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Amelioration potential of Moringa oleifera extracts against sodium arsenate induced embryotoxicity and genotoxicity in mouse (Mus musculus)

Potencial de melhoria de extratos de Moringa oleifera contra embriotoxicidade e genotoxicidade induzida por arseniato de sódio em camundongos (Mus musculus)

Abstract

Previous studies have suggested that arsenic crosses the placenta and affects the fetus development. The study under consideration aims to show comparative ameliorative effect of Moringa oleifera leaf and flower extracts against sodium arsenate induced fetus toxicity of mice. Pregnant mice (N=44) were kept in lab and divided into eleven group from (A to K) and were orally administered the doses 6 mg/kg, 12 mg/kg for sodium arsenate, 150 mg/kg and 300 mg/kg for Moringa oleifera leaf extracts (MOLE) and 150 mg/kg and 300 mg/kg for Moringa oleifera flower extracts (MOFE) comparing with control. The investigation revealed evident reduction in the fetuses weight, hind limb, fore limb, tail and snout length, crown rump and head circumferences well as malformations in tail, feet, arms, legs, skin and eyes in the negative control group (only administered with sodium arsenate). Co-administration of sodium arsenate with MOLE and MOFE ameliorate the reversed effect of sodium arsenate on the shape, length, body weight and DNA damage of fetus significantly at 95% confidence interval. However, Moringa oleifera leaf extract showed more significant results in comparison to Moringa oleifera flower extract. Hence concluded that Moringa oleifera leaf extract ameliorated the embryo toxic effects of sodium arsenate and can be used against environmental teratogens.

Keywords:
Moringa oleifera; comet assay; deformed axis; laproschisis; rump

Resumo

Estudos anteriores sugeriram que o arsênio atravessa a placenta e afeta o desenvolvimento do feto. O estudo em consideração visa mostrar o efeito melhorador comparativo de extratos de folhas e flores de Moringa oleifera contra a toxicidade fetal induzida por arseniato de sódio em camundongos. Camundongos grávidas (N = 44) foram mantidos em laboratório e divididos em 11 grupos (de A a K) e foram administrados por via oral nas doses de 6 mg/kg, 12 mg/kg para arseniato de sódio, 150 mg/kg e 300 mg/kg para extratos de folhas de Moringa oleifera (MOLE) e 150 mg/kg e 300 mg/kg para extratos de flores de Moringa oleifera (MOFE) em comparação com o controle. A investigação revelou redução evidente no peso do feto, membro posterior, membro anterior, comprimento da cauda e focinho, coroa, nádega e circunferência da cabeça, bem como malformações na cauda, ​​pés, braços, pernas, pele e olhos no grupo de controle negativo (apenas administrado com arseniato de sódio). A coadministração de arseniato de sódio com MOLE e MOFE melhora significativamente o efeito reverso do arseniato de sódio na forma, comprimento, peso corporal e dano ao DNA do feto, com intervalo de confiança de 95%. No entanto, o extrato da folha da Moringa oleifera apresentou resultados mais significativos em comparação ao extrato da flor da Moringa oleifera. Portanto, concluiu que o extrato da folha de Moringa oleifera melhorou os efeitos tóxicos do arseniato de sódio para o embrião e pode ser usado contra teratógenos ambientais.

Palavras-chave:
Moringa oleifera; ensaio cometa; eixo deformado; laprosquise; alcatra

1. Introduction

Heavy metals like arsenic, copper, cadmium, lead, zinc, nickel, and chromium are usually found in water and dangerous to human beings and environment Jaishankar et al. (2014)JAISHANKAR, M., TSETEN, T., ANBALAGAN, N., MATHEW, B.B. and BEEREGOWDA, K.N., 2014. Toxicity, mechanism and health effects of some heavy metals. Interdisciplinary Toxicology, vol. 7, no. 2, pp. 60-72. http://dx.doi.org/10.2478/intox-2014-0009. PMid:26109881.
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. Erosion of soil, weathering of rocks, industrial waste, urban sewage, pesticides, mining and other ways are common sources of heavy metals Morais et al. (2012)MORAIS, S., COSTA, F.G. and PEREIRA, M.D.L., 2012. Heavy metals and human health. Environmental Health-Emerging Issues and Practice, vol. 10, pp. 227-246.. Arsenic is one of the dangerous heavy metal and abundantly found in earth crust. It has valency of (III) or (V) and found in inorganic and organic forms. Chile, Argentina, India, Nevada, California, China, Taiwan, Thailand, Mongolia, Mexico, North Dakota, Nepal and Central Oklahoma in USA are hotspots of arsenic in world for surface or groundwater (Abdul et al., 2015ABDUL, K.S.M., JAYASINGHE, S.S., CHANDANA, E.P., JAYASUMANA, C. and SILVA, P.M.C., 2015. Arsenic and human health effects: A review. Environmental Toxicology and Pharmacology, vol. 40, no. 3, pp. 828-846. http://dx.doi.org/10.1016/j.etap.2015.09.016. PMid:26476885.
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). Arsenic is found in oxides or sulphides form of iron, calcium and sodium salts Singh et al. (2007)SINGH, N., KUMAR, D. and SAHU, A.P., 2007. Arsenic in the environment: effects on human health and possible prevention. Journal of Environmental Biology, vol. 28, no. 2, suppl., pp. 359-365. PMid:17929751.. At earth, it is 20th plentiful metal. Inorganic arsenite and arsenates are dangerous to living things and environment. 10 ppb is maximum arsenic value according to Brown and Ross (2002)BROWN, K.G. and ROSS, G.L., 2002. Arsenic, drinking water, and health: a position paper of the American Council on Science and Health. Regulatory Toxicology and Pharmacology, vol. 36, no. 2, pp. 162-174. http://dx.doi.org/10.1006/rtph.2002.1573. PMid:12460751.
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. Drinking water contamination with inorganic arsenic is globally found to be major health concern. Shakoor et al. (2015)SHAKOOR, M.B., NIAZI, N.K., BIBI, I., RAHMAN, M.M., NAIDU, R., DONG, Z., SHAHID, M. and ARSHAD, M., 2015. Unraveling health risk and speciation of arsenic from groundwater in rural areas of Punjab, Pakistan. International Journal of Environmental Research and Public Health, vol. 12, no. 10, pp. 12371-12390. http://dx.doi.org/10.3390/ijerph121012371. PMid:26445051.
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studied the concentration of as in rural areas like Chichawatni, Rahim Yar Khan and Vihari of Punjab in Pakistan. 53% out of 62 water samples showed the greater arsenic value than the WHO (World Health Organization) permissible limits 10 μg/L. Arsenic values are 15 times greater than the WHO described safe limits in the ground water in different tehsils of district vehari Shahid et al. (2018)SHAHID, M., KHALID, M., DUMAT, C., KHALID, S., NIAZI, N.K., IMRAN, M., BIBI, I., AHMAD, I., HAMMAD, H.M. and TABASSUM, R.A., 2018. Arsenic level and risk assessment of groundwater in Vehari, Punjab Province, Pakistan. Exposure and Health, vol. 10, no. 4, pp. 229-239. http://dx.doi.org/10.1007/s12403-017-0257-7.
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. In Pakistan water pollution is major problem and peoples are affected with arsenic through contaminated water, pesticides, fruits, vegetables and other edibles with concentration more than WHO described limits Bahadar et al. (2014)BAHADAR, H., MOSTAFALOU, S. and ABDOLLAHI, M., 2014. Growing burden of diabetes in Pakistan and the possible role of arsenic and pesticides. Journal of Diabetes and Metabolic Disorders, vol. 13, no. 1, pp. 117. http://dx.doi.org/10.1186/s40200-014-0117-y. PMid:25530951.
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. Inorganic arsenic consumption effects seriously to human health and causes “arsenicosis” by low and long-term exposure to arsenic McCarty et al. (2011)MCCARTY, K.M., HANH, H.T. and KIM, K.W., 2011. Arsenic geochemistry and human health in South East Asia. Reviews on Environmental Health, vol. 26, no. 1, pp. 71-78. http://dx.doi.org/10.1515/reveh.2011.010. PMid:21714384.
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. Anemia, vascular disorders, respiratory and neuropathic disorders occur due to lower chronic arsenic exposure Trevor et al. (2010)TREVOR, A.J., KATZUNG, B.G., MASTERS, S.B. and KRUIDERING-HALL, M., 2010. Pharmacology examination & board review. New York: McGraw-Hill Medical..

According to Chen et al. (2009)CHEN, Y., PARVEZ, F., GAMBLE, M., ISLAM, T., AHMED, A., ARGOS, M., GRAZIANO, J.H. and AHSAN, H., 2009. Arsenic exposure at low-to-moderate levels and skin lesions, arsenic metabolism, neurological functions, and biomarkers for respiratory and cardiovascular diseases: review of recent findings from the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh. Toxicology and Applied Pharmacology, vol. 239, no. 2, pp. 184-192. http://dx.doi.org/10.1016/j.taap.2009.01.010. PMid:19371619.
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skin lesion, circulatory dysfunction, nervous disorders, liver and kidney disorder, diabetics and death occurs due to 10 to 300 microgram per liter of As. Bladder cancer risk increased in smoker when exposed to high doses of arsenic Wang et al. (2013)WANG, Y.H., YEH, S.D., WU, M.M., LIU, C.T., SHEN, C.H., SHEN, K.H., PU, Y.S., HSU, L.I., CHIOU, H.Y. and CHEN, C.J., 2013. Comparing the joint effect of arsenic exposure, cigarette smoking and risk genotypes of vascular endothelial growth factor on upper urinary tract urothelial carcinoma and bladder cancer. Journal of Hazardous Materials, vol. 262, pp. 1139-1146. http://dx.doi.org/10.1016/j.jhazmat.2012.08.056. PMid:23009795.
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. Chronic low dose exposure could cause DNA damage and inflammation Dutta et al. (2015)DUTTA, K., PRASAD, P. and SINHA, D., 2015. Chronic low level arsenic exposure evokes inflammatory responses and DNA damage. International Journal of Hygiene and Environmental Health, vol. 218, no. 6, pp. 564-574. http://dx.doi.org/10.1016/j.ijheh.2015.06.003. PMid:26118750.
http://dx.doi.org/10.1016/j.ijheh.2015.0...
. Tabacova et al. (1994)TABACOVA, S., BAIRD, D.D., BALABAEVA, L., LOLOVA, D. and PETROV, I., 1994. Placental arsenic and cadmium in relation to lipid peroxides and glutathione levels in maternal-infant pairs from a copper smelter area. Placenta, vol. 15, no. 8, pp. 873-881. http://dx.doi.org/10.1016/S0143-4004(05)80188-2. PMid:7886027.
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in his study concluded that placental levels of arsenic were greater in arsenic contaminated area and pregnant females were at risk of oxidative damage. It has been also found that arsenic crosses the placenta and affected the fetus development Dietert and Piepenbrink (2006)DIETERT, R.R. and PIEPENBRINK, M.S., 2006. Perinatal immunotoxicity: why adult exposure assessment fails to predict risk. Environmental Health Perspectives, vol. 114, no. 4, pp. 477-483. http://dx.doi.org/10.1289/ehp.8566. PMid:16581533.
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. Preterm birth, mortality and reduced fetal weight have been observed Ahmed et al. (2011)AHMED, S., KHODA, S.M.E., REKHA, R.S., GARDNER, R.M., AMEER, S.S., MOORE, S., EKSTRÖM, E.C., VAHTER, M. and RAQIB, R., 2011. Arsenic-associated oxidative stress, inflammation, and immune disruption in human placenta and cord blood. Environmental Health Perspectives, vol. 119, no. 2, pp. 258-264. http://dx.doi.org/10.1289/ehp.1002086. PMid:20940111.
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. DNA damage and oxidative stress, epigenetic modification, unstable the genome, induced carcinogenesis by inflammation and immune modulation. Hepatic damage, dermal, pulmonary, and pancreatic cancer is linked with exposure to higher arsenic and it also produces cardiovascular and nervous disorders Rao et al. (2017)RAO, C.V., PAL, S., MOHAMMED, A., FAROOQUI, M., DOESCHER, M.P., ASCH, A.S. and YAMADA, H.Y., 2017. Biological effects and epidemiological consequences of arsenic exposure, and reagents that can ameliorate arsenic damage in vivo. Oncotarget, vol. 8, no. 34, pp. 57605-57621. http://dx.doi.org/10.18632/oncotarget.17745. PMid:28915699.
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. In genotoxicity, genetic material (DNA and RNA) losses its properties due to destructive effects of genotoxic substances which may lead to several diseases including cancer Ray (2014)RAY, S., 2014. Micronutrient, genome stability and degenerative diseases: nutrigenomics concept of disease prevention: an overview. Current Research in Nutrition and Food Science Journal, vol. 2, no. 3, pp. 159-164. http://dx.doi.org/10.12944/CRNFSJ.2.3.08.
http://dx.doi.org/10.12944/CRNFSJ.2.3.08...
. Comet assay could be used for evaluating DNA damage. In detection of DNA depletion and repairing comet assay is one of the best tool for prokaryotes and eukaryotes (Andem et al., 2013ANDEM, A.B., AGBOR, R.B. and EKPO, I.A., 2013. Review on comet assay: a reliable tool for assessing DNA damage in animal models. Journal of Current Research in Science, vol. 1, no. 6, pp. 405.; Hansen et al., 2014HANSEN, M.K., SHARMA, A.K., DYBDAHL, M., BOBERG, J. and KULAHCI, M., 2014. In vivo Comet assay-statistical analysis and power calculations of mice testicular cells. Mutation Research, vol. 774, pp. 29-40. http://dx.doi.org/10.1016/j.mrgentox.2014.08.006. PMid:25440908.
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).

Moringa oleifera Lam., synonym M. ptreygosperma Gaertn, family Moringaceae is commonly called drumstick, kelor or horse radish Anwar et al. (2005)ANWAR, F., ASHRAF, M. and BHANGER, M.I., 2005. Interprovenance variation in the composition of Moringa oleifera oilseeds from Pakistan. Journal of the American Oil Chemists’ Society, vol. 82, no. 1, pp. 45-51. http://dx.doi.org/10.1007/s11746-005-1041-1.
http://dx.doi.org/10.1007/s11746-005-104...
. It is commonly found in South Asia, Arabia, Africa, America, Pakistan, India, and Himalayan regions, islands of Caribbean and Pacific (Sreelatha and Padma, 2009SREELATHA, S. and PADMA, P.R., 2009. Antioxidant activity and total phenolic content of Moringa oleifera leaves in two stages of maturity. Plant Foods for Human Nutrition, vol. 64, no. 4, pp. 303-311. http://dx.doi.org/10.1007/s11130-009-0141-0. PMid:19904611.
http://dx.doi.org/10.1007/s11130-009-014...
). In Pakistan, Moringa concanensis and Moringa oleifera are present and widely cultured in moderate areas like, Punjab Plains, Sindh, NWFP and Balochistan. Antioxidants and nutraceuticals are present in large amount in leaves, flowers, seeds fruits and foods of Moringa oleiferaFalowo et al. (2018)FALOWO, A.B., MUKUMBO, F.E., IDAMOKORO, E.M., LORENZO, J.M., AFOLAYAN, A.J. and MUCHENJE, V., 2018. Multi-functional application of Moringa oleifera Lam. in nutrition and animal food products: a review. Food Research International, vol. 106, pp. 317-334. http://dx.doi.org/10.1016/j.foodres.2017.12.079. PMid:29579932.
http://dx.doi.org/10.1016/j.foodres.2017...
. Moringa leaves contain protein, potassium, calcium, iron, vitamin, antioxidants like ascorbic acid, flavonoids, carotenoids, polyphenols, rutin, glycosides, which could be used as food in developing countries for nourishment (Siddhuraju and Becker, 2003SIDDHURAJU, P. and BECKER, K., 2003. Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam.) leaves. Journal of Agricultural and Food Chemistry, vol. 51, no. 8, pp. 2144-2155. http://dx.doi.org/10.1021/jf020444+. PMid:12670148.
http://dx.doi.org/10.1021/jf020444+...
; Ndong et al., 2007NDONG, M., UEHARA, M., KATSUMATA, S., SATO, S. and SUZUKI, K., 2007. Preventive effects of Moringa oleifera (Lam) on hyperlipidemia and hepatocyte ultrastructural changes in iron deficient rats. Bioscience, Biotechnology, and Biochemistry, vol. 71, no. 8, pp. 1826-1833. http://dx.doi.org/10.1271/bbb.60644. PMid:17690476.
http://dx.doi.org/10.1271/bbb.60644...
). Seed and flower have been used against arsenic induce toxicity in Albino rat by (Gupta et al., 2005GUPTA, R., KANNAN, G.M., SHARMA, M. and FLORA, S.J., 2005. Therapeutic effects of Moringa oleifera on arsenic-induced toxicity in rats. Environmental Toxicology and Pharmacology, vol. 20, no. 3, pp. 456-464. http://dx.doi.org/10.1016/j.etap.2005.05.005. PMid:21783626.
http://dx.doi.org/10.1016/j.etap.2005.05...
; Chattopadhyay et al., 2011CHATTOPADHYAY, S., MAITI, S., MAJI, G., DEB, B., PAN, B. and GHOSH, D., 2011. Protective role of Moringaoleifera (Sajina) seed on arsenic-induced hepatocellular degeneration in female albino rats. Biological Trace Element Research, vol. 142, no. 2, pp. 200-212. http://dx.doi.org/10.1007/s12011-010-8761-7. PMid:20661662.
http://dx.doi.org/10.1007/s12011-010-876...
). Despite of above-mentioned beneficial effects of Moringa, its application against metallic toxicity, specifically against arsenic induced embryonic toxicity has not yet been studied. Higher intake of arsenic in drinking water may causes chronic embryo toxicity and tissue damage with genotoxic risk to the population. So, the confirmation of effectiveness of Moringa oleifera against arsenic induced embryo toxicity in mice was necessary which showed the beneficial effects of Moringa oleifera (extracts) against arsenic induced embryo toxicity during critical time of development.

2. Materials and Methods

2.1. Mice sampling

Albino Mice, Mus musculus (Temminck, 1845) (N= 44) of 7-8 week, with initial body weight of 20-25g was obtained from animal house of University of Veterinary and Animal sciences, Lahore. Ethical permission was obtained from the ethical committee of University of Veterinary and Animal sciences Lahore, Pakistan via Ref. No.161. Dated 6-2-2020.

The mice were kept under standard conditions i.e. temperature 25 °C with 12 hours, dark and light cycle and treated with normal diet (National feed No. 14) which had Energy: 2700 Kcal/Kg ± 100, Crude Protien: 16.5% ± 1, with ingredients like cereals and corn. Tap water was given in plastic bottles to animals as ad-libitum. In Pregnant female mice, timed mating was induced by placing 1 male and 2 female together and presence of vaginal plug was the indication of mating. The observation of a plug determined the gestation day zero Rodriguez et al. (2016)RODRIGUEZ, K.F., UNGEWITTER, E.K., CRESPO-MEJIAS, Y., LIU, C., NICOL, B., KISSLING, G.E. and YAO, H.H.C., 2016. Effects of in utero exposure to arsenic during the second half of gestation on reproductive end points and metabolic parameters in female CD-1 mice. Environmental Health Perspectives, vol. 124, no. 3, pp. 336-343. http://dx.doi.org/10.1289/ehp.1509703. PMid:26295903.
http://dx.doi.org/10.1289/ehp.1509703...
.

2.2. Chemicals

All chemicals like TCA, Triton X-100, Low and normal melting agarose, Tris, Ethidium bromide etc were purchased from Merck company, Germany while Sodium arsenate was obtained from Sigma chemicals, USA.

2.3. Preparation of Moringa oleifera extracts

Plant materials (leaves and flowers) were collected from the Botanical garden of UVAS, Ravi campus Pattoki. The method of extraction was given in report of Tabidi et al. (2018)TABIDI, R.H., BABEKER, E.A., and ELLAITHI, S.O., 2018. Phytochemical and antibacterial of Moringa oleifera leaves. World Journal of Pharmaceutical Research, vol. 8, no. 2, pp. 212-224.. The plant material was sun dried for 7 days, grounded into powdered form, and extracted through soxhlet apparatus with solvent. Extracted solvent air dried, stored at 40C and was used with distilled water.

2.4. Qualitative and quantitative analysis of extracts

Qualitative and quantitative analysis of Moringa oleifera leaf and flower extracts for the presence or absence of phytochemicals were carried using standard procedures given in reports of Santhi and Sengottuvel (2016)SANTHI, K. and SENGOTTUVEL, R., 2016. Qualitative and quantitative phytochemical analysis of Moringa concanensis Nimmo. International Journal of Curative and Microbiology Applied Sciiences, vol. 5, no. 1, pp. 633-640. http://dx.doi.org/10.20546/ijcmas.2016.501.064.
http://dx.doi.org/10.20546/ijcmas.2016.5...
and Adawia et al. (2016)ADAWIA, K., RAWAA, A.K. and GHALIA, S., 2016. Phytochemical screening and antioxidant activity of selected wild plants in Liliaceae family growing Syria. International Journal of Pharmacology & Phytochemical Research, vol. 8, no. 2, pp. 2025-2032..

2.5. Administration of sodium arsenate and extracts (MOFE and MOLE) to animals

Toxicant sodium arsenate to induce embryo toxicity and test extracts (leaf and flower) of Moringa oleifera as ameliorant was used with different doses at GD8-GD12. Pregnant females were divided into groups (A, B, C, D, E, F, G, H, I, J and K) with 4 pregnant females in each group. A continuous chronic dose of sodium arsenate at GD-8 to GD-12 was administered orally to the pregnant mice for induction embryonic abnormalities. Tested extract of Moringa oleifera leaf and flower was also administered orally with following doses as given below, 1hour before sodium arsenate administration in all experimental groups. Where, Group A is of control while B and C were sodium arsenate treated groups (A (0.00), B (6.00, 0.00), C (12.00, 0.00) mg/kg B.W.) Where, groups D to G were sodium arsenate and M. oleifera flower extract treated groups. (D (6.00, 150.00), E (6.00, 300.00), F (12.00, 150.00) and G (12.00, 300.00) mg/kg B.W.) Where, groups H to K were sodium arsenate and M. oleifera leafextract treated groups. (H (6.00, 150.00), I (6.00, 300.00), J (12.00, 150.00) and K (12.00, 300.00) mg/kg B.W.)All animals were observed daily to count mortality and morbidity. Gross maternal body weights were measured daily from GD0 to GD18.

2.6. Fetuses samples collection

Pregnant females were anaesthetized at GD18 using ether presented on a cotton ball and fetuses were obtained surgically. Fetuses were weighed and photographed in situ for morphological studies. Litters were observed through count, deceased, normal and abnormal fetus. Extra embryonic surroundings were cleared and formalin 10% was used to fix fetuses for 48hrs, after this kept in 70% alcohol Carson and Hladik (1997)CARSON, F.L. and HLADIK, C., 1997. Histotechnology: a self-instructional text. Chicago: ASCP Press, vol. 96, pp. 189-202.. Fetuses with more morphological defects (caraniofacial, trunk, limbs, tail and axis) were selected, recorded and tabulated. Verniercalliper was used for morphometric studies (Head circumference, nasal length, tail length, crown rump length, fore and hind limbs length).

Comet assay was used to check the DNA damage according to report of Andem et al. (2013)ANDEM, A.B., AGBOR, R.B. and EKPO, I.A., 2013. Review on comet assay: a reliable tool for assessing DNA damage in animal models. Journal of Current Research in Science, vol. 1, no. 6, pp. 405.. Briefly, 1% normal melting agarose (NMA 3 gm + 3 mL PBS) was used to precoat the slides. Embryonic tissues were chopped in 2 mL of lysis solution with ice. Homogenized solution was filtered, centrifuged at 3000 rpm for 20 minutes. Supernatants were transferred into new Eppendorf tube and pellets were discarded. Phosphate buffer saline (PBS) 1% was added and centrifuged for 5-7 min. at 3000 rpm. 10 to 20 µL of pellet was mixed with 80 to 100 µL of LMA (Low melting agarose) and spread on the precoated slides. Covered these slides with cover slips and placed on ice slab for 10 minutes, then placed upright in lysis buffer for two hour. Prepared Slides were completely dipped in alkaline buffer solution for 20 minutes. Slides were electrophoresed for 30 minutes and removed from electrophoresis apparatus. Samples stained with 1X Ethidium bromide. Comet score were obtained by the method of Ciğerci et al. (2015)CIĞERCI, İ.H., LIMAN, R., ÖZGÜL, E. and KONUK, M., 2015. Genotoxicity of indium tin oxide by Allium and Comet tests. Cytotechnology, vol. 67, no. 1, pp. 157-163. http://dx.doi.org/10.1007/s10616-013-9673-0. PMid:24337653.
http://dx.doi.org/10.1007/s10616-013-967...
. 150 nuclei per slide was visually observed and counted at magnification of 400X using a fluorescent microscope (Olympus, Japan) and scored from 0-4 where 0 used stands for undamaged and 4 for maximum damage.

2.7. Statistical analysis

Statistical analysis SAS (version 9.1) software was used. ANOVA (Analysis of variance) was used to compare the means in all groups. P<0.05 was considered significant and 5% was considered significance level for all analysis.

3. Results

Phytochemoical analyses of Moringa oleifera revealed that alkaloids, flavonoids, steroids, Phenols were rich than saponins and tanins. Currative potential of Moringa oleifera was due to huge amount of phytoconstituents like alkaloids, steroids, phenols and flavonoids as shown in Table 1.

Table 1
Phytochemical analysis of Moringa oleifera with methanol.

Phenols, alkaloids, tannins, saponins and flavonoids were found in higher concentrations in Moringa oleifera leaf extract then the Moringa oleifera flower extract while saponins were absent in Moringa oleifera flower extract (Table 1).

3.1. Gross fetus analysis

Four females were used for each group. After fetuses recovery at GD18, gross fetus analysis was done in terms of total number of fetuses obtained, normal, abnormal and resorbed fetuses (Figure 1). Significant results were obtained from all experimental groups as compared to control while Moringa oliefera leaf extract treated groups showed significant results against sodium arsenate treated groups B and C as shown in Figure 1.

Figure 1
Gross fetus analysis of fetuses recovered at GD18.

3.2. Morphometric studies

Morphometric analysis showed that fetuses weight, hind limb, fore limb, tail and snout length, crown rump and head circumference decreased significantly (P<0.05) in sodium arsenate treated groups B and C (Table 2) as compared to control group. While Moringa oleifera leaf extract treatment at a dose of 300 mg/kg B.W. showed significant amelioration against sodium arsenate induced morphometric abnormalities than the Moringa oleifera flower extract.

Table 2
Morphomatric analysis fetuses recovered at GD18 from sodium arsenate and Moringaoliefera leaf and flower extract treated groups at gestation day 8-12 in mice.

3.3. Morphological studies

All the experimental groups showed significant variation in morphological features with control group except group I (SA6 + MOLE 300 + 6 mg/kg B.W) treated with Moringa oliefera leaf extract which reduced the toxic effects of sodium arsenate as compared to Moringa oliefera flower extract treatment. Sodium arsenate at dose of 6 mg/kg B.W (group B) and 12 mg/kg B.W (groups C) showed abnormalities like hemorrhages, macroglossia, kinky tail, deformed body, cryptothalmia, low set arm, laprocrisis, club feet, omphocoel, micromelia, neck fissure, synotia, anotia, elongated snout, disstorted axis, anopthalmia and resorbed ovaries. Similarly, other groups excluding control (A) showed anomalies like macrotia, anotia, skin lesion, and open eyelids as shown in Figure 2.

Figure 2
Morphological features of mice fetuses recovered at GD18 administered with different doses of sodium arsenate and Moringa leaf and flower extracts given above. H = Head; Ey = Eye; FL = Fore Limb; HL = Hind Limb; S = Snout; T = Tail; DA = Distorted Axis; DF = Deformed; He = Hemorrhage; KT = Kinky Tail; NF = Neck Fissure; OE = Open Eyelid; Mm = Micromelia; Sl = Skin Lesion; CF = Club Feet; mg = Macroglossia; Ls = Laproschisis; Mc = Macrotia; Es = Enlarge Snout; Cr = Cryptothalmia; An = Anopthalmia; Sy = Synotia; An = Anotia; OM = Omphocoel; R = Resorbed Ovary; LSA = Low Set Arm; Ec = Exencephaly.

3.4. Comet assay

The results of comet assay (Table 3) showed dose dependent response in all groups. Statistically significant difference (P<0.05) for all treatment groups was observed as compared to control group (27.50 ± 1.29) except in Moringa oleifera leaf extract (Group I) which showed similarity (28.50 ± 1.29) with control while sodium arsenate treated groups at dose of 6 mg/kg B.W. and 12 mg/kg B.W. showed greatest DNA damage as 66.25 ± 2.21 and 85.00 ± 2.94 respectively (Table 3).

Table 3
Genotoxic effects of sodium arsenate on mice embryo and ameliorative effect of Moringaoliefera extracts (leaf and flower).

4. Discussion

This study was focused to explore the effectiveness of Moringa oleifera leaf and flower extracts against sodium arsenate induced embryo toxicity during critical time of development. Arsenic is causing embryonic abnormalities and nervous disorders during neurolation Robinson et al. (2011)ROBINSON, J.F., YU, X., MOREIRA, E.G., HONG, S. and FAUSTMAN, E.M., 2011. Arsenic-and cadmium-induced toxicogenomic response in mouse embryos undergoing neurulation. Toxicology and Applied Pharmacology, vol. 250, no. 2, pp. 117-129. http://dx.doi.org/10.1016/j.taap.2010.09.018. PMid:20883709.
http://dx.doi.org/10.1016/j.taap.2010.09...
. Mutagenic, carcinogenic, and teratogenic effects were observed when exposed to high level of arsenic Shi et al. (2004)SHI, H., SHI, X. and LIU, K.J., 2004. Oxidative mechanism of arsenic toxicity and carcinogenesis. Molecular and Cellular Biochemistry, vol. 255, no. 1-2, pp. 67-78. http://dx.doi.org/10.1023/B:MCBI.0000007262.26044.e8. PMid:14971647.
http://dx.doi.org/10.1023/B:MCBI.0000007...
. From all the teratogens, arsenic is the most common teratogen, this teratogenicity was confirmed by Hill et al. (2008)HILL, D.S., WLODARCZYK, B.J. and FINNELL, R.H., 2008. Reproductive consequences of oral arsenate exposure during pregnancy in a mouse model. Birth Defects Research. Part B, Developmental and Reproductive Toxicology, vol. 83, no. 1, pp. 40-47. http://dx.doi.org/10.1002/bdrb.20142. PMid:18186108.
http://dx.doi.org/10.1002/bdrb.20142...
. Arsenic exposure causes the DNA damage and oxidation of DNA. These DNA strand breakage were due to ROS (Reactive Oxidative Species) induced by arsenic Shi et al. (2004)SHI, H., SHI, X. and LIU, K.J., 2004. Oxidative mechanism of arsenic toxicity and carcinogenesis. Molecular and Cellular Biochemistry, vol. 255, no. 1-2, pp. 67-78. http://dx.doi.org/10.1023/B:MCBI.0000007262.26044.e8. PMid:14971647.
http://dx.doi.org/10.1023/B:MCBI.0000007...
. Phytochemicals had ability to combat with free radicals and phenolics are one of these compounds which have antioxidative property Wangensteen et al. (2004)WANGENSTEEN, H., SAMUELSEN, A.B. and MALTERUD, K.E., 2004. Antioxidant activity in extracts from coriander. Food Chemistry, vol. 88, no. 2, pp. 293-297. http://dx.doi.org/10.1016/j.foodchem.2004.01.047.
http://dx.doi.org/10.1016/j.foodchem.200...
. Moringa oliefera is a rich source of antioxidants in its leaf, flowers and seed (Morton, 1991MORTON, J.F., 1991. The horseradish tree, Moringa pterygosperma (Moringaceae): a boon to arid lands? Economic Botany, vol. 45, no. 3, pp. 318-333. http://dx.doi.org/10.1007/BF02887070.
http://dx.doi.org/10.1007/BF02887070...
; Sreelatha and Padma, 2009SREELATHA, S. and PADMA, P.R., 2009. Antioxidant activity and total phenolic content of Moringa oleifera leaves in two stages of maturity. Plant Foods for Human Nutrition, vol. 64, no. 4, pp. 303-311. http://dx.doi.org/10.1007/s11130-009-0141-0. PMid:19904611.
http://dx.doi.org/10.1007/s11130-009-014...
). Moringa bioactive compounds like polyphenolics can act against teratogens which are generating ROS (respiratory oxidative species) like arsenic which induces oxidative stress and DNA damage in mice Sikder et al. (2013)SIKDER, K., SINHA, M., DAS, N., DAS, D.K., DATTA, S. and DEY, S., 2013. Moringa oleifera Leaf extract prevents in vitro oxidative DNA damage. Asian Journal of Pharmaceutical and Clinical Research, vol. 6, no. 2, pp. 159-163.. Antioxidating mechanism of Moringa oleifera bioactive compounds was used to check the effective potential of Moringa oleifera extracts against heavy metals induced embryotoxicity.

Results of our studies showed hemorrhages, macroglossia, kinky tail, deformed body, cryptothalmia, low set arm, laprocrisis, club feet, omphocoel, micromelia, neck fissure, synotia, elongated snout, distorted axis, excencephaly, anopthalmia, resorbed ovaries and growth retardation when sodium arsenate was given orally to pregnant females. These abnormalities increased significantly with increase of dose and time which is an agreement with study of Arshad and Asmatullah (2017)ARSHAD, M. and ASMATULLAH, C.A., 2017. Embryotoxic effects of sodium arsenate in Mus musculus. Punjab University Journal of Zoology, vol. 32, no. 2, pp. 189-195. they found micromelia, anopthalmia, resorbed ovaries and cryptothalmia when treated pregnant mice with sodium arsenate. Morphometric data reveals significant decrease in fetus weight, fore and hind limb, snout and crown rump length which is compliance with studies of Hopenhayn et al. (2003)HOPENHAYN, C., FERRECCIO, C., BROWNING, S.R., HUANG, B., PERALTA, C., GIBB, H. and HERTZ-PICCIOTTO, I., 2003. Arsenic exposure from drinking water and birth weight. Epidemiology, vol. 14, no. 5, pp. 593-602. http://dx.doi.org/10.1097/01.ede.0000072104.65240.69. PMid:14501275.
http://dx.doi.org/10.1097/01.ede.0000072...
. He performed a study in cities where arsenic in water was in great amount whereas Arshad and Asmatullah (2017)ARSHAD, M. and ASMATULLAH, C.A., 2017. Embryotoxic effects of sodium arsenate in Mus musculus. Punjab University Journal of Zoology, vol. 32, no. 2, pp. 189-195. studied embryo toxic effects of arsenic and found fetuses weight, size, crown rump and head circumference reduced significantly. While Moringa oliefera leaf and flower extracts minimizes the sodium arsenate induced morphometric and morphological abnormalities of fetuses which is accordance with study of Zeng et al. (2019)ZENG, B., LUO, J., WANG, P., YANG, L., CHEN, T., SUN, J., XIE, M., LI, M., ZHANG, H., HE, J., ZHANG, Y. and XI, Q., 2019. The beneficial effects of Moringa oleifera leaf on reproductive performance in mice. Food Science & Nutrition, vol. 7, no. 2, pp. 738-746. http://dx.doi.org/10.1002/fsn3.918. PMid:30847152.
http://dx.doi.org/10.1002/fsn3.918...
who observed improved litter size, birth weight and fetus size and litter survival with Moringa oleifera leaf . In recent study leaf extract showed better results as compared to Moringa oleifera flower extract at a dose of MOLE 300 mg/kg B.W and ameliorated all the toxic effects of arsenic.

The results of comet assay to check the genotoxicity (DNA damage) induced by sodium arsenate and ameliorative potential of Moringa oleifera flower and leaf extracts showed that greatest damage was observed in only sodium arsenate treated groups (C & D). In the present study arsenic at a dose of 12 mg/kg B.W. showed significant DNA damage than the dose of 6 mg/kg B.W. It is due to greater dose concentrations which generated more ROS (Reactive Oxidative Species) and causes the DNA damage Kohen and Gati (2000)KOHEN, R. and GATI, I., 2000. Skin low molecular weight antioxidants and their role in aging and in oxidative stress. Toxicology, vol. 148, no. 2-3, pp. 149-157. http://dx.doi.org/10.1016/S0300-483X(00)00206-7. PMid:10962134.
http://dx.doi.org/10.1016/S0300-483X(00)...
. Phytochemicals can neutralize this oxidative stress acting as radical savengers Arouma (2002)AROUMA, O.I., 2002. Free radicals, oxidative stress, and antioxidants in human health and disease. Nutrition, vol. 18, pp. 872-879.. In findings that MOlE and MOFE prevents the DNA damage induced by sodium arsenate (Table 3). The extracts have free radical scavenging activity which is an agreement with study of De Bont and Van Larebeke (2004)DE BONT, R. and VAN LAREBEKE, N., 2004. Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis, vol. 19, no. 3, pp. 169-185. http://dx.doi.org/10.1093/mutage/geh025. PMid:15123782.
http://dx.doi.org/10.1093/mutage/geh025...
. These findings are in agreement with studies of Sinha et al. (2011)SINHA, M., DAS, D.K., BHATTACHARJEE, S., MAJUMDAR, S. and DEY, S., 2011. Leaf extract of Moringa oleifera prevents ionizing radiation-induced oxidative stress in mice. Journal of Medicinal Food, vol. 14, no. 10, pp. 1167-1172. http://dx.doi.org/10.1089/jmf.2010.1506. PMid:21861723.
http://dx.doi.org/10.1089/jmf.2010.1506...
and Das et al. (2012)DAS, N., SIKDER, K., GHOSH, S., FROMENTY, B. and DEY, S., 2012. Moringa oleifera Lam. leaf extract prevents early liver injury and restores antioxidant status in mice fed with high-fat diet. Indian Journal of Experimental Biology, vol. 50, no. 6, pp. 404-412. PMid:22734251.. They had checked the antioxidant efficacy of MOLE. Amelioration of MOLE at a dose of 300 mg/kg B.W. was found against sodium arsenate induced genotoxicity. Present study gives the first validation that how Moringa oleifera can provide protection with its bioactive compounds against environmental teratogens like heavy metals specifically arsenic which induces developmental toxicity like DNA damage, morphometric and morphological anomalies.

5. Conclusion

Administration of sodium arsenate during organogenesis of developing mice embryo at doses 6 mg/kg and 12 mg/kg induced morphological malformations like hemorrhages, macroglossia, kinky tail, deformed body, cryptothalmia, low set arm, laprocrisis, club feet, omphocoel, micromelia, neck fissure, synotia, anotia, elongated snout, disstorted axis, anopthalmia, resorbed ovaries, macrotia, anotia, skin lesion, and open eyelids as well as reduction in the fetuses weight, hind limb, fore limb, tail and snout length, crown rump and head circumference.. Also, the treatment of Sodium arsenate increased the DNA damage. Administration of Sodium arsenate with MOLE and MOFE ameliorates the reversed effect of sodium arsenate because of bioactive compounds like phenols, flavonoids, and alkaloids. These compounds are more concentered in leaves and that’s why MOLE shows more evident ameliorating effect in comparison to MOFE. This study presented the medicinal value of phytoconstituents of the Moringa oleifera specifically leaf against teratogenic elements as well as the hazardous effects of sodium arsenate, percolating in our environment, on the fetus development.

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Publication Dates

  • Publication in this collection
    27 Aug 2021
  • Date of issue
    2023

History

  • Received
    27 Jan 2021
  • Accepted
    09 Mar 2021
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