Recently, dependence on natural compounds for medical purposes and health maintenance is one of the basic endeavors to sustenance and improve human life. Saffron is one of these natural compounds which attract great attention. Many pieces of evidence documented that saffron’s main component such as crocin, crocetin, and safranal could inhibit neurons erosion and depression, have antitumor effects might be because of its capability to induce inhibition of nucleic acid, scavenging free radical, affecting the expression of topoisomerase 2, induce programmed cell death, reduce cancer incidence and significantly increase the lifelong of the animal. Basheeruddin and Inamdar (2010) [10] stated that saffron has cancer preventive and anti-genotoxic potential, which qualify it to play a fundamental role in cancer chemotherapy as an adjuvant.

However, several pieces of evidence have emerged during the actual use of saffron pointed to definite complications which might drive to serious problems. We tried in the present study to shed light on certain complications and avoid saffron erroneous effects in case of incessant consumption of saffron and its active components. Hence, the pivotal aim was to examine the teratogenic effect of saffron on pregnant rats upon exposure to low doses of gamma radiation.

Pregnancy is associated with momentous risks of difficulties that can affect the health of mother and newborn. One of the stern risks is the imbalance of the oxidative/antioxidant, which is manifested by discrepancies of the antioxidant defense system. Pregnancy is a physiological situation categorized by the brittleness of defense in contradiction of pro- oxidizing agents, which causes a disorder of oxidizing/antioxidant status (Derouiche et al., 2018) [18]. 

These instabilities in redox status can be accountable for significant materno-fetal difficulties. High levels of lipid peroxidation products (MDA) which are observed during pregnancy almost come from the lipid peroxidation process occurred in the placenta.  MDA produced are mainly secreted on the maternal side, pass through maternal blood, triggering damages in other tissues, and remain in maternal circulation for some time (Sainz et al. 2000) [19]. In a normal pregnancy, the natural antioxidant can cope with this increase in the production of ROS. However, the disturbances of maternal antioxidants during pregnancy can increase reactive oxygen species which could act as primary or secondary messengers on both cell death and growth. The oxidative stress raised because of disturbances in the balance between antioxidants and reactive oxygen species could alter several reactions that affect fetal growth.

During completion of the intact investigations, we observed significant modifications of redox status (MDA, GSH concentration, and GSHPx activity) in placental and fetus tissues of rats that received saffron and exposed to low doses of gamma irradiation. These changes were less pronounced compared to pregnant rats exposed to saffron (Figure1). The results got might be attributed to the antioxidants aptitudes of saffron active constituents which could play a vital role in the adjustment of redox tone in both placental and fetal tissues during normal pregnancy or during exposure of pregnant animals to gamma radiation. Rahaiee et al., (2014) [20] depicted that spices comprising phenolic and flavonoid compounds possess antioxidant activities and are commonly consumed as antioxidants food supplements. The antioxidant activities of saffron could be ascribed to its phenolic content and to its active ingredients such as safranal, crocin, crocetin, and carotene (Karimi et al. 2010) [21].  The saffron extract of C. sativus flower contains many polyphenol components which might successfully decrease the activity of free radicals and provide a robust defense for the different organs in contradiction of various oxidative damages under a dose-dependent behavior (Makhlouf et al., 2011) [22].

Despite saffron could protect the fetus against oxidative stress, the challenge here is the neutralization of ROS plenty, which might require exerting a central role in the fetus growth were ROS act as primary or secondary messengers on cell death and on growth. This vital and direct role of ROS on growth is because of the redox status that turns on the arrangement of certain transcription factors which affect pathological cellular signaling during proliferation which detour the flawed differentiation towards apoptosis (Dennery 2007) [23]. Also, our data revealed increases of HIF-1α and sFLT-1 in both placenta and fetus tissues in rats supplemented with saffron compared to untreated pregnant rats. There are no detectable changes recorded in pregnant rats exposed to gamma radiation for the 2 parameters, even in placenta or fetus tissues. Whereas, significant decreases of HIF-1a and sFLT-1 are observed in the SRP group compared to the SP group (Figure 2: A-C). Tal et al (2010) [24] reported that the transcription factor HIF-1α (hypoxia-inducible factor-1α) accomplished a certain important function during placental development. HIF-1α arbitrates adaptive responses to oxidative trauma by nuclear translocation and regulation of gene expression. Mitochondrial variations are serious for this adaptive response.

As ordinary pregnancy is characterized by a state of oxidative stress in which placental mitochondria generate reactive oxygen species (Fisher et al., 2019) [25], the saffron might bother this operation because of overproduction of HIF-1a that sync the highest concentration of ROS. Thus, placental and fetal tissue alteration of hypoxia hallmarked by overexpression of HIF-1a paved to pregnancy disorder in rats received saffron supplement because of angiogenic instabilities which handles the development of placental cord and substantially the fetal development. Also, Khalil et al. (2008) [26] reported that the antiangiogenic soluble fms -like tyrosine kinase 1 (sFLT-1) which is a non-membrane associated variant of VEGF receptor binds the angiogenic factors VEGF (vascular endothelial growth factor) and PlGF (placental growth factor) and prevent them from doing normal functions. Therefore, the rises in sFLT-1 in rats supplemented by saffron might alter the process of blood vessel growth through alteration of angiogenic/ antiangiogenic balances present during normal pregnancy and causing circulating low level of free VEGF and PIGF. Gayatri et al. (2016) [27] showed that the abnormal concentration of HIF-1α, the preliminary factor for the hypoxic conditions, causes the inequality in the expression of the angiogenic protein. Increasing tissue oxygen accompanied with saffron treatment may induce part of the antiangiogenic effect, as hypoxia is one of the most important stimulators of angiogenesis (Mousavi et al., 2014) [28]. The hypoxia stimulated the expression of sFLT-1 in normal pregnancies where, despite the placental growth in a hypoxic milieu, leads to a 20-fold rise in sFLT-1 expression (Shibata et al., 2005) [29]. The placenta is assumed to be the major source of sFLT-1 during pregnancy (Maynard et al., 2003) [30]. It seemed that the HIF-1α increases in rats who received saffron extract could be contributed to the increased expression of sFLT-1 in both placental and fetus tissues.

The placenta is one of the richest sources of both pro-angiogenic and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is the most important aspect for all steps of placental vascular formation and development. VEGDF stimulates endothelial expression of proteases that break down the extracellular matrix and release endothelial cells from anchorage, permitting them to migrate and proliferate (Pepper et al., 1991) [31]. The current study showed that the administration of saffron to pregnant rats significantly decreases the concentration of VEGF in maternal plasma and VEGFR in the placenta compared to untreated pregnant rats. No significant changes in rats exposed to gamma-ray as applied in the present study. Interestingly, the changes for these parameters are less expressed in the SRP group of rats when compared to SP rats, despite significant differences from P rats (Figure 3: A-B). These results might premise irregular pregnancy with rats treated with saffron. Ferrara (2001) and Shalaby et al (1995) [32,33]. stated that deactivation of a single VEGF allele or interruption of genes encoding VEGF receptors such as VEGFR1 causes embryonic lethality because of anomalous blood vessel development during embryogenesis, signifying a crucial role of VEGF/VEGFRs in vascular formation. Zheng et al. (2008) [4] reported that VEGF powerfully arouses placental artery endothelial production of nitric oxide (NO). Nitric oxide, one of the most multifunction signaling molecules at systemic and cellular levels, is actively taking part in trophoblast invasion and placental development (Krause et al., 2011) [34]. Our results pointed to the increased level of NO in placental and fetus tissues of pregnant rats supplemented by saffron, while the level of NO decreased in rats exposed to gamma radiation compared to pregnant rats. Despite significant increases recorded in pregnant rats treated by saffron and exposed to gamma radiation compared to pregnant rats, the increases are less pronounced compared to the PS group (Figure 3: C-D). The increases in NO in pregnant rats treated with saffron could show pregnancy problems. Herr et al. (2010) [35] stated that vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (FLT-1) (as signaling molecules which are expressed during angiogenesis) exert their effects in part through NO synthesis. High NO levels can prevent angiogenesis and its effect on cell survival and proliferation depends on its concentration (Ouyang et al., 2008 and Thomas et al., 2008). Tikvica et al. (2010) and Myatt (2010) [36-39] reported that some pregnancy diseases, such as IUGR and PE, display a high concentration of placental NO and nitrosative stress deprived of acceptable placental vascularization.

Besides, our data reveals that the apoptotic regulators caspase3 and granzyme B endures significant increases in both placental and fetus tissues of rats supplemented with the saffron extract. In rats treated with saffron and exposed to gamma irradiation, the increases are less manifested compared to saffron treated rats (Figure 4: A-D). Bartlett (2017) [40] showed that apoptosis is essential for development, including neural development, reduction of oocytes at birth and shaping of fingers and vestigial organs. Some cells quickly proliferated during development undergo apoptosis, the process necessary for many steps in development, including neural development, reduction in egg cells (oocytes) at birth, and the shaping of fingers and vestigial organs in humans and other animals (Bartlett, 2017) [40]. Therefore, understanding the mechanisms involved in physiological and in disturbed or dysregulated apoptosis may lead to the development of alternative methods of preventive treatment of various developmental abnormalities.

The development of obstetric complications and underlying pathogenesis of different diseases of the fetus could be attributed to the disruption of apoptotic regulatory mechanisms which results in apoptosis inhibition or hyper-activation (Straszewski et al., 2005) [41].  

The apoptotic regulators caspase 3 and Granzyme B are notably over-expressed in both placental and fetal tissues of pregnant rats treated with saffron, pregnant rats exposed to gamma radiation, or rats treated with saffron and exposed to gamma radiation. However, the increase is less pronounced with rats treated with saffron and exposed to gamma radiation. The increases in apoptotic regulators accompanied with decreases in angiogenic regulators might be mutually pointed to pregnancy problems because of saffron. Saffron reestablished the superoxide dismutase, catalase, and malondialdehyde levels and increased the expression of active caspase 3 and granzyme as observed in our results. These results are consistent with the finding of Fikry et al. (2018) [42] who reported that saffron administration enhanced the formation of the active form of caspase3 and inhibited the formation of MMP9. Cleaving of caspase 3 plays a vital role in the induction of apoptosis. Caspase activation occurs through the release of cytochrome c from the mitochondria. The mitochondrial outer membrane permeability and cytochrome c release are directly activating effector caspase (caspase 3 and 7) which execute the apoptotic program (Bahashwan et al., 2015) [43]. The increased granzyme in the PS group triggers caspase-independent target cells. Granzyme enforces mitochondria to generate reactive oxygen species (ROS) that disrupt the transmembrane potential but does not perambulate the mitochondrial outer membrane leading to mitochondrial damage which is critical to granzyme-induced cell death (Denis et al., (2008) [44].

The assessment of LDH protein expression in placenta and fetus tissues revealed increases in LDH expression in both placenta and fetus tissues of pregnant rats supplemented with saffron extract compared to pregnant rats. No significant changes were observed in PR and PRS rats. Exposure to the low dose of gamma radiation in sync with supplementation with saffron extract could reduce by the certain way the damages induced by saffron on placental and fetus tissues (Figure 5: A-C). LDH is expressed widely in body tissues, an enzyme found in almost all living cells. It is a sign of common impairment and disease because it is released during tissue damage. He et al. (1995) [45] reported that the low birth weight of infants is associated with an increase in serum LDH levels. However, Qublan et al. (2005) [46] stated that there is no significant overtone between pregnancy disorders and LDH level. In our study, the increases in placental and fetus tissues, and plasma LDH, could prove the existence of certain pregnancy difficulties which might be related to saffron intake.

Based on all the previous, it could be suggested that diet including saffron could be dangerous on maternal or fetus health and increase the incidence opportunity of miscarriage or incomplete development of the embryo. This could be attributed to the effect of saffron in placental angiogenesis and apoptotic regulators, and that joined to the disturbances of redox tone and NO concentration in placental and fetus tissues. The synchronized exposure to the low dose of gamma irradiation and saffron supplementation could reduce the effect of saffron in the certain process as observed in the current study. It could be recommended avoiding using saffron during pregnancy stages.

Authors’ Contributions:

The 2 authors have participated in the design, interpretation of the studies, and analysis of the data and review of the manuscript, conducting the experiments and NMElF wrote the manuscript and GElT contributed in reformatting the main document following the author instruction.

Acknowledgments:

The authors thank Prof. Dr. Khaled Shaaban Azab for reviewing the manuscript and advice in statistical analysis".

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors and authors depend on themselves exclusively to balance all research costs.