International Journal of Pure and Applied Zoology

All submissions of the EM system will be redirected to Online Manuscript Submission System. Authors are requested to submit articles directly to Online Manuscript Submission System of respective journal.
Reach Us +44-1518-081136

- International Journal of Pure and Applied Zoology (2016) Volume 4, Issue 2

Effect Of Maternal Caffeine And Retinoic Acid Intake On Liver During Pre- And Postnatal Development Of Mice, Mus Musculus

Fakhr El-Din M. Lashein*, Amin A. Seleem and Abeer A. Ahmed
Department of Zoology, Faculty of Science, Sohag University, Egypt
Corresponding Author: Fakhr El-Din M. Lashein, E-mail: e.tyokumbur@mail.ui.edu.ng
Received 10th March 2016; Accepted 08th April 2016; Published 18th April 2016
Visit for more related articles at International Journal of Pure and Applied Zoology

Abstract

The present study was conducted to evaluate the effect of caffeine and retinoic acid as the active metabolite form of vitamin A on the liver at pre- and postnatal periods of development. In either the pre- or the postnatal period of study, caffeine was able to perturb carbohydrates, increase calcium and decrease iron those accompanied with down regulated expression of TGFβ2 in hepatic tissue as compared to control. Treatment with retinoic acid at both doses in the present study either separate or combined with caffeine upregulate calcium against suppression of iron. Moreover, TGFβ2 show biphasic that severely down regulated during gestation and extended to express intranuclear at postnatal in liver tissue. Decreased iron overload of liver with concomitant down regulation of TGFβ2 expression represent the most important points of this study that protect the liver of young age from oxidative damage and suppress the gene-dependent extracellular matrix deposition. These data may contribute to the therapeutic uses of these substances in preventing the progression of liver damage in hepatitis diseases.

Keywords
Caffeine, Retinoic acid, TGF β2

INTRODUCTION
Caffeine is a methylxanthines of secondary plant metabolite derived from purine nucleotides (Ashihara and Crozier, 1999). The most well known methylxanthines are caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine), which occur in tea, coffee, cacao and a number of other non-alcoholic beverages of plant origin. In 2001 the Committee on Toxicity of Chemicals in Food, UK, concluded that although caffeine intake >300 mg/day might be associated with low birth weight and spontaneous miscarriage, the evidence was inconclusive (COT, 2001). In pregnancy, caffeine is the most widely consumed xenobiotic with the potential to adversely affect the developing fetoplacental unit. Caffeine intake of ≥ 300 mg/day has been associated with fetal growth restriction (Grosso et al., 2001; Bracken et al., 2002; Clausson et al., 2002). The amount of caffeine and metabolites available to the fetoplacental unit depends on the maternal caffeine metabolism and clearance, which shows marked variation between individuals because of genetic and environmental factors (Rasmussen et al., 2002; Grosso et al., 2006; Boylan et al., 2008). The mean caffeine intake throughout pregnancy was recommended to be reduced than the limit of 300 mg/day as recommended (COT, 2001; Bech et al., 2007). However, the consumption of caffeinated beverages during pregnancy is quite common (Brent et al., 2011). Both clinical data and animal experiments indicate that caffeine has the potential to induce reproductive and embryonic toxicity (Vik et al., 2003; Momoi et al., 2008; Huang et al., 2012). Epidemiological investigations also indicate that caffeine intake during pregnancy is associated with impaired fetal growth that preferentially and adversely affect fetal skeletal (Bakker et al., 2010). In addition, prenatal caffeine exposure before and during pregnancy were found to increase the incidence of fetal absorption and still birth in mice, also the bodyweights and body lengths of the liveborn offspring were severely retarded (Huang et al., 2012). However, other studies report that caffeine intake during pregnancy is not associated with changes in birth weight or length (Clausson et al., 2002; Bech et al., 2007).
Moreover, the requirement of the developing mammalian embryo for retinoic acid is well established. Retinoic acid (RA) belongs to the retinoid family and includes the isoforms alltrans retinoic acid (ATRA), 9-cis retinoic acid (9-cis-RA), and 13-cis retinoic acid (13-cis-RA). Retinoic acid, the active form of vitamin A, can be generated from retinol and retinyl ester obtained from food of animal origin, and from carotenoids, mainly β-carotene of vegetables and fruits. The mammalian embryo relies on retinol, retinyl ester and β-carotene circulating in the maternal bloodstream for its supply of vitamin A. RA is involved in various physiological processes, such as embryonic development, reproduction, vision, cell growth, differentiation, apoptosis, inflammation and maintenance of epithelial tissues (Marletaz et al., 2006; WHO, 2009;Tanumihardjo, 2011; Zhou et al., 2011; Zhou and Qin, 2012). It has been shown also that intake marginally above the recommended dietary intake with low clearance during pregnancy is associated with embryonic malformations. The classical signs of hypervitaminosis A occur in skin, nervous system, musculo-skeletal system, circulation, and internal organs, as well as in the fetus following an excessive dietary intake or an intake of drugs containing large doses of specific retinoids (Blomhoff and Blomhoff, 2006).
From the above mentioned data, it is clear that both of caffeine and vitamin A sharing:- 1) A rapid absorption and decreased clearance during pregnancy and in new born 2) Ease of placental transfer without degradation 3), Adverse effects on fetoplacental unit, skleton and visceral organ. So, the aim of the present study was to find out further details relationship effects to resolve the controversy between the psychoactiveergogenic food item (caffeine) and hypervitaminosis A in developing embryos and neonates as their dams consume these substances during pregnancy and lactation. Also, early feed pups were included as they become fed independently.

MATERIALS AND METHODS
Caffeine: Caffeine, anhydrous pure crystals (Merck, C5H10N4O2, 194.2 g/mol) was obtained commercially. Stock solution in saline was prepared and renewed as required during the experimental period.
Retinoic acid (the active metabolite of vitamin A): Retinoic acid (C20H28O2, 300.4 g/mol), the active metabolite of vitamin A in the form of 13-cis form (Isotretinoin) product of Sigma 500 mg/package was obtained. In olive oil (Wang et al., 2007), stock solution was prepared and renewed as required during the experimental period.
Animals and experimental design: Immature mice, Mus musculus, after weaning were obtained from animal house of Assiut University. Animals were acclimatized in laboratory under normal light and temperature conditions with free access of food and water. Males and females were kept separately till maturity in cages. For the prenatal study at days14, 18 of pregnancy, one male was mixed with two females in several cages. After the insurance of vaginal plug following copulation in one round of gestation, the pregnant females were classified into six groups from the 7th day of gestation (the first day of the organogenesis in mice) (Stromland et al., 1991), G1 a control (intraperitoneally vehicle treated), G2 administered orally with caffeine at dose (2 mg/100 g bw), G3, G4 intraperitonally injected with retinoic acid at doses 2, 4 mg/kg bw, respectively. G5, G6 a combined groups caffeine-administered at morning (2 mg/100 g bw) and at evening intraperitonally injected with retinoic acid (2, 4 mg/kg bw), respectively. Caffeine dosing of pregnant females was carried out according to Case et al., (1996). Meanwhile, isotretinoin dosing was carried out at 2, 4 mg/kg bw, a range of doses that induce different abnormalities on injected at the seventh day of gestation (Sulik et al., 1995). Treatment of pregnant females with caffeine was conducted daily, while retinoic acid treatment was conducted at day after the other day for either the separate or combined treatments from the 7th to the 18th day of gestation. Pregnant females at day 14 and at day 18 of pregnancy for different groups were dissected.
For postnatal study, from pups delivery up to six week of age after birth, newborns were classified into six groups. G1 control (intraperitoneally vehicle treated), G2 caffeineadministered through lactating dams at dose (2 mg/100 g b.w) up to the 3rd week of pups age and then through oral administration up to the six week (the period of independent feeding). G3, G4 intraperitonally injected with retinoic acid at doses (2, 4 mg/kg b.w), respectively for six weeks. G5, G6 combined groups treated with caffeine and retinoic acid. Caffeine-administered through suckling up to the third week and then by oral administration up to the 6th week. Treatment of pups with caffeine was conducted daily, while retinoic acid treatment (2, 4 mg/kg bw) was conducted at day after the other day for either the separate or combined treatments similar to the treatments of groups, G3 and G4, respectively. At the 3rd and 6th week of postnatal period, pups of various experimental groups were dissected and fixed for histological, histochemical and immunohistochemical investigations.
Histological and histochemical study: For prenatal study, mice embryos at E14 and E18 of previous groups were fixed in Carnoy's fixative, dehydrated in ethyl alcohol, cleared in methyl benzoate and infiltrated with paraffin wax. Serial sections of embryos at 7 μ thick in paraffin was mounted on glass slides and dried at 40ºC. Sections were stained with Haematoxylin and Eosin for general histology, Periodic acid Schiff's (PAS) reaction for polysaccharide detection, Prussian blue for iron detection, Alizarin red S-stain for calcium detection (Drury and Wallington, 1976), Acridin orange/Ethidium bromide stain for nuclear fluorescence (Kasibhatla et al., 2006). For postnatal study at the end of 3rd (suckling period) and the end of the 6th week (independent feeding), liver of different groups were processed for section preparation. Mounted sections were stained with the different stains as indicated above in addition to Masson's stain for collagen at the 6th week postnatal. Sections were dehydrated in ascending grades of ethanol, cleared in xylene and mounted with DPX. Selected sections of the developing liver at 14, 18 day of gestation were photographed and processed as required. Also, sections of the liver at the 3rd and the 6th week were selected, photographed and processed for evaluation in different groups compared to control. Also, the percentage of binucleated hepatocytes at the 3rd and the 6th week were evaluated and compared to control. The comparison of this last evaluation was made by counting cells with binuclear nucleus in 500 hepatocytes per section in various treatments. Counting and imaging were made using a light microscope (Axiolab Standart 20, Carl Zeiss, Jena, Germany) coupled to a video camera (AxionCam, Carl Zeiss, Jena, Germany).
Immunohistochemistry of TGFβ2: In immunohistochemical study, deparaffinized Superfrost/ Plus slides-mounted sections of embryos at 14, 18 day of gestation and those of liver at the 6th week postnatal of the different experimental groups were retrived for reantigenicity using 10 mM citrate buffer at pH6 in 100˚C for an hour (Buchlowalow and Bocker, 2010). After cooling at room temperature, sections were treated for 10 minutes with 0.3% hydrogen peroxide block and then with protein block (phosphate buffer solution, pH 7.6, with 0.5% BSA, 0.5% casein and less than 0.1% sodium azide) for 10 minutes to block nonspecific background staining, then sections were incubated with primary antibody (Rabbit Anti-human TGF β2 polyclonal antibody, Spring Bioscience, USA) and then washed using phosphate buffer and incubated with secondary antibody, Biotinylated Goat Antipolyvalent (Antipolyvalent HRP DAB detection system, Spring Bioscience, USA) according to the manufacture protocol. Reactions and colour were visualized by using chromogene mixed with 3, 3ʹ-diaminobenzidin (1:10) (DAB substrate, chromogen, Spring Bioscience). In all cases, negative control sections in which the primary antibody not applied to tissue sections were carried out. Sections were dehydrated in ascending grades of ethanol, cleared in xylene and mounted with DPX mounting media.
Processing: In either the histological, histochemical or the immunohistochemical preparations, sections were examined microscopically to evaluate the effect of caffeine, retinoic acid and the co-administration of both in experimental groups compared to the control. Sections of the developing liver of pre- and postnatal periods of study were selected, photographed and processed for evaluation in different groups compared to control. Male embryos were considered for prenatal period of study based on serial sectioning and staining with H&E at gonadal region to identify the testicular tissue. While males of postnatal study were selected following dissection.

RESULTS
14 Days old embryos
At E14 of gestation in control, the liver has the characteristics of hematopoietic tissue in Hematoxylin and Eosin-stained sections. Three types of cells, hemopiotic, hebatoblasts and immature megakaryocytes are distinguished. High density of hematopoietic cells is present throughtout the developing organ. Among the hematopoietic tissue, proliferative hepatoblasts are scattered that characterized by large basophilic vesicular euchromatic nuclei of variable size and shape making it difficult to appreciate individual cell shape. The hematopoietic cells present at this stage are nucleated of the erythroid lineage that contains uniformly hyperchromatic nuclei scattered among the hepatoblasts. The third type of cells that can be easily distinguished in Hematoxylin and Eosin-stained sections is the immature megakaryocytes that are characterized with their large size and regular outlines (Figure 1A). In acridin orange/ethidium bromide-stained sections proliferated hepatoblasts have variable flourescencs while the hematopoietic cell nuclei are dense and homogenously fluorescent (Figure 1B). PAS-stained sections revealed the positive cytoplasm of megakaryocytes (Figure 1C). TGFβ2 stained section revealed intense expression around the developing blood vessels against relatively pale expression throughout the developing parenchyma (Figure 1A and Figure 1D).
In caffeine treated embryos, the developing liver revealed the existence of hyperchromatic hemopoietic and vesicular euchromatic nucleated hepatoblasts similar to those observed in control. The most observed effect of caffeine treatment is a distorted nuclei of the megakaryocytes compared to control (Figure 1E). Acridin Orange/Ethedium bromide-stained sections revealed a well fluorescence of hepatoblasts and hyperchromatic hemopoietic cells similar to control (Figure 1F). Decrease of PAS-stained cytoplasm of the distorted megakaryocytes and sever dowenregulation of TGFβ2 expression were noted (Figure 1G and 1H) as compared to control.
In retinoic acid treated embryos, the effect seems to be a dose-dependent. Megakaryocytes are the most affected of the constituent cells (hyperchromatic hemopoietic and hebatoblasts) in H&E stained tissue. In 2 mg/kg b.w treated with retinoic acid, embryos distorted nuclei of megakaryocyte were observed against the hyperchromatic hemopoietic cells and hepatoblasts, which look like to those observed in control (Figure 1I). Acridin orange/ethedium bromide stained sections revealed a little effect concerning the fluorescence of hepatoblasts that appeared dark against a well fluorescence of hemopoietic cells (Figure 1J). The effect of 2 mg/kg b.w of retinoic acid is best manifested in PAS stained sections since a decreased stainability of megakaryocytes was noted (Figure 1K) as compared to those obsereved in control and caffeine treated embryos. In contrast to caffeine effect, TGFβ2 expression was best detected around the developing blood vessels (Figure 1L). In 4 mg/kg b.w of retinoic acid treated embryos, deformed megakaryocytes (Figure 1M), decreased fluorescence of both the hepatoblasts and hemopoietic cells (Figure 1N), accumulation of PAS positive granules and negatively stained macrophages were noted (Figure 1O) as compared to those of control, caffeine and 2 mg/kg bw treated embryos, respectively. In addition, the dose dependent effect of retinoic acid is best manifested in immunostained sections since severe dowenregulation of TGFβ2 expression was observed around the developing blood vessels (Figure 1P) similar to caffeine effect.
In combined treatment of caffeine and retinoic acid at dose 2 mg/kg b.w megakaryocyte with branched cytoplasm and well organized nuclei, hyperchromatic hemopoietic cells and hepatoblasts are well observed (Figure 1 Q). However, depressed fluorescence of hepatoblasts (Figure 1R), PAS-staining of megakaryocytes (Figure 1 S) and severe dowenregulation of TGFβ2 expression (Figure 1T) were noted. In combined treatment with 4 mg/kg b.w lobulated megakayocyte nuclei (Figure 1U), depressed tissue fluorescence (Figure 1 V), decrease in PAS-posive tissue granules (Figure 1W) were the most observed differences as compared to the effects of 4 mg/kg b. w of retinoic acid treated embryos. Also, the expression of TGFβ2 was severely inhibited (Figure 1X).
18 Days old embryos
At 18th day of gestation, H&E stained sections of control embryos revealed a decline in erythropoietic activity as the hepatocytes gain contact with each other and continue to form the hepatic cords. At this age of development the hematopoietic populations have been restricted to reduced hematopoietic islands, individual cells and regressed megakaryocytes scattered throughout the developing parenchyma of the liver (Figure 2A). PAS-stained sections of control revealed regularly distributed macrophages throughout the hepatic parenchyma. The contents of these cells are heavily stained that make it difficult to differentiate between their nuclei and cytoplasm (Figure 2A/1). While the hepatocytes are stained with a little PAS-positive inclusions. Iron and calcium and TGFβ2 expression homogenously detected throughout the developing hepatic parenchyma (Figures 2A/2,/3,/4) respectively. In caffeine treated embryos hepatocyte vacuolation, congested blood vessels and sinusoids were the most observed effects (Figure 2B) compared to control. PAS stained sections revealed inhibition of macrophage content (Figure 2B/1). Also, caffeine treatment results in decreased iron (Figure 2B/2), increased calcium (Figure 2B/3) and down regulated expression of TGF β2 (Figure 2 B/4).
Retinoic acid treatment was found to provoke decrease in hemopoietic cells, hepatocyte vaculation and nuclear pyknosis in a dose-dependent manner (Figures 2C and 2D), induced PAS positive contents throughout the hepatic parenchyma (Pl 2 C/1, D/1) and decrease in iron content (Figure 2C/2 and 2D/2) and calcium (Figure 2C/3 and 2D/3) while TGF β2 expression was not changed (Figure 2C/4 and 2D/4) as compared to control.
In combined treatments, hepatocyte vacuolation and nuclear pyknosis were less observed (Figures 2E and 2F) compared to caffeine or retinoic acid treatments. PAS stained sections revealed intense reticular framework between hepatocytes at G6 (Pl 2 F/1) compared to the different treated groups. In addition, reduced iron (Figures 2 E/2 and 2F/2), calcium (Figures 2E/3, 2F/3) and down regulation TGF β2 expression (Figure 2 E/4, F/4) were noted.
Liver of suckling period
At the end of suckling period (3 weeks post natal) in which the newborn receive the food through lactating control and treated mother's the liver was studied at histological and histochemical levels. In control, the characteristic organization of the hepatic parenchyma was noted. Hepatic cords are radiated around central vein those interspersed with sinusoids. Kupffer's cells and lymphocytes are recognized within the lumen of sinusoids (Figure 3A). Binucleated hepatocytes are recognized that represents about 20% of the constituting cells. In all treatments the lobular organization was the same as observed in control (Figures 3B–3F). The most observed difference was confined to the percentage of binucleated hepatocytes in caffeine and in the combined treated groups with the two doses of retinoic acid. The recorded percentages were 12, 11, 10%, respectively. Meanwhile the recorded percentages of the binucleated hepatocytes in retinoic acid treatments at 2, 4 mg/kg b.w of treated mother's look like to that of control that represents 18, 20%, respectively. In PAS stained sections caffeine was found to provoke carbohydrate deposits (Figure 3H) in contrast to retinoic acid at both doses of treatments (Figures 3I and 3J) those are negatively stained similar to control (Figure 3G). PAS positive deposits in combined treated groups appeared to be retinoic acid dependent. At 2 mg/kg b.w of retinoic acid treatment combined with caffeine carbohydrate deposits appeared (Figure 3K) similar to that of caffeine treatment only. Meanwhile negatively stained deposits at 4 mg/kg b.w of retinoic acid treatment combined with caffeine (Figure 3L) similar to both of the control (Figure 3G) and retinoic acid treatments at both doses (Figures 3H and 3I), respectively was noted.
Liver of independent feeding
At independent feeding, that begins after weaning up to the 6th week postnatal during the direct treatments the liver was studied. In control the binucleated tetraploid hepatocytes percentage increased (30%) compared to that observed (20%) at the 3rd week of postnatal during suckling. Decreased percentage of the binucleated hepatocytes was encountered in caffeine treatment (14%) and in combined treatment with caffeine and both doses of retinoic acid (15, 11%, respectively) at the same age as compared to control. In retinoic acid treatments, the percentage of binucleated hepatocytes was severely decreased at 2 mg/kg b.w (5%) as compared to the percentage at 4 mg/kg b.w (15%) and to that of control at both the 3rd and the 6th week. The decreased percentages of binucleated hepatocytes were markedly correlated with hepatocyte hypertrophy and reduced of sinusoidal lumen in all treatments.
Histological examination of H&E stained liver sections revealed the general architecture of hepatic parenchyma in both of the control and all treated groups (Figure 4). Lobular organization, central vein, radiated hepatic cords, sinusoids and complementary cells (Kupffer's and lymphocytes) were recognized. The most observed effect of treated groups including infiltrated foci near the central vein and deeper in the hepatic parenchyma of caffeine treatment was noted (Figure 4B), dose-dependent induction of giant nuclei that accompanied with cell hypertrophy in retinoic acid treatments (Figures 4C and 4D) and vacuolation in combined treatments with caffeine and retinoic acid (Figure 4E, F) as compared to control (Figure 4A).
In acridin orange/Ethedium bromide stained sections, regular hepatocyte nuclei were noted throughout the liver tissue that intensely fluorescent in control (Figure 4A insert). In caffeine, in addition to infiltrated foci around the central vein and deeper in hepatic parenchyma, pale fluorescence of some hepatocyte nuclei were noted against a well fluorescent inflammatory cells (Figure 4B insert) as compared to control. Retinoic acid treatments results in a dose dependent giant nuclei induction with well fluorescence (Figures 4C and 4D inserts) in contrast to caffeine treatment. Giant nuclei induction was concomitant with a well-recognized Kupffer's cell that lines the sinusoidal cavity. Combined treatments with caffeine and both doses of retinoic acid don’t reveal either the paleness or giant nuclear induction (Figures 4E and 4F inserts) as compared to caffeine or retinoic acid treatments.
In Masson's stain for collagen detection, thicker wall of central veins was noted in caffeine (Figure 5B) and retinoic acid treatment (Figure 5D) at 4 mg/kg b.w as compared to both the control (Figure 5A), retinoic acid treatment (Figure 5C) at 2 mg/kg b.w and in combined treatment with caffeine and retinoic acid at both doses (Figures 5E and 5F), respectively. In addition, caffeine treatment results in fibrotic foci (Figure 5B) in the inflammatory foci that observed in H&E stain. From the other hand, retinoic acid treatment at 4 mg/kg b.w however, did not initiate the fibrotic foci it induce intense collagen stainability around the hypertrophoid hepatocytes (Figure 5D) as compared to either the control or to the combined treatments.
PAS stained sections revealed uniform distribution of carbohydrates within the hepatocytes of control (Figure 6A). Caffeine (Figure 6B), retinoic acid at 2 mg/kg b.w (Figure 6C) or in combination treatment with retinoic acid at 2 mg/kg b.w (Figure 6E) result in decreased carbohydrate store of hepatocytes as compared to control. In contrast, positive carbohydrate detection was noted in retinoic acid at 4 mg/kg b.w (Figure 6D) and in combined treatment with caffeine (Figure 6F) however, the carbohydrate content was decreased as compared to control. Calcium detection in Alizarin red S stained section revealed high stainability of calcium in the hepatic parenchyma in caffeine treatment (Figure 6H). Increased satiability of calcium was also noted in retinoic acid treatment at 4 mg/kg b. w (Figure 6J) and in the combined treatment with caffeine and retinoic acid at both doses (Figures 6L and 6M) as compared to control (Figure 6G) and retinoic acid treatment at 2 mg/kg b. w (Figure 6I). In contrast to calcium detection, prussian blue stained sections for iron revealed a decrease in iron in hepatic parenchyma in caffeine (Figure 6 N), retinoic acid at both doses separately (Figures 6O and 6P) or in combination with caffeine (Figure 6Q and 6R) as compared to control (Figure 6M).
Concomitant changes in TGFβ2 expression was noted since caffeine treatment results in compete inhibition (Figure 6T), upregulation in retinoic acid treatments (Figures 6U and 6V) and differential cytoplasmic/neuclus expression in combined treatments with caffeine and retinoic acid (Figures 6W and 6X) compared to the faint expression of control (Figure 6S) that show faint expression.

DISCUSSION
The present study revealed that the fetal liver at 14th day of gestation at histological level can be compared to adult bone marrow, hyperchromatic hemopoietic progenitor and the polyploid megakaryocytes (the platelet forming cells) were well observed. Caffeine and retinoic acid dosing during pregnancy results in altered and dose-dependent deformation of megakaryocyte differentiation and function as expressed in H&E and PAS stained sections. Concomitant fluorescence depression of hepatoblastes and dowenregualated expression of TGFβ2 in the developing liver were noted that relatively recovered in combined treatment with caffeine and retinoic acid in a dose-dependent manner in the prenatal period of development. Studies on the development of hematopoietic system have revealed the initiation sites of hematopoiesis and proposed a migration of stem cells during development of the mouse embryo (Huang and Auerbach., 1993; Medvinsky and Dzierzak, 1996; Cumano et al., 1996). Cells with longterm marrow repopulating activity have been detected in the yolk sac and the aorta-gonadmesonephric region at days 9 to 10 of gestation for the first time in an embryonic life of mice (Muller et al., 1994; Yoder et al., 1997). Hematopoietic stem cells are believed to migrate into the liver around day 11, and subsequently into the bone marrow and spleen, whereas the fetal liver remains as a main organ of definitive hemopoiesis during the embryonic period as has been noted in the present study. It has been also reported that the fetal liver hematopoietic stem cells have a greater proliferative capacity than do adult bone marrow stem cells (Rebel et al., 1996; Harrison et al., 1997). The frequency of hematopoietic stem cells in day 14-fetal liver cells is comparable to that in adult bone marrow cells similar to the observed histology as indicated in the present study that regressed at 18th day of gestation as the development proceed and the liver acquired the characteristic cellular architecture of hepatocytes at 18 day of gestation. The observed effects of either caffeine or retinoic acid treatments indicates their ability to cross the placenta to exert their effects. In this context, Ikeda et al., (1982) and Kimmel et al., (1984) concluded that no placental barrier to caffeine and unusually high levels of caffeine have been reported in premature infants born to women who are heavy caffeine consumers as has reported (Khanna and Somani, 1984). The authors also reported that the cytochrome P450 1A2 which is the principal enzyme involved in caffeine metabolism is absent in the placenta and the fetus. For retinoic acid excess during development, also results in major embryonic defects which often overlap with those observed in retinoid deficiency (Morriss-Kay and Ward, 1999). Patterns of retinoid accumulation in embryonic liver indicate the onset of vitamin A storage occurs by midorganogenesis that exceeds the retinoids in placenta of the developing concepts (Shah et al., 1987) and exerts their effects as indicated in the present study concerning specially the differentiation of megakaryocytes at 14th day of gestation and altered the stain ability of polysaccharides and macrophages at18 day of gestation. The defective differentiation of megakaryocytes in either caffeine or retinoic acid treatments was recovered in cotreatments with caffeine and retinoic acid. Megakaryocytes in these treatments are similar to those observed in control since the hallmarks of megakaryocyte maturation that include polyploidization and expansion of cytoplasm were noted as reported by Patel et al., (2005) and Richardson et al., (2005). Also, regardless the altered reactivity of megakaryocytes toward PAS and the down regulated expression of TGFβ2 at the prenatal period of development, proper differentiation of megakaryocytes were noted in co-treatments which may indicate the involvement of other growth factors of the transforming superfamily that need further investigation.
At postnatal during suckling and after weaning in control increased percentages of binucleated hepatocytes from the 3rd to the 6th week were noted. Decreased percentages of binucleated hepatocytes were observed in all treatments with concomitant polyploid giant nuclei induction compared to control. Binucleation was considered as an interesting feature in adult hepatocytes that begins from the neonatal liver (Celton-Morizur et al., 2010). In this context, weaning was found to increase the amount of circulating insulin that induce incomplete cytokinesis to generate binuclear hepatocytes during liver maturation (Celton-Morizur et al., 2009, 2010; Gentric et al., 2012). Hence, the observed decrease in binucleated hepatocytes in various treatments during suckling or after weaning suggests that cells under caffeine, retinoic acid or combined treatments failed to divide properly and tend to polyploidization as clearly indicated in H&E and acridin orange stained nuclei of hepatocytes at the 6th week postnatal. Polyploidy, the state of having greater than a diploid DNA content (tetraploid, octoploid, etc.), is a widespread physiological phenomenon observed particularly in plants, fish, and amphibians (Otto and Whitton, 2000). Although it is less common in mammals, some tissues including heart muscle cells, platelet progenitor megakaryocytes, and liver parenchyma develop a certain degree of polyploidy during their normal life cycle. The appearance of polyploid cells is often associated with late fetal development and coincides with terminal differentiation and restricts hepatomrgalogenesis (Gerlyng et al., 1993; Gupta, 2000). Polyploidy is a characteristic feature of mammalian hepatocytes (Celton-Morizur and Desdouets, 2010). During postnatal growth, the liver undergoes substantial changes, including gradual polyploidization. This process generates the successive appearance of tetraploid and octoploid hepatocytes with 1 (mononucleated; e.g., 4n, 8n) or 2 nuclei (binucleated; e.g., 2 × 2n, 2 × 4n). In adults, 70% of all hepatocytes in rodents and 40% in humans are tetraploid (Seglen , 1997; Toyoda et al., 2005). Interestingly, in the adult liver, the generations of polyploid cells were re-induced following a variety of cellular stressors. Polyploid cells were detected in response to stress and injury, as has been described for heart muscle cells; VSMCs during hypertension; and thyroid cells in hyperthyroidism (Auer et al., 1985; Vliegen et al., 1995; Hixon et al., 2000; Storchova and Pellman, 2004). Moreover, in many human carcinomas, cells with polyploidy, DNA content arise as an early step in tumorigenesis and precede the formation of a neuploid cells (Ganem et al., 2007). Liver re-growth after partial hepatectomy or following oxidative damage and metabolic overload (copper/iron) is associated with a pronounced increase in the proportion of cells that are polyploid (Sigal et al., 1999; Gorla et al., 2001).
In the present study, giant nuclei inductions in either caffeine, retinoic acid or in the combined treatments reflect their ability to suppress cell proliferation through inducing incomplete cytokinesis resulting in polyploidy. For many years, caffeine has been generally believed to suppress cell proliferation (Levi-Schaffer and Touitou, 1991). Caffeine has also been shown to inhibit ultraviolet B (UVB)- induced skin cancer in mice (Lou et al., 1999), suppressed epidermal growth factor (EGF)-induced malignant cell transformation (Nomura et al., 2005) and perturb key cell cycle regulatory proteins (Bode and Dong, 2007). Also, retinoids have been shown to exert an anticarcinogenic effect through suppression of the cell cycle, induction of apoptosis, suppress liver regeneration and/or differentiation. Meanwhile giant nuclei were noted in BrdU incorporation indicating polyploidy induction similar to the observed findings in the present study indicating DNA replication in endomitosis of polyploid induction (Ledda- Columbano et al., 2004). Also, RA induces cell cycle arrest and differentiation exerts in different cell including ES, F9, or HL60 cells (Rochette-Egly and Chambon, 2001; Altucci et al., 2007; Mongan and Gudas. 2007) and in the present study in both caffeine and retinoic acid either in the separate or in the combined treatments resulting in polyploidization a mechanism by which hepatomegally and direct mitosis in proliferative disease can be controlled.
The present study revealed that caffeine administration from the 3rd to the 6th week during the independent feeding induces inflammatory cell infiltration and deposition of collagen. A drawback effect of caffeine that is controlled in cotreatment with retinoic acid at both doses used in the present study. In contrast, beneficial effects of caffeine were reported in preventing liver dysfunctions in several investigations (Tanaka et al., 1998; Wells, 2000; Gressner et al., 2002; Ruhl and Everhart, 2005; Gressner et al., 2008). Still, however, conflicting data about the effect of caffeine were reported. In the present study the inflammatory infiltrated foci that detected closely to the blood vessels or deeper in hepatic tissue with collagen deposits can be attributed to the chronic administration, the young age at which the treatment was conducted and the species differences. In this context, various studies previously reported that RA regulates the expression of ECM and plays a significant role in fibrotic diseases (Wang et al., 2007; 2008; Hisamori et al., 2008; Aguilar et al., 2009; Ye et al., 2010; Xiao et al., 2011; Zhou et al., 2013). However, divergent and contradictory effects are reported in the literature that can be attributed to the RA doses used in various studies for RA treatment in fibrotic disease.
Concomitantly, in either the pre- or the postnatal period of study, caffeine was able to perturb carbohydrates, increase calcium and decrease iron those accompanied with down regulated expression of TGFβ2 in hepatic tissue as compared to control. Treatment with retinoic acid at both doses in the present study either separate or combined with caffeine upregulate calcium against suppression of iron. Moreover, TGFβ2 show biphasic that severely down regulated during gestation and extended to express intranuclear at postnatal in liver tissue. In chronic administration of caffeine to young rats an increase in intestinal calcium absorption, which compensated for the urinary loss was reported (Yeh and Aloia, 1986; Yeh et al., 1986). Also, Massey and colleagues (Massey and Wise, 1984; Massey and Hollingbery, 1988; Bergman et al., 1990) showed that caffeine-induced diuresis increased urinary calcium loss acutely. So the increased absorption of calcium in young age during caffeine administration contributes to the elevation of circulating calcium with consequent upregulation in hepatic tissue before excretion. In contrast, decreased hepatic store of iron in caffeine administration was noted. This observation is in accordance with earlier study since a cup of coffee reduced iron absorption from hamburger meal by 39% (Morck et al., 1983). Retinoides were also included in calcium and iron metabolism in bone as the main site of calcium deposition and liver as the main organ of iron store. In this concern, calcium mobilization resulting in bone fragility (Melhus et al., 1998) and depletion of hepatic store of iron were reported in retinoid administration (Tsuchya et al., 2009). So, in cotreatments calcium overload and iron depletion were noted accompanied with down regulation of TGFβ2 expression regardless the intranuclear expression noted in the present study. Retinoic acid is so antagonize the down regulated effect of caffeine on TGFβ2 gene may be in part due to its receptor interference with TGFβ (Pendaries et al., 2003).
In conclusion, the present study contribute to the benefits of both caffeine and retinoic acid as the active metabolite of vitamin A during pregnancy, suckling and independent feeding of young age. Decreased iron overload of liver with concomitant down regulation of TGFβ2 expression represent the most important points of this study that protect the liver of young age from oxidative damage and suppress the gene-dependent extracellular matrix deposition. These data may contribute to the therapeutic uses of these substances in preventing the progression of liver damage in hepatitis diseases.

Figures at a glance
Figure Figure Figure
Figure 1 Figure 2 Figure 3

Figure Figure Figure
Figure 4 Figure 5 Figure 6

References

  1. Aguilar, R.P.,Genta, S.,Oliveros, L.,Anzulovich, A.,Gimenez, M.S. and Sanchez, S.S., (2009). Vitamin A deficiency injures liver parenchyma and alters the expression of hepatic extracellular matrix. J. Appl. Toxicol.,29: 214-222.
  2. Altucci, L.,Leibowitz, M.D.,Ogilvie, K.M.,de Lera, A.R.andGronemeyer,H., (2007). RAR and RXR modulation in cancer and metabolic disease. Nat. Rev. Drug Discov.,6:793-810.
  3. Ashihara,H. and Crozier,A., (1999). Biosynthesis and metabolism of caffeine and related purine alkaloids in plants. Adv.Bot.Res., 30:117-205.
  4. Auer, G.U.,Backdahl, M.,Forsslund, G.M., and Askensten, U.G., (1985). Ploidy levels in nonneoplastic and neoplastic thyroid cells. Anal. Quant. Cytol. Histol.,7:97-106.
  5. Bakker, R.,Steegers, E.A.,Obradov, A.,Raat, H.,Hofman, A. andJaddoe, V.W., (2010). Maternal caffeine intake from coffee and tea, fetal growth, and the risks of adverse birth outcomes:The Generation R Study. Am. J. Clin. Nutr.,91:1691-1698.
  6. Bech, B.H.,Obel, C.,Henriksen, T.B. and Olsen, J., (2007). Effect of reducing caffeine intake on birth weight and length of gestation: randomised controlled trial. B. M. J.,33:409- 412.
  7. Bergman, E.A., Massey, L.K., Wise, K.J. and Sherrard,D.J., (1990). Effects of oral caffeine on renal handling of calcium and magnesium in adult women. Life Sci.,47:557-564.
  8. Blomhoff,R. and Blomhoff, H.K., (2006).Overview of retinoid metabolism and function. J. Neurobiol., 66:606- 630.
  9. Bode, A.M. and Dong, Z., (2007). The enigmatic effects of caffeine in cell cycle and cancer. Cancer Letters,247: 26-39
  10. Boylan, S.M., Cade, J.E., Kirk, S.F., Greenwood, D.C., White, K.L., Shires, S.,Simpson, N. A.,Wild, C.P. andHay, A.W., (2008). Assessing caffeine exposure in pregnant women. Br. J. Nutr., 100:875-882.
  11. Bracken,M.B.,Triche,E.W., Belanger,K.,Hellenbrand,K. and Leaderer,B.P., (2002).Association of maternal caffeine consumption with decrements in fetal growth. Am.J.Epidemiol., 155:429-436.
  12. Brent, R.L., Christian, M.S. and Diener, R.M., (2011). Evaluation of the reproductive and developmental risks of caffeine. Birth Defects Res. B. Dev. Reprod. Toxicol.,92:152-187.
  13. Buchlowalow, B.I. and Bocker, W., (2010). Immunohistochemistry. Basics andmethods. Springer Verlag Berlin Heidelberg, PP: 48.
  14. Case, T.S., Saltzman, M.J.,Cheuk, J.,Yazdani, M.,Sadeghpour, A., Albrecht, D.,Rossowska, M. J. and Nakamoto, T., (1996). Combined effects of caffeine and alchol during pregnancy on bones in newborn rats. Res. Exp. Med.,196:179-185.
  15. Celton-Morizur, S.,Merlen, G.,Couton, D.,Margall-Ducos, G. and Desdouets, C.,(2009). The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleatedtetraploid liver cells in rodents. J.Clin. Invest., 119: 1880-1887.
  16. Celton-Morizur,S.,Merlen, G.,Couton , D. andDesdouets,C.,(2010).Polyploidy and liver proliferation: central role of insulin signaling. Cell Cycle, 9: 460-466.
  17. Celton-Morizur, S.,and Desdouets, C., (2010). Polyploidization of liver cells. In Polyploidization and cancer. Adv.Exp.Med.Biol., 676:123-135.
  18. Clausson, B.,Granath, F.,Ekbom, A., Lundgren, S.,Nordmark, A.,Signorello, L. B. and Cnattingius, S., (2002). Effect of caffeine exposure during pregnancy on birth weight and gestational age. Am. J. Epidemiol.,155:429-436.
  19. Committee on Toxicity (2001). COT statement on the reproductive effects of caffeine. London: Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment.
  20. Cumano, A.,Dieterlen-Lievre, F. andGodin, I., (1996). Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonicsplanchnopleura. Cell.,86:907-916.
  21. Drury, R.A.B. and Wallnigton, E.A., (1976). Carleton’s Histological Technique. Oxford University Press, London.
  22. Ganem, N.J.,Storchova, Z.and Pellman, D., (2007). Tetraploidy, aneuploidy and cancer. Curr. Opin.Genet. Dev. 17:151-162.
  23. Gentric, G.,Celton-Morizur, S. and Desdouets, C.,(2012). Polyploidy and liver proliferation. Clin. Res. Hepatol. Gastroenterol., 36: 29-34.
  24. Gerlyng, P.,Abyholm, A.,Grotmol, T.,Erikstein, B.,Huitfeldt, H.S.,Stokke, T. and Seglen, P.O., (1993). Binucleation and polyploidization patterns in developmental and regenerative rat liver growth. Cell Prolif., 26: 557-565.
  25. Gorla, G.R.,Malhi, H., and Gupta, S., (2001). Polyploidy associated with oxidative injury attenuates proliferative potential of cells. J. Cell Sci.,114:2943-2951.
  26. Gressner, A.M.,Weiskirchen,R.,Breitkopf,K. andDooley, S., (2002). Roles of TGF-beta in hepatic fibrosis. Front. Biosci.,7:793-807.
  27. Gressner, O.A., Lahme, B., Rehbein, K., Siluschek, M., Weiskirchen, R., Gressner, A.M., (2008). Pharmacological application of caffeine inhibits TGF-beta-stimulated connective tissue growth factor expression in hepatocytes via PPAR gamma and SMAD2/3-dependent pathways. J. Hepatol., 49:758-767.
  28. Grosso,L.M., Rosenberg,K.D., Belanger,K.,Saftlas,A.F.,Leaderer,B. and Bracken,M.B., (2001).Maternal caffeine intake and intrauterine growth retardation. Epidemiol.,12: 447-455.
  29. Grosso,L.M.,Triche,E.W., Belanger,K.,Benowitz,N.L.,Holford,T.R. and Bracken, M.B., (2006). Caffeine metabolites in umbilical cord blood, cytochrome P-450 1A2 activity, and intrauterine growth restriction.Am.J.Epidemiol.,163:1035-1041
  30. Gupta, S., (2000). Hepatic polyploidy and liver growth control. Semin. Cancer Biol.,10:161-171.
  31. Harrison, D.E.,Zhong, R.K., Jordan, C.T.,Lemischka, I.R. and Astle, C.M., (1997). Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term. Exp. Hematol.,25:293.
  32. Hisamori, S.,Tabata, C.,Kadokawa, Y.,Okoshi, K.,Tabata, R., Mori, A.,Nagayama, S., Watanabe, G., Kubo, H. and Sakai, Y., (2008).All-trans-retinoic acid ameliorates carbon tetrachloride-induced liver fibrosis in mice through modulating cytokine production. Liver Int.,28: 1217-1225.
  33. Hixon, Obejero-Paz, D.,Muro-Cacho, C., Wagner, M.W.,Millie,E.,Nagy, J.,Hassold, T.J. andGualberto, A., (2000). Cks1 mediates vascular smooth muscle cell olyploidization. J. Biol. Chem.,275:40434-40442.
  34. Huang, H. and Auerbach, R., (1993). Identification and characterization of hematopoietic stem cells from the yolk sac of the early mouse embryo. Proc. Natl. Acad. Sci. U S A.,90:10-110.
  35. Huang, J., Zhou, S., Ping, J., Pan, X., Liang, G.,Xu, D., Kou, H.,Bao, C. and Wang, H., (2012).Role of p53-dependent placental apoptosis in the reproductive and developmental toxicities of caffeine in rodents. Clin. Exp. Pharmacol. Physiol.,39:357-363.
  36. Ikeda, G.J.,Sapienza, P.P.,McGinnis, M.L.,Bragg, L.E.,Walsh, J. J. andCollins, T.F., (1982). Blood levels of caffeine and results of fetal examination after oral administration of caffeine to pregnant rats.J. Appl. Toxicol.,2:307-314.
  37. Kasibhatla, S., Amarante-Mendes, G.P., Finucane, D., Brunner, T., Bossy-Wetzel, E. and Green, D.R., (2006). Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis. CSH Protoc.,1: 4493.
  38. Khanna, N.N. andSomani, S.M., (1984). Maternal coffee drinking and unusually high concentrations of caffeine in the newborn. J. Toxicol. Clin. Toxicol.,22: 473-483.
  39. Kimmel, C.A.,Kimmel, G.L.,White,C.G.,Grafton, T.F.,Young, J.F. andNelson, C.J., (1984). Blood flow changes and conceptal development in pregnant rats in response to caffeine. Fundam. Appl. Toxicol., 4:240-247.
  40. Ledda-Columbano, G.M.,Pibiri, M.,Molotzu, F.,Cossu, C.,Sanna, L. Simbula, G.,Perra, A and Columbano, A., (2004).Induction of hepatocyte proliferation by retinoic acid. Carcinogen.,25: 2061-2066.
  41. Levi-Schaffer, F. and Touitou, E., (1991). Xanthines inhibit 3T3 fibroblast proliferation, Skin Pharmacol., 4: 286-290.
  42. Lou, Y.R., Lu, Y.P.,Xie, J.G., Huang, M.T. and Conney, A.H., (1999). Effects of oral dministration of tea, decaffeinated tea, and caffeine on the formation and growth of tumors in high-risk skh-1 mice previously treated with ultraviolet b light.Nutr. Cancer, 33:146-153.
  43. Marletaz, F., Holland, L.Z.,Laudet, V. and Schubert, M., (2006). Retinoic acid signaling and the evolution of chordates. Int. J. Biol. Sci., 2:38-47.
  44. Massey, L.K. and Hollingbery,P.W., (1988). Acute effects of dietary caffeine and sucrose on urinary mineral excretion of healthy adolescents. Nutrit. Research, 8:1005-1012.
  45. Massey, L.K. andWise, K.J., (1984). The effect of dietary caffeine on urinary excretion of calcium,magnesium, sodium and potassium in healthy young females. Nutrit. Research, 4: 43-50.
  46. Medvinsky, A. and Dzierzak, E., (1996). Definitive hematopoiesis is autonomously initiated by the AGM region. Cell.,86:897-906.
  47. Melhus, H.,Michaelsson, K.,Kindmark, H., Bergstrom, R., Holmberg, L.,Mallmin, H,Wolk, A, and Ljunghall, S., (1998). Excessive Dietary Intake of Vitamin A Is Associated with Reduced Bone Mineral Density and Increased Risk for Hip Fracture.Ann. Intern. Med.,129: 770-778.
  48. Momoi, N.,Tinney, J.P., Liu, L.J.,Elshershari, H., Hoffmann, P.J.,Ralphe, J.C., Keller, B.B. and Tobita K., (2008). Modest maternal caffeine exposure affects developing embryonic cardiovascular function and growth. Am. J. Physiol. Heart Circ. Physiol.,294:2248-2256.
  49. Mongan, N.P. and Gudas, L.J., (2007). Diverse actions of retinoid receptors in cancer prevention and treatment. Differentiat., 75:853-870.
  50. Morck, T.A., Lynch, S.R. and Cook, J.D., (1983). Inhibition of food iron absorption by coffee. The Amer. J. of Clinical Nutrit., 37: 416-420.
  51. Morriss-Kay, G.M. and Ward, S.J., (1999). Retinoids and mammalian development. Int. Rev. Cytol., 188:73-131.
  52. Muller, A.M.,Medvinsky, A.,Stouboulis, J.,Grosveld, F. and Dzierzak, E., (1994). Development of hematopoietic stem cells activity in the mouse embryo. Immunity, 1:291-301.
  53. Nomura, M., Ichimatsu, D., Moritani, S.,Koyama,I.,Dong Z., Yokogawa, K.,Miyamoto, K., (2005).Inhibition of epidermal growth factor-induced cell transformation and Akt activation by caffeine. Mol. Carcinog., 44:67-76.
  54. Otto, S.P., and Whitton, J., (2000). Polyploid incidence and evolution. Annu. Rev. Genet., 34: 401-437.
  55. Patel, S.R.,Hartwig, J.H and Italiano, J.E., (2005). The biogenesis of platelets from megakaryocytes proplatelets. J. Clinical Investigation, 115: 3348-3354.
  56. Pendaries, V.,Verrecchia, F., Michel, S.and Mauviel, A., (2003). Retinoic acid receptors interfere with the TGF-b/Smad signaling pathway in a ligand-specific manner. Oncogen, 22: 8212-8220.
  57. Rasmussen, B.B., Brix, T.H.,Kyvik, K.O. and Brosen, K., (2002). The interindividual differences in the 3-demethylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors. Pharmacogenet.,12:473- 478.
  58. Rebel, V.I., Miller, C.L.,Thornbury, G.R.,Drogowska, W.H., Eaves,C.J. and Lansdorp, P. M., (1996). A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Exp. Hematol.,24: 638.
  59. Richardson, J.L.,Shivdasani, R.A., Boers, C.,Hartwig, J.H. and Italiano, J.E., (2005). Mechanism of organelle transport and capture along plateletsduring platelet production. Blood, 106: 4066-4075.
  60. Rochette-Egly, C., and Chambon, P., (2001). F9 embryocarcinoma cells: a cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling. Histol. Histopathol.,16:909-922.
  61. Ruhl, C.E. and Everhart, J.E., (2005). Coffee and caffeine consumption reduce the risk of elevated serum alanine aminotransferase activity in the United States. Gastroenterol., 128:24-32.
  62. Seglen, P.O., (1997). DNA ploidy and autophagic protein degradation as determinants of hepatocellular growth and survival. Cell Biol. Toxicol., 13:301-315.
  63. Shah, R.S.,Rajalkshmi, R.,Bhat, R.V.,Hazra, M.N., Patel, B.C.,Swamy, N.B. and Patel, P.V., (1987).Liver stores of vitamin A in human fetuses in relation to gestational age, fetal size and maternal nutritional status. Br. J. Nutr., 58:181-189.
  64. Sigal, S.H.,Rajvanshi, P.,Gorla, G.R.,Sokhi, R.P.,Saxena, R.,GebhardJr, D.R., Reid, L.M., Gupta, S., (1999). Partial hepatectomyinduced polyploidy attenuates hepatocyte replication and activates cell aging events. Am. J. Physiol., 276: 1260-1272.
  65. Stromland, K., Miller, M.,Cook, C., (1991). Ocular teratology. Surv. Ophthalmol., 5:429- 446.
  66. Storchova, Z., and Pellman, D., (2004). From polyploidy to aneuploidy, genome instability and cancer. Nat. Rev. Mol. Cell Biol., 5:45-54.
  67. Sulik, K.K., Dehart, D.B., Rogers, J.M. and Chernoff, N., (1995). Teratogenicity of low doses of all-trans retinoic acid in presomite mouse embryos. Teratol.,51:398-403.
  68. Tanaka,K., Tokunaga,S.,Kono, S., Tokudome, S.,Akamatsu, T. and Moriyama, T., (199
    Get the App