Assessment of Protective Role of Quinoa against Sodium Fluoride Induced Skeletal Deformities in Mice Fetuses

 

Shilpa Choudhary, Priyanka Mathur

Department of Life Sciences, IIS (deemed to be University), Gurukul Marg, SFS, Mansarovar, Jaipur - 302020, Rajasthan, India.

 

ABSTRACT:

Rajasthan has the highest number of habitations where groundwater contains fluoride specifically in the region of Thar Desert. Infact, Pan India is affected by fluoride problem, but Rajasthan is the only state where almost all the districts are affected by high fluoride. High Fluoride concentration in drinking water has been shown to cause skeletal and dental abnormalities, besides others such as osteosclerosis. It has been shown through medical research that almost all the vital systems and organs in the body including thyroid, kidney, cardiovascular, gastrointestinal, endocrine, neurological, reproductive, developmental, molecular level, immunity etc. gets affected by concentration of Fluoride when it exceeds WHO standard values. This finding facilitated us to go in further and find the effect of Sodium Fluoride (NaF) when given to pregnant mice through drinking water at various fetal developmental stages with focus on study of developmental pattern of the skeletal system of growing fetuses followed by a protective role of phytomaterials against it. In this study Quinoa seed, were selected because of its high nutritive value and latest findings indicating its antiteratogenic nature. The study was planned in such a way that females in question were subjected to fluoride treatment prior to mating, during pregnancy and post partum period when weaning process is on. The observations clearly indicates that post Sodium fluoride treatment there was a reduction in size and general body weight of fetuses. Besides this, there was an increase in the number of immature fetuses. Various skeletal anomalies were also observed, such as partially ossified ribs, reduced skull ossification, reduced sternebrae etc. Chenopodium Quinoa, also called Superfood due to its high quality of protein and wide range of minerals and vitamins was used for its protective role as mentioned earlier.In  this study NaF along with Quinoa seeds were administered to treated animals. These animals showed improvement in maternal weight, increase in body weight and size of fetuses as well as bone ossification. These findings suggest that Quinoa seeds possibly have a protective effect against Sodium Fluoride induced teratogenicity.

 

 

INTRODUCTION:

Fluoride is found abundantly and naturally in soil, water and foods and human body essentially requires it. The main source of human intake to fluoride is drinking water, appropriate amount of which prevents dental caries, promotes calcium sediment in the bone, increase bone growth and maintains bone health.¹ If in excess concentration, it causes skeletal and dental abnormalities.

 

Fluoride readily crosses the placenta and also reaches the maternal milk affecting the bones of fetuses.² It even accumulates in bones and may affect their structure and strength. The effect of prenatal Fluoride exposure are long lasting and persist until adulthood.³ Fluorosis, a progressive degenerative disorder from excessive intake of Fluoride causes damage of soft tissue viz: brain, liver, kidney and spinal cord,  thus developing animals are more sensitive to Fluoride compared to adults.⁴ The skeletal system of fetuses is especially susceptible to fluoride due to free radical oxidative damage. The findings of Butterfield that further gives strong opinion.⁵

 

The present study was planned to use a plant product that has ameliorating effects on Fluoride induced teratogenicity due to ample supply of nutritional sources of antioxidants in it, and wide range of proteins, vitamins, and minerals. Quinoa, a member of family Chenopodiacea,  as rightly said a super food is a very good source of complete proteins, minerals, vitamins, fibers, unsaturated fatty acids and antioxidants.⁶ Previous studies observed that bioactive compound of Quinoa could change antioxidant status in the organism by preventing oxidative stress and also helps reduce the risk of various chronic diseases thus active as anti-inflammatory, immunomodulatory and anticarcinogenic plant product.^7,8 However, its protective role against teratogenicity has not been studied much, hence it attracted the attention  and the study was carried forward to test Quinoa for its protective role against teratogenicity caused by Fluoride.

 

MATERIAL AND METHOD:

Test Animal:

As mammalian model, Swiss albino female mice were used, which were obtained from the registered animal house of the University, Jaipur. The females were housed in plastic cages with sawdust to soak the excretory fluids and ejected debris. The mice had free access to water and food, they were housed in a controlled condition with a constant temperature (26ºC), and photoperiod (lights on from 7.00  to 19.00 h). All procedures were performed using protocols approved by the Animal Ethical Committee (IAEC) – (CPCSEA – Regd No: 1689/PO/Re/S/13/CPCSEA)

 

Test substance:

The test substance Sodium fluoride(HiMedia Laboratories Pvt. Ltd.) was used as a solution by dissolving in distilled water and was given ad libitum.

 

Quinoa seed powder:

The dried seeds were packaged in moisture-proof containers, and stored in  cold but were brought to normal room temperature before use. The constituents of seed were not studied in this work. The air dried seeds were powdered mechanically.⁹ After standardizing, the Quinoa seed powder was orally administered to mice at a dose of 250 mg/kg body weight.

Animal treatment:

Females were exposed toNaF in different concentrations (100ppm and 250ppm) for 14 days prior to mating and throughout gestation and lactation period. Mice were randomly divided into four groups– The 1st group served as the control and the animals were given normal tap water ad libitum. 2nd and 3rd groups were given water containing 100ppm and 250ppm of Sodium fluoride respectively; 4th group was given 250ppm of NaF and ground Quinoa seeds during gestation and lactation periods.

 

Fertility test:

Animals were observed daily from the 1st day of exposure to NaF for symptoms of toxicity. Their water consumption was measured every day and the body weight was taken on alternate days. Parous female mice from the control as well as experimental groups were housed with a sexually mature untreated males in separate cages. Every morning females were checked for presence of vaginal plug, which appear after copulation by the coagulation of a secretion of the seminal vesicles of male and this day was considered as day 0 of pregnancy. The pregnant females were weighed on alternate days throughout the experimental period. The pregnant mice were sacrificed on 18th day of gestation. During autopsy the following parameters were recorded – number of implantations, number of resorptions, number of viable fetuses and dead fetuses, number of stunted fetuses, maternal body weight and placental weight, fetal body weight and size.

 

Preparation of the fetuses for skeletal examination:

All the experimental mice were sacrificed using ether and 50% fetuses were eviscerated through a small midline incision in the anterior abdominal wall. Fetuses were skinned and rinsed in acetone for 1 day then 95 % ethyl alcohol for seven to ten days. The specimen were cleared in a solution of 1% KOH until the bones were clearly visible. They were then stained with a fresh solution of alizarin red stain in 1% KOH. Finally the specimens were transferred to solutions containing 30%, 50% and 70% glycerin respectively and stored in pure glycerin.^10,11,12 Skeletal stained fetuses were examined with the aid of a stereoscope microscope (Olympus SZ61; Olympus Corporation, Tokyo, Japan). Ossification was scored as being complete ossification (CO), partial ossification (PO), or absent ossification (A).

 

Statistical analysis:

The data was analysed with the student ‘t’ test.

 

Observations:

It was observed during the study that not even a single animal died from the control group or the first treated group (NaF 100ppm) but in the second treated group (NaF 250ppm) 20% animals showed mortality. It was also observed that in this group pregnant mice showed weakness, decreased water and food intake during the treatment period in both 100ppm and 250ppm NaF treated groups as compared with other groups. Maternal body weight during the whole gestation period (Table-1) got significantly decreased at 100ppm and 250ppm as compared to control value (control – 21.13±0.67; 100ppm – 16.63±0.37; 250ppm – 12.25±0.37) whereas control and NaF + Quinoa treated animals showed weight gain. Table-1 shows no mortality in pregnant females at low dose of NaF and Quinoa group. There was no significant difference between the NaF groups and the control group in the average number of corpora lutea, implantations and the resorptions but there was a significant difference in live fetuses, dead fetuses, fetal weight and fetal size compared to the control as depicted in Table-2.

 

Table 1 :Effect of Sodium Fluoride and Sodium Fluoride + Quinoa on maternal weight and mortality

Student’s ’t’ test *Significant difference(P<0.05)**Highly significant difference (P<0.01) ***Extremely Significant difference (P<0.001)

·       LD- Low dose

·       HD- High dose

 

Table 2: Effect of administration of NaF and Quinoa at prenatal stage on albino mice (n = 8 in each group)

Student’s ’t’ test *Significant difference(P<0.05)**Highly significant difference (P<0.01) ***ExtremelySignificant

difference (P<0.001)

   

Figure 1. Lateral view of 18 day-old mouse fetus from control group showing complete ossification of frontal (F), parietal (PT), interparietal (IP), supraoccipital (SO), exo- occipital (EO), basioccipital (BO), tympanic bulla (T), squamosal (SQ), zygomatic arch (ZA), maxilla (MX), premaxilla (PMX), mandible (MN), lacrimal (L) and nasal (N) bones using Alizarin Red stain.

 

Figure 2 and 3. Lateral view of 18 day-old mouse fetus from Sodium Fluoride  treated group showing partial ossification of parietal (PT), interparietal (IP), squamosal (SQ), and tympanic bulla (T) bones. (Alizarin red stain). Affected bones are marked in the figure

 

Figure 4. Lateral view of 18 day-old mouse fetus from Sodium Fluoride  and Sodium Fluoride + Quinoa treated group showing  partial ossification of supraoccipital(SO) bone.

 

 

Table  3: Effect of Sodium Fluoride and Sodium Fluoride + Quinoa on skeletal system of fetuses after maternal exposure during gestation period (n = 8 in each group)

 

 

Figure 5. Poster-dorsal view of 18 day-old albino mouse fetus from control group showing complete ossification of scapula (S), humerus (H), radius (D), ulna (U), metacarbal (MC), ribs (R), vertebral arches (A), centra (C), ilium (IL), ischium (IS), femur (FM), tibia (T), fibula (FB) and metatarsal (MT) ossification centers.

 

The alizarin preparations indicated that in control group the skull bones showed normal ossification. The ribs, sternebrae, vertebrae and limb bones did not show any malformations, whereas in treated mice NaF affected the ossification of bones compared to the control and NaF + Quinoa group. In NaF (100ppm) 6.66% fetuses showed reduced ossification in skull bones and widened cranial sutures.

 

Figure 6. Sodium Fluoride treated group showing defects in Ribs (A) Wavy and rudimentary ribs (B) Wide gaps between ribs (C) partially ossified ribs and rudimentary ribs (D) Fetus with reduced sternebrae. However no defects were seen in limb bones.

 

Figure 7.Sodium Fluoride + Quinoa treated group showing complete ossification of ribs, sternum, and limbs.

 

The rib defects like wide gaps between ribs, and absence of ribs were not found in 100ppm dose of NaF group, whereas in the treated group of NaF (250ppm) total 56% of fetuses had skeletal abnormality, that is increased number of malformation as compared to control, and low dose and NaF+Quinoa groups (Control – 8.57%, low dose group – 26%, High dose group – 56%, and NaF + Quinoa group – 16.66%). In low dose group of NaF 3.33%  fetuses showed ribs defects like  partially ossified ribs, rudimentary ribs and wavy ribs. In high dose group highly reduced skull ossification, widened cranial structure, ribs defects, and reduced sternbrae were clearly observed.

 

In NaF + Quinoa treated group the percentage of skeletal malformation is very low as compare to NaF group. There was only 3.33% fetuses showed reduced ossification in skull bones, widened cranial sutures and wavy ribs.

 

DISCUSSION:

In the present study, effect of fluoride on the developing stage of an embryo was studied. It was thought on the basis of research that fluoride crosses placental barrier and thereby cause harm to the developing fetus.¹³. Armstrong et al., have reported that it even reaches mother’s milk. Infact, the most sensitive stage of exposure is when developing animal is in-utero and the transplacental passage is from maternal to fetus or from the mother’s milk to the nursing baby causing deformities during the most crucial fetal life.¹⁴

 

Research shows that by interfering with calcium metabolism and enzyme mechanisms, fluorides act as direct cell poisons. It has also been reported that Fluoride combines in the stomach with hydrochloric acid to form a powerful corrosive hydrofluoric acid. Moreover Fluoride ion scan combine with serum cations after absorption, especially calcium and magnesium leading to hypocalcaemia and hypomagnesaemia.

 

In the present study two dose of NaF (low and high) and a high dose of NaF in combination with Quinoa seeds were given to three different groups of albino mice during the gestation and lactation periods. Mice were given two different doses of Fluoride 100ppm and 250ppm through drinking water ad libitum and a dose of 250 ppm of NaF + Quinoa (250mg/kg/day) orally through gavage pipe.

 

The observations of this research work showed that NaF treated mice showed decrease in weight as compared to control, while in NaF + Quinoa treated mice there was no change in weight as compared to control. A 20% mortality was observed at 250ppm of NaF group however, there was no effect on implantation; resorptions were also not seen. Number of dead fetuses in high dose of NaF treated group got increased when compared to the control group. In allow dose group, no major effect on the weight of fetuses was observed and no remarkable anomalies were observed. The treatment of NaF in this study also affected the average body weight of pups and the placental weight. This effect was more significant due to high dose of NaF. Though, study similar to this has been conducted worldwide with different results.¹⁵ On the other hand, some studies showed teratogenic effect after exposure of NaF at higher doses.¹⁶ Several authors reviewed a dose dependent relationship between NaF exposure and embryonic growth retardation in mice (and also suggested some protective effects of vitamins and antioxidants to ameliorate these teratogenic effect and also warn to excessive intake of Fluoride containing drinking water during pregnancy because of various teratogenic effect.

 

Vitamin C and D and Calcium have shown significant improvements in skeletal, clinical and biochemical parameters in children consuming water with 4.5ppm of Fluoride.¹⁷ Similar results have also been reported by Susheela and Bhatnagar¹⁸ where consumption of vitamin rich foods and antioxidants That scavenge free radicals and thereby eliminates them . Further this treatment also reduces the fluoride levels in the body.

 

The alizarin preparation of the skeletal structure of developing fetus at gestation day 18, in the present study indicated a dose dependent decrease in the ossification of the fetal bones due to administration of 100ppm and 250ppm of Fluoride. The ossification of treated fetus showed highly reduced skull ossification, wavy ribs, and reduced ossification of sternebrae when compared with control and Quinoa group.

 

The role of fluoride ions is important because even its low concentration can either inhibit or stimulate enzymatic processes, thereby can disrupt normal physiology of the body.¹⁹ Bones are more prone to its effect primarily because fluoride replaces hydroxides and itself gets deposited in bones; the chronic effect of which is called Fluorosis of the skeleton.²⁰ Such a finding of Hussain can be correlated with the finding of Brittany et al., 2006, in which people have developed bone related problems, in those geographical areas where Fluoride concentration is high.^21,22 Quinoa seeds are able to reduce the oxidative stress, which may help alleviate the free radicals generation during pathological state.

 

Quinoa is a rich source of proteins and antioxidants. Antioxidants are said to help neutralize our bodies ' free radicals, and this is supposed to boost overall health.²³ These substances  prevent or slow down cell damage caused by free radicals, unstable molecules that are produced by the body in response to environmental pressures and other pressures.

 

Such findings have also been reported  in which quinoa seeds can act as a protective agent against fructose-induced changes in rat by reducing LPO and enhancing blood plasma and heart, kidney, testis, lung, and pancreas antioxidant capacity. They concluded that when these seeds are present in the diet, the plasma and selected tissue antioxidant system is more effective.

 

Quinoa, a superfood as it is called and its products are rich not only in macronutrients, such as protein, polysaccharides, and fats, but also in micronutrients such as polyphenols, vitamins, and minerals. Besides this, it also contains 10 essential amino acids.²⁴

 

Quinoa is also reported to have flavonoids. These Bioflavonoids are widely distributed in seeds and reported to exert multiple biological effects including free radical-scavenging activity. It has also been reported that the bioflavonoids have a protective effect on the DNA damage which is  induced by the hydroxyl radicals²⁵or Quercetin 5 is a flavonol, it is found in quinoa seeds known to protect DNA fromoxidative damage resulting from the attack of OH, H2O2, and O2 on the DNA oligonucleotides.²⁶

 

Thus, the present study observed that Quinoa seed powder showed significant antioxidant property and supported the use of Quinoa as a potent anti-teratogenic agent.

 

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Accepted on 17.11.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(5): 2129-2134.