The Effects of Different Concentrations of Retinoic Acid on the Histological Structure of Liver of Adult Albino Female Mice and Their Prenatal Fetuses

Retinoic acid (RA) an active metabolite of vitamin A, plays essential signaling roles in mammalian embryonic development and adult homeostasis through concentration-dependent activity but in excess, RA is teratogenic. The present study was designed to evaluate the harmful effect of RA on the: liver tissues of the adult pregnant female albino mice and their prenatal fetuses and adult morphological and behavior changes. Forty-five pregnant adult female albino mice were divided into three groups (n=15 for each group). Group I, the control group orally received 0.1ml of olive oil. Group II and III received 25 and 50mg RA/kg B.W. respectively. RA was dissolved in 0.1ml of olive oil and orally given on 7 th , 8 th , and 9 th days of pregnancy. On the 18 th day of gestation, these females were sacrificed and the liver from mother and their prenatal were removed, weighed, and prepared for histological study. The RA treated adult group exhibited behavior changes including general fatigue and loss of appetite, in addition to the morphological changes which included weight loss, change in normal red eye color towards black, and redness in the mouth and chin area associated with loss of hair (fur) around this area. These observations were showed to be more severe in the 50 mg RA group. The results showed that treatment with 25 and 50mg RA caused no significant decrease in the adult liver weight, while in their prenatal fetuses 50mg RA caused a higher significant decrease in the liver weight, but 25 mg RA caused no significant effects on this weight. Treated with 50mg/kg RA caused a highly significant increase in the number of aborted and dead fetuses as compared with other groups. The results of the histological study showed that treatment with both concentrations of RA caused several degrees of the liver damage with disrupted the normal architecture pattern along with hepatocellular steatosis, hypertrophy, and inflammation. In conclusions, RA caused variable degrees of degeneration and destruction of the liver tissues of adult female (mother) and their prenatal. The most important effects of RA are causing hepatocellular steatosis, hypertrophy, and inflammation, and when RA giving during the critical periods of embryonic development, caused harmful effects on the developing liver, therefore treatment with RA should be avoided at any stage of gestation.


Introduction
Vitamin A and its physiologically active derivative, retinoic acid (RA), participate in many biological conditions, that control normal growth, embryo development, hormonic function, vision, reproduction, the maintenance and modulation of the immune feedbacks, and epithelial tissues and mucosa homeostasis (Cassani et al., 2012;Saeed et al., 2018;Haaker et al., 2020). The human body cannot produce vitamin A in adults nor the embryo (Blomhoff & Blomhoff, 2006). Therefore, maternal nourishing is the only route of vitamin A intake throughout embryonic development. From the human diet, two pre-cursor forms of vitamin A are obtained, retinol and retinyl ester from meat, and carotenoids (β-carotene, α-carotene, and β-cryptoxanthin) from plants. Both are ultimately metabolized into either RA or retinal (retinaldehyde) which is the active metabolite of vitamin A, or they are stored as inactive retinyl esters in the liver or adipose tissue but to a lesser degree (Schreiber et al., 2012).
Retinoic acid is a morphogen derived from retinol (vitamin A). Retinol is metabolized to RA through successive enzymatic reactions catalyzed by distinct sets of dehydrogenases. First, the retinol is oxidized into retinal, which is then oxidized into RA. The RA reacts with the retinoic acid receptor (RAR) and retinoic acid X receptor (RXR). both types encompass three isotypes (α, β, and γ) which affect transcription of several genes during vertebrate development (Linney et al., 2011;Kam et al., 2012).
The normal developing embryo requires retinoids (vitamin A and its derivatives) in controlled concentrations, any excess or deficiency at the wrong stage and time could potentially have an adverse effect on embryonic development (Sporn & Roberts, 1991). RA administration can cause a wide range of defects in the central nervous system and neural tube, in addition to defects in the urogenital system, heart, thymus; and limb skeletal, cleft palate, ear, and some other craniofacial defects. This broad range of malformations is observed in the offspring of different species such as humans (Lammer et al., 1985), and rodents (Kessel & Gruss, 1991).
Retinoid toxicity can be caused by either topical or oral administration. Each one with its own set of adverse effects. Toxicity by oral retinoids can be acute (adults administration of doses as 150-1200 mg over a brief period), chronic (adults administration of doses as > 30 mg over a longer duration of time), and teratogenic (malformation

3.2
The effect of retinoic acid on the liver weight of adult female albino mice (mother) and their prenatal fetuses No significant effects of 25 and 50 mg RA were observed on the liver weight (P>0.05) of the adult female mice (mother) as compared to each other or with the control group. While the liver weight in prenatal, the result showed that 50mg/kg RA, caused a highly significant decrease (P<0.01) in their liver weight as compared with the control group. On the other hand, 25mg/kg RA had no significant effects (P>0.05) on their liver weight in comparison with other groups (Table 1). NS ** Similar letters within each column refer to the non-significant (P>0.05), while different letters in the same column refer to significant differences (P<0.05). NS= non-significant; ** P<0.01= highly significant.

The effects of retinoic acid on the pregnancy outcome
As shown in Table (2), there were no significant differences (P>0.05) in the total number of pregnancy outcomes among all groups. But as the results of the RA treatment, the concentration of 50mg/kg RA caused the death of these fetuses and resulted in a highly significant increase (P<0.01) in the number of aborted fetuses if compared with other groups. This table also showed that treatment with 25mg RA caused a highly significant increase in the number of aborted fetuses compared with the control group. NS ** Similar letters within each column refer to the non-significant (P>0.05), while different letters in the same column refer to significant differences (P<0.05). NS= non-significant; ** P< 0.01= highly significant.
3.4 Histological study of the RA effects on the liver tissues of adult female mice (mother) Microscopic examination of the liver sections of the adult group showed normal histological architecture of the liver with no pathological appearance ( Fig. 2 A, B, and C). showing normal appearance of the liver structure; Liver parenchyma (red star), central vein (black arrows) with flat endothelial lining (blue arrows), polygonal hepatocytes radiating from the central vein with rounded vesicular nuclei (white arrows), Binucleated cells (small head red arrows), Kupffer cells (green arrows), narrow radiating blood sinusoids in between liver cords (long red arrows) and their lining endothelial cells (yellow arrows) (H&E, A: 100 X, B and C 400 X).
The result of the present study showed that treated with 25mg RA caused several damages to the adult liver tissue; these damages are presented in figures (3, 4, and 5). The most effective of this concentration was causing hepatic steatosis (fatty liver diseases) (Fig. 4A, B, and D and 5A). This fatty degeneration included the formation of macrovesicular steatosis which appeared with large lipid droplets and this droplet displacing the nucleus of the hepatocyte to the periphery of the cell in addition to the presence of microvesicular steatosis appeared with small lipid droplets are present in hepatocytes (Fig. 4D). Ballooning hepatocytes (hypertrophy and vacuolated cytoplasm) and macrovesicular steatosis with large lipid droplets are present in hepatocyte also were detected (Fig. 5D).
Despite these changes, it has been observed the presence of healthy binucleated hepatocytes (which looked in large numbers in the liver tissue) (Fig. 3D, 4C and 5A), normal hepatocytes ( Fig. 3C) and (4B and D), megakaryocyte cells ( Fig. 3B and C), and hematopoietic cells (Fig. 4C). These findings indicate the ability of Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.10, No.18, 2020 28 liver regeneration and its hematopoietic function. Treatment with 50mg RA caused severe degeneration in the adult liver tissues; this degeneration was illustrated in figures (6, 7, and 8).
As in the group of 25mg, the liver sections of this group showed the presence of some of the normal hepatocytes and binucleated cells but, in less number, if compared with the group of 25 mg RA (

Histological studies of the effect of RA on the liver of prenatal fetuses
In the control group, the liver sections of the prenatal showed normal appearance as indicated in figures (9A, B, C, D, E, and F). These figures showed that the liver in this stage of development consists of hepatocytes, blood sinusoids, central and portal veins. As demonstrated in (Fig. 9C and E), the hepatocytes gain increased contact with each other to form hepatic cords. Figure (9A) showed these hepatocytes are normal, large in size, and flat in shape. Generally, in mammals, it is well known that the liver is the main site of hematopoiesis during prenatal development, therefore the figures of the control group showed many hematopoietic cells found scattered individually and in foci among liver parenchyma.
Histological evaluation of the prenatal liver after exposing their mothers to 25mg RA revealed that this concentration caused adverse effects on the liver tissue; these effects are presented in figures (10 and 11). This concentration also caused fatty degeneration in the liver tissue ( Fig. 11C and D); these figures showed the formation of foamy hepatocytes in which the cell's nuclei were located within the center of the cells without the displacement that commonly occurs.
Normal and healthy mononucleated and binucleated hepatocytes and lymphocytes also were detected in these sections (Fig. 10D).
More histological alterations were detected in the liver of the prenatal of 50mg RA. All the prenatal of this group were aborted except one of them. The liver sections of this animal revealed the degeneration of the liver tissue ( Fig. 12 A, B, C, and D). From the liver results of adult (mother) and their prenatal, it appears that treatment Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.10, No.18, 2020 34 with RA (both concentrations) caused hepatocellular steatosis, hypertrophy, and inflammation.

Discussion
In the present study RA was given orally on 7 th , 8 th , and 9 th days of gestation, these days as indicated by Rugh, (1968) and Stromland et al., (1991) are considered to be a critical time for organogenesis in mice, since liver diverticulum is reported to be formed by 9 th days, and proliferates cells rapidly beginning of 9 1/2 days gestation shortly thereafter epithelial cords appear in the liver primordial (Rugh, 1968).
Many authors such as Okano et al., (2012), Kelly & Drysdale, (2015), Canete et al., (2017), khaksary-Mahabady et al., (2018, Zasada & Budziz, (2019) and Roberts, (2020) reported the important role of RA during embryogenesis and for tissue homeostasis in both embryo and adult. In the embryo, RA homeostasis in a regionspecific manner is in many ways the reason determining proliferation and differentiation of the developing cells. For this reason, in the developing embryo, the concentration of RA must be cautiously controlled in both time and space. In general, RA overdosing is well known as a teratogen and results in particular patterns of abnormalities implicating many individual organ systems eventually causing birth defects (Ross, 2002;Piersma et al., 2017;Noori & Waheed, 2020 (unpublished data)). Therefore, in the present study when the pregnant female was treated with RA during gestation periods mentioned above, this treatment caused damage to the liver tissue in both mother and prenatal fetuses. This result is in agreement with the results of Morriss-Kay and his colleague (1991), they showed that mouse administered 12mg/kg RA dose on day 7 or 8 of gestation caused overall developmental retardation.
The result of the present study showed the exposure of adult female mice to the RA (25 and 50 mg) caused suppression in their activities and decreases of food intake, these observations are in agreement with the result of Aguwa et al., (2016), Their results revealed that when the rats were exposed to an overdose (400 and 500mg/kg) of vitamin A for 14 days resulted in general body fatigue, decreased food and water uptake and the rats become smaller, shrunk, weak, and waif. The loss of appetite is a symptom seen in many case reports of hypervitaminosis A and during the administration of medication analogs to vitamin A metabolites (NIH, 2017;Vallerand et al., Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.10, No.18, 2020 38 2018; Babatola et al., 2019). Another effect of RA was the loss of hair (fur) around the mouth, this finding is in agreement with Okano et al., (2012), this report demonstrated that hair loss is associated with excess administration of RA. The dark eye was also noticed during the morphological examination of treated adult mice, all-trans-retinoic acid (atRA) a metabolite of RA has been detected in the retina. In the outer connective tissue shell of the eye, the sclera it is speculated that atRA controls the expression of numerous genes that influence changes in scleral extracellular matrix readjusting ocular size and refraction (Summers, 2019). Adverse effects on the visual system have been documented with confidence from the medication containing RA (Suuberg, 2019).
Results of the present study also showed that treatment with RA leads to the death and abortion of the prenatal fetuses, this was well documented in the group of 50mg RA which caused a higher significant increase in the number of aborted adults and death of fetuses compared with the other groups. Furthermore, the entire live adult (mother) in this group had dead fetuses except one of them was found alive, this finding is in agreement with Ross, (2002), their result indicated that RA is being toxic and leading to the spontaneous abortion. Result of Mao et al., (2000) are found in agreement with the result of the recent study, their results indicated that retinol dosage of 20 or 40 mg/kg did not increase embryo lethality or alter litter size, while the dosage of 60mg/kg of retinol increased in embryo lethality, and dosage of 80mg/kg caused high embryo lethality (92.5%). In 2016, Aguwa et al. also reported treatment with an overdose of vitamin A (400 and 500 mg/kg) caused spontaneous abortion and death of the fetuses.

Histological studies of the RA effects on the liver of adult female mice (mother) and their prenatal fetuses
The liver is the largest gland in the body and it is composed principally of epithelial cells (hepatocytes), which are bathed in blood-derived from the hepatic portal veins and hepatic arteries. Therefore, there is a continual chemical exchange between the blood and the cells (Moore & Dalley, 2018). The liver is the main site of detoxification and is principally the major site of intense metabolism by being very critical in the breakdown and removal of toxic or potentially toxic material through the blood. Therefore, the liver has a tendency to various disorders as a result of its exposure to various toxins in extrinsic and intrinsic forms (Guyton & Hall, 2006).
Result of the present study has shown that both concentrations of RA caused a non-significant decrease in the weight of the liver of the adult female mice if compared with the weight of the control, while in their prenatal fetuses, group of 50 mg RA showed a higher significant decrease in the liver weight compared with the other groups. This result is in agreement with Barandeh et al., (2019), who demonstrated that RA applies toxic effects on the development of the embryonic organ in a dose and time-dependent manner. But the present result was disagreement with Mahassni & Al-Shaikh, (2014) they showed no connection between oral intake of vitamin A and liver weight in rats.
Treatment with RA caused several histological alterations in the liver tissues of RA treated female mice and their prenatal fetuses so its caused vasodilation, elongation, and congestion of the blood vessels, sinusoids, and central veins, associated with severe hemorrhage and inflammatory cells infiltration in the liver parenchyma. These results are in agreement with other studies such as Mehrotra & Shah, (2004), their results showed that intraperitoneally administration to low dose of vitamin A (7.5 mg/kg B.W.) once as a single dose in day 11 th of gestation caused elongation and disruption of the central vein, and this resulted in severe hemorrhage. Yousef & Azizzadeh, (2010) also showed that RA caused disruptive effects on the vascular systems, such as an increase in the diameter of the sinusoids in embryonic liver tissue. In 2019, Barandeh et al. showed that when pregnant female mice were treated in day 10 th of gestation with 60mg/kg all-trans RA via diet caused cavity formation in the liver tissue, increase in sinusoids diameter, and disruption of epithelial cells arrangement.
Another effect of RA which recorded in the present study was the degeneration of hepatocytes, damaged and severs inflammation among the portal area and liver parenchyma, in addition to the formation of necrotic foci, multinucleated cells, and apoptotic bodies. These findings are in agreement with Tanumihardjo, (2004), who demonstrated that vitamin A might be very harmful and act as a teratogen for pregnant women especially those who depend on food rich in this vitamin. So, they recommended a routine monitor of liver serum, reserve, and milk vitamin A concentration in pregnant women to prevent both hypo-and hypervitaminosis A in fetuses. In the same year (2004), Hodgson, also showed that the exposure to RA caused severe damage to the liver cells and produced hepatotoxicity. Barandeh et al., (2019) results are also in agreement with the results of the current study, they recorded disruption of embryonic liver cells and parenchyma caused by RA intake. But the present results are in disagreement with the results obtained by Aguwa and his colleagues, (2016), they demonstrated that 400 and 500 mg/kg vitamin A treated group recorded non-significant differences in the liver histological features of as compared to the control group.
The present results can be explained as demonstrated by Erkelens & Mebius, (2017), that the effect of RA is mediated by two major groups of peptides; these are nuclear receptor proteins and cytoplasmic binding proteins. It is well known that there are two members of RA receptors, the RA receptors (RARs) and the retinoid X receptors (RXRs). RAR and their ligands may be involved in normal and abnormal embryonic development (Kam et al., 2012). Also, both RA receptor's families exhibit a very definite spatial and temporal distribution within Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.10, No.18, 2020 39 developing embryo (Mark et al., 2009). Therefore, Kam et al., (2012) suggested that RA binds to the cytoplasmic receptors to enter the nucleus and bind to RAR to promote gene expression required for embryonic liver cell growth and differentiation.
The result of the current study also showed that RA caused fatty degeneration in the liver tissue and resulted in hepatocellular steatosis (fatty liver disease), hypertrophy of hepatocyte, vacuolar degeneration of hepatocytes, and inflammation of the liver. Steatosis as shown by Green and Hodson (2014), Mulder et al., (2015), and Nassir et al., (2015), meaning the accumulation of lipid droplet within hepatocytes and is considered pathologic condition when it affects more than 5% of hepatocytes. Morphologically, steatosis hepatocytes are classified into macrovesicular steatosis in this one large lipid droplets filling up the hepatocyte and displacing the nucleus to the cell periphery and microvesicular steatosis when multiple small lipid droplets are present in the hepatocyte and giving the cytoplasm a foamy appearance. These two types of cells were recorded in the present study.
In the normal liver, retinol (which is mainly dietary-derived fat-soluble signaling molecule of vitamin A) is stored as a characteristic lipid droplet by hepatic stellate cells (HSCs) in the space of Disse and from which they are released and transported regularly to the target tissues as retinol-binding protein (RBP). In the normal animal, HSCs account for 50-60% of the total retinoid's storage in the entire body, these cells may be triggered in response to different kind of injuries, and may form a myofibroblast-like phenotype. (Shirakami et al., 2012;Mawson, 2016). Reynaert, (2002), showed that when HSCs are activated, these cells express the gene of smooth muscle α actin and functionally produce an elevated quantity of extracellular matrix components and matrix-degrading enzymes. In the liver, excess vitamin A is stored in HSCs, and accumulation can cause their activation and hypertrophy, increase in collagen formation, fibrosis, and liver damage the toxicity is dose-related and can be reproduced in animal models (Seegmiller et al., 1997).
Furthermore, present study results indicated that RA caused fatty change which noticed among other hepatotoxic injuries, this observation is in agreement with Thoolen et al., (2010), in both animals and man, they revealed that fatty changes can be produced along with a combination of other hepatotoxic damages such as, chronic liver toxicity, inflammation, degeneration, and necrosis; or nutritional disorders such as, diet and excess vitamin A.

Conclusions
, RA caused variable degrees of degeneration and destruction of the liver tissues of adult female (mother) and their prenatal. The most important effects of RA are causing hepatocellular steatosis, hypertrophy, and inflammation, and when RA giving during the critical periods of embryonic development, caused harmful effects on the developing liver, therefore treatment with RA should be avoided at any stage of gestation.