Aloe Vera Protects Fluoride Induced Teratogenic Effects During Pre- and Postnatal Development in Mice

Pregnancy and feto-gestational toxicities on exposure to uoride (F) and its possible amelioration on co-administration with Aloe-vera were studied in pregnant Swiss albino mice. Once the conrmed pregnancy was tested, animals were equally divided into four groups and were given following treatment. Group I was given no treatment and served as Control, Group II and III were administered sodium uoride, 100 and 300 ppm respectively while group IV was co-administered with sodium uoride, 300 ppm and Aloe-vera (300mg/kg) daily for 14 days prior to gestation and continued till the 18 th day of gestation. Animals were sacriced `on the 19 th day of gestation for prenatal observations. Maternal body weight, the gravid uterine weight, number of corpora lutea in both the ovaries, number of implantations and resorptions, number of live (mature and immature ) male and female fetuses as well as number of dead fetuses were examined in each dam. The treatment continued in another set of animals till the completion of weaning period to observe postnatal changes due to test substances on the mother and pups. Sodium uoride treated animals showed morphometric and skeletal changes which were more pronounced in the high dose group showing signicantly decreased body weight gain in pregnant mothers; and dead/immature fetuses. Morphometric changes included open eyelids, limb defects, wrinkles on whole body, anophthalmias, pulmonary edema, enlarged esophagus and decreased body weight of fetuses and pups. Alizarin prepared skeletal structures of fetuses of such female mice showed delayed ossication or bending in number of bones of skull, thoracic and limb regions. However, concomitant exposure to Sodium Fluoride and Aloe-vera treated animals, there was a marked improvement in all the prenatal and postnatal variables. The study suggests that Sodium uoride at the high concentrations may be teratogenic while co-administration of Aloe-vera during uoride exposure might be benecial in reducing these toxic effects. We thus recommend use of aloe vera as preventive agent or as a complimentary agent during uoride treatment.


Introduction
High Fluoride concentration in groundwater is a common problem in number of countries globally and also a serious environmental health issue in India and in particularly the state of Rajasthan, India (Perumal et al., 2013). Increased uoride concentration in soil and water is known to cause wide array of toxic effects and health issues in human and most common among them are tooth defects, skeletal and visceral malformations, brain injury etc. (Jing et.al, 2004). Fluoride induced teratogenic effects are less studied. Developing animals are more susceptible to uoride as it crosses the placental barriers and enter the fetus causing malformations. Fluoride concentration has been reported in saliva, sweat and milk though, tough at low concentration (Narayanaswami and Basha, 2010).
Infant mortality due to congenital malformation is now ranked second most important cause of death in developing countries. (Madu, 2015). A common teratogen is known to increases the incidence of structural and/or functional abnormalities in offspring, if consumed in higher concentration by parents before conception, the female during its pregnancy period or if it directly reaches the developing organism through exposure. Various stages of the development of the fetus are also a key factor that determine the susceptibility to teratogenic agent because a speci c damage occurs readily during the organogenesis phase of embryonic development. (Inamdar et al., 2012).
Antioxidants and chelators are commonly used in minimizing uoride induced toxic effects and/or stress, besides documenting the function of minerals and vitamins speci cally A, D, C, E against uoride toxicity (Dharani et al., 2011). Herbal or Plant extracts including their constituents have recently been reported to be of immense bene t as preventive agents, or being used as complimentary or as adjuvant in reducing metal induced toxic effects. With minimal side effects, the medicinal value of these extracts have assumed an important dimension in the past few decades owing largely due to being a rich source of antioxidants that combat oxidative stress through their redox active secondary metabolites and the rising concerns about the side effects of synthetic drugs (Flora et al., The test substance Sodium Fluoride (HiMedia Laboratories Pvt. Ltd.) was used after making a solution in normal potable water (Fluoride content in the water was in the range of 1 -2 ppm).

Plant
Mature, healthy and fresh leaves of Aloe-vera were washed with fresh water, cut transversely into pieces and thick epidermis was selectively removed. The solid gel in the center of the leaf was taken after homogenizing the leaves (Madhusudhan et al., 2009). The water extract of Aloe-vera gel was prepared mechanically and kept in a clean and air-tight bottle. Each time a dose was prepared by taking 0.7g of leaf gel in 10 ml water, which was properly ground.

Teratological evaluation
Mice were used for studying the teratogenic and anti-teratogenic evaluation. They were divided into 4 groups of 8 mice in each and kept in an air-conditioned room with 12 h of daylight and 12 h of night. Estrous cycle was monitored for all animals before mating. Two females with one male animal per cage were kept overnight. Next day, in the early hours, the females were examined for signs of mating by ushing the vagina with saline using a pipette, and then examining the obtained solution microscopically. Mating was con rmed by the presence of sperm cell/plugs. The presence of spermatozoa was recorded as day zero of pregnancy, daily increase in weight further con rmed pregnancy (Hussein and Mahmoud, 2013).

Experimental design
Animals were randomly divided into four groups with 8 pregnant mice in control group and 24 pregnant mice in treatment groups. Group 1 was given no treatment and served as Control; Group 2 was administered a dose of 100 ppm NaF; Group 3 was administered 300 ppm NaF while, Group 4 was administered 300 ppm Sodium uoride along-with Aloe-vera. Doses were selected based on its concentration present in many polluted areas and what has been reported in the literature. Doses were given through drinking water ad libitum from 14 days prior to mating and continued till 18 th day of gestation.
Postpartum procedure -The animals were autopsied one day after the completion of dose. Following laparotomy, the uterus was exteriorized and the number and location of fetuses, resorption, live and dead fetuses and number of corpora lutea in each ovary were noted, then their weight and length were measured.
Living fetuses were distinguished from the dead by re ex movement initiated by gently touching the fetus. Live fetuses were weighed and examined by a hand lens, to observe any malformations. Individual fetuses were examined carefully for external anomalies, then 50% fetuses were examined for skeletal malformation; rest 50% fetuses were kept in Bouin's solution for soft tissue examination.
Data analysis -Statistical analysis was performed on parametric values. The values have been expressed as Standard Error of mean (S.E). Maternal weight gain, fetal weight, fetal length and neonatal weights was analyzed statistically using student's t-test.

Results And Observations
Maternal toxicity 20% mortality was observed during the treatment with 300 ppm dose of Sodium uoride. The general body weight gain in mother during the three phases viz: 14 days prior to mating, gestation and lactation periods was signi cantly reduced at 300 ppm of Sodium uoride relative to controls ;while the maternal body weight in Sodium uoride+ Aloe vera group increased in all three phases compared to 300 ppm Sodium uoride exposed group . No mortality was observed in any of the females in this group. Water and food consumption decreased signi cantly in the animals exposed to 300 ppm Sodium uoride.

Fetal toxicity
The prenatal teratological observations in were recorded after performing autopsy on 19 th day of gestation. The rst parameter which was assessed was the number of corpora lutea counts in animal under study. The average number of corpora lutea in all the animals were found to be insigni cantly different from the values recorded in control. In total 77 live fetuses were observed from all the eight animals in the control group; the number of fetuses obtained from all the eight animals in animals exposed to 100 ppm NaF were reduced (non-signi cant) compared to the number noted in control group. The value were signi cantly decreased to 53 in 300 ppm NaF exposed group indicating signi cant toxic effects during the developmental phase of the animals. The increase in litter size in NaF (100 ppm) + Aloe vera group was close to litter size in control indicating positive effects of Aloe vera co-administration. Table 1 indicates signi cant toxic effects of 300 ppm NaF where out of 67 implantation sites, only 53 live fetuses were counted but this included 45 mature fetuses while 8 fetuses were found to be immature (Figure I) (Eii); Number of resorptions and dead fetuses were high in this group of animals. Aloe vera, in view of reported antioxidant effects and other medicinal properties provided signi cant protective value which were evident from the elevated number of mature fetuses as 65 with just 2 immature fetuses out of the total 67 live fetuses . The resorptions and other parameters were also not signi cantly on the higher side. The average fetal body weight was recorded at 1.162 ± 0.045 g in control, which was signi cantly decreased in the 300 ppm NaF (0.716±0.054). In the other experimental groups there was no signi cant decrease in average fetal body weight and the average placental weight compared to control.. When 50% fetuses of each female were sectioned through head, thorax and abdomen; the sections showed normal structure of various organs in the control group However, in 300 ppm NaF exposed group, sections in soft tissue depicted anophthalmias, pulmonary edema (enlarged air spaces in lungs) and enlarged esophagus ( Figure 2). Structures seemed normal in female fetus which were co-administered Aloe vera with NaF.
The alizarin staining in remaining 50% of the fetus which were processed to observe defects of the skeletal system indicates normal ossi cation in the skull bones of the control group. The ribs, Sterne brae, vertebrae and limb bones did not show any malformations, the observations were similar in the Aloe vera treated group. Sodium uoride (300 ppm) fetuses showed Craniofacial disorganization, reduced ossi cation in skull bones, and widened cranial sutures ( Figure 3). The rib defects like wide gaps between ribs, and absence of ribs were not found in low dose of NaF group, whereas in the treated group of NaF (300 ppm) a signi cant number of fetuses had skeletal abnormalities. In this group highly reduced skull ossi cation, widened cranial sutures, ribs defects like partially ossi ed ribs, wide gap between ribs, wavy ribs, and reduced Sterne brae, partial ossi cation of metacarpals (Mc) and metatarsals (Mt), absence of 13th rib were clearly observed ( Figure 4).
The postnatal observations of each mother and pups was recorded following parturition till 21 days of weaning period. Table 2 shows consistently decreased in the average number of alive pups in NaF (HD) treated group from day 1 through 21 days of weaning period compared to control. (Control 8.40 ± 0.51 on day 1 and 7.60 ± 0.24 on day 21, NaF (300 ppm) -7.00 ± 0.45 on day 1 and 3.60 ± 0.51*** on day 21) while in NaF (100 ppm) and NaF (300 ppm) + Aloe-vera group there was insigni cant difference in number of alive pups compared to control. The trend was the same in viability index and weaning index also. Average pup weight which is also expressed as Growth index was calculated for different postnatal days ndings. It was not much affected in NaF (300 ppm) and NaF(300 ppm)+Aloe-vera, compared to control but in NaF (300 ppm) it got signi cantly affected.

Discussion
Epidemiological studies conducted in the areas with high levels of naturally uoridated water shows increase in birth defects placing uoride into the same category as toxic metals like lead, mercury, methyl mercury etc. (Guth et al., 2020). Exposure to high uoride concentration in drinking water during in utero development may result in skeletal uorosis which becomes evident in childhood. Adverse effects on reproduction have been reported in animals exposed to 100 ppm or greater concentrations of uoride in their diet or through drinking water. Number of previously reported animal studies suggest that exposure to low concentrations of uoride do not result in impaired fertility and reproductive capabilities. The present study is an effort to give reason to and correlate the ndings obtained through experiments with the various mechanisms by which Sodium uoride reaches the tissues of the body and in uences the normal physiological function of the body; thereby causing developmental defects during the pregnancy period. The study also attempts to nd the possible reason / mechanism for averting the teratogenic effect of uoride by using Aloe vera gel which is taken as a supplement during the early development of a fetus Animal studies have reported that uoride crosses the placenta and gets incorporated in fetal issues; increasing its concentration in fetal blood two folds. Fetus is not protected against uoride that circulates in the maternal blood. The study further reports that uoride gets transferred to the infant through problems, in those geographical areas where Fluoride concentration is high. The high accumulation of NaF in brain tissues after passing through placenta and mother's milk to its newborns has also been reported (Sun et al., 2018) affecting the prenatal and postnatal development of mice. These effects were more signi cant following exposure to a high concentration of 300ppm of NaF causing teratogenic effects which got reduced by taking nutritive plant products like Quinoa during the whole experimental period (Choudhary and Mathur, 2020). Several authors reviewed a dose dependent relationship between NaF exposure and embryonic growth retardation in mice and have suggested protective effects of vitamins and antioxidants to ameliorate these teratogenic effects besides cautioning against excessive intake of Fluoride in the drinking water .
Acemannan, an acetylated glucomannan that makes up the major active component of the mucilaginous Aloe vera possess multifunctional properties and therefore acts as a bioactive molecule, exerting an immunostimulatory effect by activating macrophages, (Callaghan et al., 2012) and possessing wound healing actions. (Osefo et al., 2009, Hull andBeale, 1985). It also stimulates broblast proliferation, (Lee et al., 2017) tissue regeneration (Rauws et al, 1990) and bone marrow stromal cell proliferation and differentiation in vitro. (Hull and Beale, 1985). Thus, it is an interesting preventive agent for tissue repair. Aloe vera sap has also been found to have favorable effects on estrogen synthesis due to its phytoestrogen components such as beta sitosterol that can increase the estrogen level, thus bringing hormones to support normal development of fetus (Atik et al., 2019). More detailed studies are required to validate and explain the action mechanism of acemannan in bone building. .
It is also an established fact that nutritional supplements such as vitamins and antioxidants prevent the toxicity that is induced by uoride because these supplements reduce the oxidative stress, decreases the lipid peroxidation, and augments the activity of antioxidant enzymes. (Elshama et al., 2018). One of the strategies to protect the body from oxidative injury and to prevent from many disorders is to increase the levels of antioxidant enzymes by increasing the dietary intake of supplements rich in antioxidant enzymes (Godoy et al., 2018). Aloe vera has very strong antioxidant nutrients. Glutathione peroxide activity, superoxide dismutase enzymes, and a phenolic antioxidant have been reported to be present in Aloe vera gel, which may be responsible for these antioxidant effects (Atik et al, 2019). Aloe vera also has unique ability to improve the absorption of natural antioxidants like vitamin C and vitamin E that in turn enhances antioxidant status to alleviate uoride induced ROS (Vinson et al., 2005).Vitamin E which is an essential component of Aloe vera must have exerted antioxidant effects by scavenging lipid peroxyl radicals that are free radicals in vivo as well as in vitro (Niki, 2014). Ayurveda also mentions that Aloe-vera can been taken as a Naimittika Rasayanas during pregnancy; because, it being rich in nutritional contents and iron supplements that are very essential to promote the health of the pregnant women and facilitates full growth and development of progeny in the womb (Shastry,1999;Singh, 2000).
The ndings in the present study suggest the multifaceted qualities of Aloe vera. The present study also suggests marked e cacy of Aloe vera in reducing uoride induced teratogenic effects. However, still more detailed studies are required using variable doses of Aloe vera post ouride exposure

Conclusion
The present study suggest that pregnant mice exposed to Sodium uoride may be at the risk of congenital malformations. Aloe vera, a promising herbal product with its various clinical applications in medicine could be useful if co-administered during uoride to minimize uoride induced toxic effects including teratogenicity.

Declarations
Ethics approval: Institutional Ethics committee gave its approval to work on the given experimental model (vide Sl 2-18-5-18).

Consent to Participate:
Authors have given consent to participate in the given research work Consent to publish: Authors have given consent to publish the work which includes gures and tables in between the text in the journal.

Con ict of interest
The authors declare that there is no con ict of interest related to this study.

Availability of data and materials : Not applicable
Author's funding The author(s) received no speci c funding for this work.
Authors' contribution PM conceptualized the experimental plan, supervised the experiment, prepared the rst draft of the manuscript and the main author., SC performed the experiment, collected data and analyzed. PB prepared the nal draft, participated in the discussion during the experiment, and overall supervision of the experiments. Values are mean ± SD; ***p<0.001, **p<0.01, *p< 0.05 compared to control as evaluated by Student's 't' test using IBM SPSS statistics 22 software.