxidative stress. The prescribed dose of desferal was adopted after confirming the occurrence of oxidative stress.

Statistical analysis
All data were expressed as mean ± S.E, independent t-test was employed for determining the significant between pre and post induction of oxidative stress. Whereas, paired t-test was employed for determining the significant between studied groups by using SPSS program (Allen et al., 2014). P-value 0.05 was considered significant.

Experiment of oxidative stress induction. Effect of desferal injection on glutathione level.
The injection of 100 mg desferal/kg body weight led to significant decline in serum glutathion level after 14 and 20 days of injection between pre and post injection at P-value 0.001, 0.002 respectively. Whereas, injection of DFO up to 7 days showed non significant decline in glutting at P-value 0.055. (Figure 1).

Effect of desferal injection on lipid peroxidation marker.
Lipid peroxidation marker (MDA) was significantly higher in post injection duration compare to pre injection period after 14 and 20 days of continuous desferal injection to induce oxidative stress at P-value 0.001, 0.004 respectively. In the time, the first 7 continuous days had no significant elevation in lipid peroxidation level at P-value 0.064. (Figure 2).

Experiment of effect of induced oxidative stress in both pregnant rats and their fetuses.
Effect of induced oxidative stress on glutathione level after 7, 14 and 20 days of pregnancy.
The statistical analysis showed no significant differences for the glutathione level in serum and uterine tissue after injecting female rats with desferal for 7 days at significant level the 0.064 and 0.18 for both serum and uterine tissue, respectively.   The induced oxidative stress showed significant changes represented by a decrease in the glutathione level in serum and embryonic tissues at the P-value was 0.032 and 0.001, respectively. ( Effect of induced oxidative stress on MDA level after 7, 14 and 20 days of pregnancy. The induced oxidative stress by desferal did not showed non-significant elevation in the MDA level of both serum and uterine tissue after ip injection for 7 days at the P-value was 0.076, 0.085 for both serum and uterus respectively. ( Figure  6). The MDA level in the serum of pregnant rats showed a significant increase compared to the control group at the level P-value 0.032, While, the mean value level of MDA of belongs to induced oxidative stress group was not significant compared to the its value of the control group P 0.084. (Figure 7 Induced oxidative stress up to 20 days during pregnancy showed a significant increase in the MDA level in serum and fetal tissues between studied groups at the P-value was 0.006 and 0.001, respectively. (Figure 8).

Experiment of the effect of induced oxidative stress on prenatal development and weight of fetuses at day 20 of pregnancy.
The statistical analysis of the fetal development data of studied females' rats showed a non-significant decrease in the number of implantations (P 0.104) between control and stressed females. Whereas, the oxidative stress led to a significant decrease (P 0.043) in the average number of blastocysts in the uterus of the stressed females compared with the control females. (Figure 9). The induced oxidative stress showed no significant decrease in both the total number of embryos and the number of alive embryos of stressed females compared to control females (P 0.105 and 0.051, respectively). Whereas, the induced oxidative stress resulted in a significant increase in the number of dead embryos and the number of absorbed embryos in the stressed females compared with the control females (P 0.037, 0.001). (Figure 10). The induced oxidative stress in female rats led to a significant decrease in the total number of adult fetuses and the number of alive fetuses / female compared with the control group females (P 0.001). Whereas, the rate of the number of dead fetuses and the rate of the number of fetal anomalies / female showed a non-significant increase of the intrauterine stressed fetuses compared with the non-stressed fetuses (P 0.813, 0.264, respectively). The weight of the fetuses at day 20 of pregnancy did not show difference between the intrauterine stressed fetuses and non-stressed fetuses (P 0.061). (Figure 11 and 12).

Experiment of the effect of induced oxidative stress on certain postnatal development parameters and weaning day.
The postnatal development of the newborns of stress subjected rats during a period of 7 and 14 days of pregnancy showed a difference between the two studied groups of some studied criteria. For instance, there was a significant increase in the number of days required for the appearance of hair and teeth in the newborns from the stresses dams compared with the newborns from the normal dams (P 0.004 and 0.001 respectively). While, the other criteria did not show any significant differences between the two groups. (Figures 13 and 14). While, the induced oxidative stress up to 20 days of pregnancy lead to clear changes in some of the studied growth parameters. For instance, the appearance of hair, teeth and the appearance of the eye opening was delayed in the pups of mothers subjected to intrauterine stress up to 20 days compared with the same criteria for the pups of dams of the control group (P 0.045, 0.006, 0.03, respectively). (Figure 15). Statistical analysis using independent t-test for evaluating the data of the weaning age of the pups from both stressed and non-stressed dams resulted in a significant increase in the number of weaning days required for weaning in the intrauterine stressed pups group compared with the control group (P 0.001) ( Figure  16).

DISCUSSION
Excessive oxidative stress as a result of an increase in the level of free radicals, especially ROS and hydroxyl, and a decrease in the activity and efficiency of antioxidants within the uterine tissue and the placenta. This gives an important explanation for many cases of pregnancy failure and defects in the growth and development of fetuses during and after pregnancy in a certain period (Silveira et al., 2018;Wu et al., 2017(. For the purpose of inducing oxidative stress in rats, Desferal was used in a high dose for two consecutive weeks. Injecting intraperitoneal resulted in oxidative stress induced through apparent changes in the levels of oxidative markers. desferal works to oxidize iron in cytosol and convert it from the Fe +2 to the Fe +3 by reacting with hydrogen peroxide because of the generation of free hydroxyl radicals in the cell. This indicates that Desferal, in addition to its role as an antioxidant, acts as a prooxidant when used in a high dose for a long time. The stimulation of the lipid peroxidation process resulted from Desferal may be with the presence or absence of iron through the production of highly reactive hydroxyl radicals that are toxic to cells, and this opinion is identical to what was reported by Mukosera et al. (2020); Tada et al. (2015 ); Kim et al. (2007); Poot et al. (1989); Morehouse et al. (1988) ; Morehouse et al. (1987).
The current results are in agreement with a previous study in which researchers demonstrated that 6 mmol of desferal when incubated for 2 hours with erythrocytes led to oxidative stress events by reducing the redox potential of NAD in erythrocytes (Niihara et al.,2002). The other cause of the oxidative stress induced by desferal may be attributed to its role as an electron transport medium from ascorbic acid in cells to oxygen to produce the superoxide anion, which in turn leads to the production of the active nitroxide radicals, as well as the role of desferal in the Fenton reaction (Jiang et al., 2016).
The current study resulted in clear changes in the levels of oxidative stress parameters for pregnant rats and their fetuses at different stages of pregnancy. This was evident in the second week of pregnancy and continued until the end of the trial 13 period in both mothers and their fetuses at the level of blood and tissue. The reason for this is due to the insufficiency of the action or the low level of antioxidants, for example glutathione peroxidase, as well as the overproduction of free radicals of various kinds. Where, glutathione is a strong and effective reducing substance in the cell and has a distinct role in the regeneration and maintenance of moderate levels of the enzyme glutathione reductase very important in vital tissues and developing, including the pregnant uterus (Wickens, et al., 1981;Yu, 1994). The current results showed an increase in the level of malondialdehyde and a decrease in the level of glutathione peroxidase, which is due to an increase in the level of free radicals and a decrease in the level of glutathione in the tissue and blood, and this corresponds to previous studies that illustrate the role of oxidative stress in pregnancy (Lu, et al., 2018;Al-Gubory et al., 2010;Patil et al., 2007). Tissue glutathione depletion directly affects the level of antioxidants, making cells more vulnerable to oxidative stress even under physiological conditions (Sekhon et al., 2010) .this mechanism is attributed to a series of interconnected enzymatic processes associated with cellular oxidative stress, which may result in cell death or failure in its basic function. Among these enzymatic processes is the stimulation of arachidonic acid by activating the enzyme phospholipase A2 associated with the release of arachidonic acid, which works to destroy cells that have depleted glutathione because of metabolism (Balsinde et al., 2002). Arachidonic induction by the action of the enzyme lipoxygenase, which in turn stimulates the synthesis of the active superoxide radicals during the metabolism of arachidonic acid, which plays a dangerous toxic role as a result of depletion of glutathione leading to cell damage and death (Mytilineou et al., 2002).
This cellular pathway is one of the most important pathways associated with glutathione depletion, which induces a series of cellular events that end with an increase in the level of free radicals and their attack on cellular molecules, causing sufficient oxidative damage to cell death or loss of its basic function (Katsuki et al., 1995). The increase in malondialdehyde in the body is an important indicator of the severity of the activity of the free radicals produced and their effect on the lipid peroxidation resulted from the occurrence of oxidative stress (Ruder et al. 2009). The active aldehydes are the second messengers of the deleterious activity of the active oxygen species, which are also by-products of the lipid peroxidation of unsaturated fatty acids (Ishii, et al., 2006). Malondialdehyde is also produced as a byproduct of the inactive thromboxane formation from its inactive form, inactive thromboxane TXA2, which is a vasoconstrictor agent (Bauer et al., 1999). This in turn supports our hypothesis about the lack of oxygen supply to the placental tissue on the one hand and the uterine tissue in the areas of growth and development of fetuses due to the lack of blood flow in them. On the other hand, a second byproduct of lipid peroxidation is the proliferation of smooth muscle cells of the lining of blood vessels that contribute to vascular injury (Watanabe et al., 2002).This results in early loss of embryos as well as impaired growth and development of embryos.
With regard to previous studies, it has been shown that oxidative stress is closely related to early miscarriage and many conditions related to pregnancy, which in turn occurs as a result of lack of oxygen in the uterine tissue during the early stages of pregnancy. Where the placenta are characterized by low antioxidant activity as ‫الــرافـديــن‬ ‫زراعـــة‬ ‫مـجـلـــة‬ ( ‫المجلد‬ 49 ( ‫العدد‬ ) 1 ) 2021 ISSN: 2224 -9796 (Online) ISSN: 1815 -316 X (Print) well as their low concentrations, especially in the first trimester of pregnancy (Schoots et al., 2018). The blastocysts are more susceptive to damage resulting from variation in oxygen levels and free radicals resulting from the biological processes accompanying the implantation of embryos and their growth (Aouache et al., 2018;Mujica et al., 2018). The current study demonstrated that early exposure to oxidative stress in pregnant rats led to many undesirable effects, represented by a decrease in the number of blastocysts in the first week, an increase in the number of dead and absorbed embryos in the second week, and a decrease in the number of live embryos in the third week of pregnancy. The reason for these changes may be attributed to the enzymatic activity associated with the growth and development of the embryos and its increased influence on the metabolic processes in the placenta due to the imbalance of oxygen levels in the placental tissue, which results in an excessive increase in the synthesis and production of free radicals (Schoots et al., 2018). Whereas, it is known that placental hypoxia is closely related to the conversion of xanthine dehydrogenase into xanthine oxidase, the main source and important in generating cytotoxic free superoxide radicals (Poston et al., 2011). Burton et al. (2009) observed that lack of oxygen in the placental tissue causes stress of the endoplasmic reticulum stress and stimulates the formation and accumulation of misfolded proteins in the cell, leading to stimulation unfolded protein responses to try to restore the balance of the endoplasmic reticulum. This activity is mainly related to the functioning of placental cells in accordance with their levels of antioxidants and the level of oxygen in order to prevent tissue damage leading to premature death of the fetus or the inability of the uterine tissue to receive a fertilized egg (Torres-Cuevas et al., 2017).
Conclusion Our study suggest that the desferal injection has a pro-oxidant capacity on rats' model at dose of 100 mg/kg BW. An oxidative stress plays an important role during pregnancy especially on prenatal development as well as persist up to postnatal life.