Morphological Study of The Effects of Caffeine Beverages Can Cause Birth Defects on Swiss White Mice Embryos

The current study was conducted to identify the effects of different doses of Arabic coffee, black tea and Coca-Cola syrup on the possibility of birth defects in Swiss white mice embryos. (25) pregnant female mice were used in the study, divided into five experimental groups, including the control group dosed with distilled water, and four experimental groups were dosed with concentrations (4000-2500-2000-1000) mg/kg body weight for Arabic coffee and black tea and (2,3,4,6 ml/kg) for Coca-Cola syrup; once a day, from the seventh day until the eighteenth day of pregnancy. The results of the current study showed cases of abortion and fetal death at varying degrees and many abnormalities in white mouse embryos due to the different concentrations of the beverages used were characterized by the appearance of elongated and enlarged embryos. The elongation and deformation of the fetus, And the embryos mutated. Small embryos folded like a ball and curved in the shape of letter C, and the severity of the deformities increased by the appearance of mutated embryos resembling the body of a fish and deformed embryos similar to a water mermaid and not fully developed and the appearance of the head in the shape of a blurred triangle, as well as forward with a pointed end resembling a bird's beak, and brain deformation through depression in the posterior cranial region of the skull, and for the first time a new deformation is a cerebral Meningomyelocele in the form of a cystic


Introduction
In most countries around the world, social beverages are now one of the most popular drinks, but how does it look when consumed in large amounts during pregnancy, causing miscarriages, damages, deformities, and birth defects (AL-Rasheedi,2009). Currently, commercial advertisements in various media outlets are widely observed by companies and factories that produce such drinks strikingly in order to highlight their nutritional importance, which has caused a large number of people to over-consume them without any reference to the negative effects of unusually using them. Caffeine increases the amount of energy in the body and stimulates the muscles to oxidize fat and protein (Yen et al.,2005). Caffeine crosses the placental wall to the fetus and increases the likelihood of spontaneous abortion, as well as a lack of fetal growth and congenital malformations within the womb, which sometimes leads to fetal loss and death (Weng and Odouli,2008). There are very few studies on the effect of caffeine beverages on fetus levels and fetal development, especially during pregnancy (Bech et al.,2005). Three types of coffee beverages, Arabic coffee, black tea and Coca-Cola syrup, have been selected in the current study. Many studies have focused on the benefits and importance of these drinks in the first place (Greenberg et al.,2006). Very small studies (Bech et al.,2007).

Scientific Journal for the Faculty of Science-Sirte University (SJFSSU)
This matter has stopped embryologists from paying attention to studying the various deformities that occur during the embryonic development process, their types, causes and mechanisms of formation under the name of Congenital malformations. Teratology (Ashmead,2003), that many pregnant women frequently drink coffee and tea and over-drink various soft drinks without concern. The health damage caused by these high doses of used drinks. The fact that women who drank four or more cups of coffee increased the function of the placenta and thus the death of the fetus (CARE,2008) (Bierrnacki et al.,2000). Given the lack of studies to clarify the effects of caffeine beverages on birth defects, this study was conducted. Therefore, the aim of study to identify the teratogenic effects of caffeinated beverages on the fetuses of pregnant albino mice

Materials and Methods
Mice were selected at (9-12) weeks of age for both sexes, their average weight was (2 ± 23) g, and the mice were healthy and healthy. The mice were placed under standard laboratory conditions throughout the study period. Females ready for fertilization have been placed with males at the rate of one male with three females in each cage. And the fertilization was confirmed by the observation of the vaginal plug in the morning of the following day. The date of mating is day zero of pregnancy, and the next day is the first day of mating (10).

Drinks Used
I used Arabic coffee that was bought from the local market. The aqueous solution was prepared for coffee by dissolving (1-4) grams of coffee in (50) ml of distilled water, boiling the solution for two minutes and stirring continuously to simulate what is being prepared and used by humans, use caffeine black tea purchased from the local market and prepare the aqueous solution of black tea by boiling (1-4) grams of black tea in (50) ml of distilled water And boiling the solution for three minutes, as a simulation of what is prepared and used by humans, as for the Coca-Cola drink, is to use a ready-made drink packed in 330 ml metal cans.

Concentrations used
(25) pregnant female mice, which were divided into five groups and included in the control group and were dosed with distilled water, and four experimental groups were dosed with an aqueous solution of Arabica coffee at an increase in body weight (4000,2500,2000,1000) mg/kg In comparison to the study's median lethal dose (LD50) of (5 g / kg body weight) orally (43.5 mg caffeine / kg) which was reached through this study, Black tea with progressive concentrations (4000,2500,2000,1000) mg/kg body weight in comparison to (LD50) and its amount (6 g/kg body weight) orally (39 mg caffeine / kg), which was achieved in this study and Coca-Cola syrup, the concentrations used are (2 ml, 3 ml, 4 ml, 6 ml) In comparison to the median lethal dose (LD50) of (200 ml/kg body weight) orally (0.86 mg caffeine/kg) obtained in this research, daily and once orally with a gavage absorbent needle starting on the seventh day of pregnancy (Organogenesis stage) until the 18 th day of pregnancy (The day of the autopsy).

Results
The results of the study showed the incidence of partial and total abortion to varying degrees. The results of the current study showed miscarriage and fetal death at varying degrees in experimental groups dosed with the drinks used, as observed when aqueous solution of Arabic coffee was administered at a concentration of 4000 mg/kg body weight; the rate of abortion was 33 per cent and the death of some pregnant mice on the 15th day of pregnancy. The experimental group fed an aqueous solution of black tea at a concentration of 4000 mg/kg of body weight had a 29 percent miscarriage rate. On the 14th day of pregnancy, several pregnant mice had partial miscarriages and died.used, the current study found several abnormalities in white mouseembryos due to the various concentrations of the drinks use. In terms of the findings of the study of abnormalities in embryos at various doses of the drinks used, the current study found several abnormalities in white mouse embryos due to the various concentrations of the drinks used. The embryos were elongated and enlarged, the fetus was elongated and deformed, and the embryos were mutated (Figures b, c). Indicating that regular dosing of the aqueous solution of black tea caused deformities in pregnant mouse embryos and it curved in the shape of the letter C, and the emergence of mutated embryos that resemble the body of a fish (Figure d).   Results also showed small, deformed, mutated and incomplete embryos when dosed at a concentration of 6 ml/kg of body weight of Coca-Cola syrup ,Results also showed that the head was deformed by 75 %-85.5 % when dosed with an aqueous solution of Arabic coffee, and that the appearance of a cleft lip (Figure g (1), congestion (CON) and enlargement of the head (2), deformation of nasal facial features (3), exophthalmos (4), and bleeding (BL) in the brain area (5) are all seen in this lateral view of a tiny white mouse fetus. Aqueous black tea solution at a concentration of 1000 mg / kg of body weight was given.
In addition to the formation of a head with a pointed end that resembles the beak of a bird, the brain is deformed by depression in the posterior cranial region of the skull (Figure k). Whereas, the severity of phenotypic head anomalies, when taken with Coca-Cola syrup, increased to a higher rate of 88% and was represented for the first time by the development of a new malformation, which is a cerebral meningocele in the form of a cystic tumor in the posterior brain region (Figure m)

Discussion
The study's findings revealed varied degrees of partial and entire abortion. The present study's findings revealed varying degrees of miscarriage and fetal mortality in experimental groups given the drinks. Excessive coffee intake and its caffeine content, which occurred at higher rates than other beverages, resulted in a doubling of the risk to the fetus within the uterus and a rise in the incidence of spontaneous and complete abortion during pregnancy (Hey ,2007), which could be due to the increase in contractions induced by stimuli containing caffeine. (Ullman et al., 1992).
The current study discovered several abnormalities in white mouse embryos due to various concentrations of the drinks used. The embryos were elongated and enlarged, the fetus was elongated and deformed, and the embryos were mutated, according to the findings of the study of abnormalities in embryos at various doses of the drinks used these findings backed up what was previously stated (Ormerod ,2001). Regular dosing of an aqueous solution of black tea produced abnormalities in pregnant mouse embryos, according to the findings. It curled in the shape of the letter C, as well. And the appearance of mutant embryos that resemble a fish's body and the reason may be attributed to the amount of caffeine in excessive doses of black tea, as indicated by the emergence of mutated embryos that resemble the body of (Grosso and Bracken,2005). When dosed with Coca-Cola syrup, the consequence was the emergence of malformed embryos that resembled a water mermaid, and distorted metamorphosed embryos. This may be due to the effect of Coca-Cola syrup, which contains caffeine in inhibiting growth during the stage of organogenesis, causing a lack of growth (Jana et al.,1994), or perhaps explaining the reason for increased fetal resorption and stunted growth of the fetus during the period of organogenesis on the sixth day of pregnancy (Al-Mamouri,2001), When Coca-Cola syrup was dosed at a concentration of 6 ml/kg of body weight, the results revealed tiny, malformed, mutated, and incomplete embryos and the cause may be attributed to damage to developing placenta that begins to function on the ninth day of pregnancy. Decreases in the exchange of nutrients between the fetus and the mother and reduces the process of protein building in the embryos, leading to delayed growth, low weights and the appearance of small embryos ( Padmanabhan et al.,1981).
When given an aqueous solution of Arabic coffee, the head was deformed by 75 percent to 85.5 percent, and the appearance of a cleft lip was also seen this was similar to that indicated by (Matijasevich et al.,2005). The head appears to be in the shape of a blurring triangle and this combined with what the researcher pointed out (Hammoudi, 2005). When given an aqueous solution of black tea, it causes congestion and growth of the head in relation to the rest of the body, distortion of the facial characteristics of the nose, bulging eyes, and hemorrhage in the brain region, as well as deformation of the skull (70 %). The brain is deformed by depression in the posterior cranial region of the skull, in addition to the creation of a head with a pointed end that resembles a bird's beak, and that's in line with what reported in Fazal and Jalali, 2002. When combined with Coca-Cola syrup, the severity of phenotypic head malformations grew to an 88 percent rate, which was represented for the first time by the development of a novel malformation, a cerebral meningocele in the shape of a cystic tumor in the posterior brain region this perhaps the explanation is due to the combination of maternal consumption of Caffeine-containing stimuli and the risk of developing brain myeloma (Rogwei et al., 2011).

Conclusion
The study showed that caffeinated drinks have congenital anomalies on the fetus and the pregnant mother should be careful when consuming.