EVALUATION OF THE EFFECTIVE SAFE DOSE OF VITAMINA ADMINISTRATION AGAINSTEHRLICH ASCITES CARCINOMA (EAC) IN MICE.

Nesreen Ahmed Hassaneen 1 , Soad Shaker Ali 2 , Sabah M. Hassan 1,3 and Samar Rabah 1 . 1. Biology Department, Science College, king Abdulaziz University, Jeddah Saudi Arabia. 2. Anatomy Department, king Abdulaziz University, Jeddah Saudi Arabia. 3. Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 12 October 2018 Final Accepted: 14 November 2018 Published: December 2018

Vitamin A is well known as fat soluble vitamin with marked beneficial effects for human health. However, there is contradictory data regard its safety in cancerous cases. Purpose: The main objective of the present study was to highlight the importance of searching for the optimum dose of vitamin A that could be considered safe and effective in controlling cancerous growth using Ehrlich ascitescarcinoma (EAC) in mice model. Experimental Design: Healthy 45 male Swiss albino mice, weighting from 30 to 35 g and aged from 8 to 10 weeks old wereacclimatized to laboratory condition for 10 days. Solid tumors were induced by subcutaneous injection of EAC cells (2.5x10 6 cells/ mice) in the right thigh of themice. Different doses of Vitamin A (0.2214, 0.4428 and 0.8856 µl) were dissolved in olive oil and given to experimental mice. Body weight and tumor size were recorded at 0, 2,3and 4 weeks and statistically analyzed. Histological studies for tumor tissuesweredone after 4 weeks. Results: Insignificant differences in body weight were found between untreated and treated animals in all groups. Solid tumor size was significantly increased in non-treated mice throughout the experimental duration. Administration of olive oil (O) before and after tumor found to significantly reduce the tumor size compared to untreated mice. The O+T+O group found to has the most decreased tumor size followed by O+T+A, O+T+2A and O+T+4Agroups. Histological studies showed degenerative apoptotic changes in all treated groups with mild response in animals receiving 4doses of Vitamin A. Conclusion: Administration of vitamin A by cancerous casesmust becritically controlled but not decreas tumor size. In the current study, recommended daily dose was found to be controlling EAC solid tumor growth as evidenced by both gross and microscopic examination. High doses must be avoided as it will act as pro-oxidant agents hindering the action of anticancer drugs .The gene expression analysis using some cancer and apoptotic related genes will be done using the same tumor samples for further investigation of vitamin A potential role in tumorigenesis.
Vitamin A is the name of a group of fat-soluble retinoid, including retinol, retinal and retinyl esters .Vitamin A is involved in immune function, vision, reproduction and cellular communication [4,5,6].
Botanical and natural dietary agents have been used for the treatment of cancer throughout history. These dietary agents also can be useful in the prevention of cancer. Epidemiological studies suggest that reduced risk of cancer is associated with high consumption of vegetables and fruits. The major part of total antioxidant activity is from the combination of photochemical [7].
Facts that must be taken in mind regarding some previous studies, which find out that, antioxidants accelerate the growth of some cancers [8]. A clinical study showed that supplementing the diet with the antioxidants Nacetylcysteine (NAC) andvitamin E increases the tumor progression and reduces survival in mouse models of B-RAF and K-RAS-induced lung cancer [9].The authorsreported that, RNA sequencing revealed NAC and vitamin E, which are structurally unrelated, produce highly coordinated changes in tumor transcriptome profiles, dominated by reduced expression of endogenous antioxidant genes. NAC and vitamin E increase tumor cell by reducing ROS, DNA damage and P53.Antioxidants accelerate tumor growth by disrupting the ROS-p53 axis [9].
Smokers and other people at high risk for lung cancer could make it worse if they take antioxidant supplements when they developed lung cancer. Mice with lung cancer was found to develop more aggressive tumors and die faster when their diet is supplemented with antioxidants via deactivation of P53, the protein that neutralizing free radicals effect on tumors [10].
Several randomized controlled trials have investigated the effect of antioxidant supplements during cancer treatment and reported that they alters the effectiveness or reduce the toxicity of specific therapies and worsen the outcome of cancer especially in smoker persons [11].
In some preclinical studies, antioxidants have been found to promote tumor growth and metastasis in tumor-bearing mice and increase the ability of circulating tumor cells to metastasize [12]. Therefore, antioxidant supplements should be used with caution. Cancer patients should inform their doctors about their use of any dietary supplement.

Research Objective:-
The main objective of the present study was to highlight the importance of searching for the optimum dose of vitamin A and its dosage regimen that could beConsidered safe and effective in controlling cancerous growth using Ehrlich carcinoma in mice model.

Statistical Analysis:-
The data were expressedas mean +/-standard error of differences between the groups were calculating by SPSS Version 21 using Person correlation between body weight and thigh size.  Changein body weight in all groups compared to untreated mice was shown in (Table 1). Marked effect was observed in animals receiving the recommended dose of vitamin A compared to other used doses.   (2) showed tumor circumference (TC) and its correlation to body weight at different periods of experiment. There was positive significant reduction of TC was observed in all treated groups with more response in O+T+O Group followed by O+T+A compeer to control group. OtherVitamin A regimens showed partialdecrease in tumor size compared with untreated mice.      Table (3), the tumor circumference (mm) at day 30showed decrease in all group campers to untreated group, howeverthe inhibition rate was higherin O+T+O (30.87%, 21.82% respectively) followed by O+T+A camper to other treated groups.

Histological study of solidTumormass:
In the present study tumor inoculated into mice thigh was observed by light microscope for features of cellular proliferation or degeneration.

Discussion:-
Dietary supplements such as vitamins and minerals have become widely available to be taken for a variety of reasons. Some people are advised by their doctors or dietitian to take dietary supplements especially if they have certain medical conditions such as pregnancy or those who have a restricted dietary intake. Many people also take vitamins and supplements while they are being treated for cancer; others take them to prevent cancer.Cancer patients are increasingly turning to supplements to strengthen their immune systems and ease nausea from chemotherapy 771 [15]. Contradictory data regard beneficial versus unwanted potentiating carcinogenic effect of vitamin A. Epidemiological studies have suggested an inverse correlation between cancer development and dietary consumption of vitamin A.Pharmacological concentrations of vitamin A decrease the incidence of chemically induced experimental tumors [16]. Natural and synthetic retinoid have been demonstrated to inhibit the growth and the development of different types of tumors, including skin, breast, oral cavity, lung, hepatic, gastrointestinal, prostatic, and bladder cancers [17][18].Clinical evidence shows that vitamin A helps to prevent cancer. The role of vitamin supplements in preventing breast cancer still remains unclear.The association of vitamin A and cancer was initially reported in 1926 when rats, fed vitamin A-deficient diet, developed gastric carcinomas. The first investigation showing a relationship between vitamin A and human cancer was performed in 1941 by Abelsetal who found low plasma vitamin A levels in patients with gastrointestinal cancer. [19] Moon et al reported that daily supplemental doses of 25,000 IU of vitamin A prevented squamous cell carcinoma. Studies that use animal models have shown that retinoids (including vitamin A) can act in the promotion-progression phase of carcinogenesis and block the development of invasive carcinoma at several epithelial sites, including the head and neck and lung. [19].Vitamin A supplementation likely does not reduce chemotherapy efficacy, and for many patients, is unlikely to cause harm. [20].
Vitamin A was advised by cliniciansfor its antioxidant activity once thought to help preventing orpromoting cancertherapy. However, more recent studies Showedthat high levels of these supplement may increase cases of gastrointestinal cancers, such as stomach, colon, and esophageal, in some populations. [15] In the present study 3different doses of Vitamin A were tested for their effect on EAC cells inoculated in mice thigh to develop solid tumors. Recommended dose in literature was reported to be ranged from (900 mcgRAE for male-700 mcg RAE for female). [21] Alsodouble and 4times of this dose were tested to define if they are safe to be given in case of neoplastic status.
The results of the present study showed that the recommended dose of vitamin A was apparently safe and also potentiate the effect of olive oil as an anti-cancer agent most probably to Polyphenol group [22].
A suicidalcell program toeliminate damaged or cancerous cellsknown asapoptosis. It is a pathway of cell death induced by activates enzymes that degrade the cell nuclear DNAviaactivate caspases pathways. In the present study, histological characteristics of cell apoptosis [23] were observed in EACtumors masses of both oliveoil treated animals alone or those given vitamin A after tumor induction, however, in the latter group, some tumor cells appeared swollen and vacuolated as if being entered into necrotic changes. Necrosis is another pathway of cell death Mainlyresults from cell membrane damage following lipid peroxidation [24]. Here in the present study, although shrinkage of cells , dark staining of nuclei could point to apoptotic changes but needs to be confirmed by immunohistochemicallystaining apoptotic markers (caspase 3/P53 specific markers). Investigation is going on for the same samples to determine P53 /P16 using quantitative real time (qrt-PCR) Accord to methodology used by [25].

Conclusion:-
while dietary factors may be crucial in modifying cancer risk, the role of vitamin supplements in preventing cancer still remains unclear.In the present study,recommended doses of Vitamin A seemed to be safe and did not interfere with olive oil suppression of cancer cell proliferation in case of EAC .However high doses must be avoided to protect against any prooxidant effect that may enhance cancer cell proliferation.Further investigations are warranted to elucidate the mechanisms by which vitamin A supplementation may modify cancer development.