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Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 AENSI Journals Advances in Environmental Biology ISSN-1995-0756 EISSN-1998-1066 Journal home page: http://www.aensiweb.com/AEB/ Antibacterial and antitumor effects of bromobenzaldehyde-4-iminacetophenone) tetraaquocobalt(ΙΙ) sulphate complex bis-(4- 1,2Amani F. H. Noureldeen, 1Safaa Y. Qusti, 1Wafa A. Alamoudi, 1Al-Anoud I. Rawas and 3Ramadan M Ramadan 1 Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt. 3 Chemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt. 2 Address For Correspondence: Amani F. H. Noureldeen, Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Received 12 August 2016; Accepted 17 December 2016; Available online 31 December 2016 ABSTRACT The present study aimed to evaluate the antibacterial and anticancer activities of bis-(4-bromobenzaldehyde-4-iminacetophenone) tetraaquocobalt(ΙΙ) sulphate complex (BBIA-Co). The antibacterial activity of the synthesized complex was tested against two bacterial strains; Staphylococcus aureus and Escherchia coli. HepG2, A549 and PC3 carcinoma cell lines were used for evaluating in vitro cytotoxicity of the complex. For the in vivo study, 125 mice were classified equally into 5 groups: normal, EAC bearing mice, vehicle, BBIA-Co (healthy mice) and treated (mice inoculated with EAC cells) groups. Results indicated that the BBIA-Co complex has the capacity to inhibit the metabolic growth of the tested microorganisms. In vitro study demonstrated medium cytotoxicity against tested cell lines with IC50 values equivalent to 25.0 µg/ml, 17.5 µg/ ml and 21.7 µg/ ml for HepG2, A549 and PC3 respectively. In vivo study indicated that BBIA-Co complex treatment had successfully increased mean survival time which in turn resulted in prolonged the life span of EAC-tumor bearing mice (77%). The complex has effectively reduced the accumulated ascites fluid and decreased cell viability. The hematological parameters and other biochemical parameters were significantly restored towards normal level post BBIA-Co-treatment, indicating the effectiveness of the complex as an anticancer agent. KEYWORDS: Cobalt complexes; Biological activity; Ehrlich tumors; In vitro cytotoxicity; In vivo cytotoxicity. INTRODUCTION Cancer or malignant neoplasm is a class of diseases in which a group of cells display uncontrolled growth, invasion and even sometimes metastasis [16,55]. It continues as a serious public health problem throughout the world as the most feared diagnosis. It is the second leading cause of human death after cardiovascular diseases in developing and undeveloped countries [7]. Treatment for cancer primarily includes surgery and chemotherapy, but the curative effects of the existing chemotherapeutic drugs are not good enough and they have plentiful side effects. The development of more effective drugs for treating patients with cancer has been a main attempt over the past 50 years [25,27,4]. Researches have shown significant progress in the utilization of metal complexes as drugs to treat several human diseases like carcinomas, lymphomas, infection control, anti-inflammatory, diabetes and neurological disorders. Schiff base ligands readily coordinate with a range of metal ions yielding stable complexes which exhibit interesting physical, chemical, biological and catalytical properties [44]. Schiff base metal complexes formed with metals like copper, gold, gallium, germanium, tin, ruthenium, iridium have shown significant Copyright © 2016 by authors and Copyright, American-Eurasian Network for Scientific Information (AENSI Publication). 160 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 antitumor activity in animals [45]. There has been considerable interest in the chemistry of the metal complexes of Schiff bases containing O, N and S donor [52]. Despite the success of cisplatin; one of the most active and clinically useful agent used in the treatment of cancer, however, it lacks selectivity for tumor tissue, which leads to severe side effects. Various tumor cell lines are now growing resistance to cisplatin [51]. For this reason, new research area are currently directed towards the development of more effective drugs with minimum side effects for treating patients with cancer. Recently, Ramadan et al., [46] reported the existence of medium in vitro cytotoxicity for a novel cobalt complex; bis-(4-bromobenzaldehyde-4-iminacetophenone) tetraaquocobalt(ΙΙ) sulphate against human liver carcinoma cell line (HepG2). The present study aimed to evaluate the biological activity as well as in vitro and in vivo cytotoxicity of that reported complex (BBIA-Co), Scheme 1. Scheme 1: Structure of the [Co(BBIA)2(H2O)4] SO4 complex MATERIALS AND METHODS a) Preparation of bis-(4-bromobenzaldehyde-4-iminacetophenone )tetraaquocobalt(II) sulphate: The complex (BBIA-Co) under investigation was prepared according to the method described by Ramadan et al., [46]. b) Biological activity: Antibacterial activity of the synthesized complex was tested using paper diffusion disc procedure. The activity was screened against two bacterial strains; Staphylococcus aureus as a gram-positive bacteria and Escherchia coli as a gram-negative bacteria. Comparison of the obtained data was carried out with the two standards: Amikacin and ciprofloxacin antibacterial agents. The nutrient agar medium; containing peptone, beef extract, NaCl and agar-agar bacteriological grade B&V (purchased from the agar company; Italy); and 5mm diameter paper discs of Whatman No.1 were used. The test compound was dissolved in DMSO (Sigma Chemical Company) in 0.1-0.4% concentrations. The paper discs were soaked in different solutions of the compound, dried and placed in the Petri plates (9 cm diameter) previously seeded with the test organisms. The plates were incubated for 24-30 h at 27 ±1°C and diameter of the inhibition zone (mm) was measured around each disc. c) In vitro study: Liver (HepG2), lung (A549) and prostate (PC3) carcinoma cell lines were used for in vitro screening experiments of the tested complexes. The cancer cells were obtained frozen in liquid nitrogen (-180 °C) from the American Type Culture Collection, Manassas, UA, USA.The sensitivity of the human tumor cell lines to thymoquinone was determined by the sulforhodamine B (SRB) assay. This method was carried out according to that described by Skchan et al. Cells were used when 90% confluence was reached in T25 flasks. Adherent cell lines were harvested with 0.025% trypsin (Sigma Chemical Co., St. Louis, Mo., USA). Viability was determined by trypan blue exclusion using the inverted microscope. RPMI-1640 medium (Sigma Chemical Co., St. Louis, Mo, and USA) was used for culturing and maintenance of the human tumor cell lines. Cells were seeded in 96-wells microtiter plates at a concentration of 5×10 4-105 cells/well in a fresh medium and was left to attach to the plates for 24 h. Growth inhibition of cells was calculated spectrophotometrically using a standard method with the protein-binding dye SRB, Monks et al. [38]. The optical density (OD) of each well was measured at 564 nm with an ELIZA microplate reader (Meter Tech. R 960, USA). The percentage of cell survival was calculated as follows: Survival fraction = O.D of treated cells / O.D of control cells. The IC50 values are the concentrations of thymoquinone required to produce 50% inhibition of cell growth. 161 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 d) In vivo study: Materials: EAC cells: Cells were obtained from American Type Tissue Culture Collection, Manassas, VA, USA. Cells were maintained in vivo in Swiss albino mice by intraperitoneal transplantation of 2 x10 6 cells/mouse after every 8 days [47]. Vehicle: The BBIA-Co complex was freshly dissolved, directly before use, in a vehicle containing dimethyl sulfoxide (DMSO) and distilled H2O (4:1 v/v). Animals Management and Groups: 125 Male Swiss albino mice (8-10 weeks of age, 30-33 g body weight) were obtained from King Fahd Medical Research Center, King Abudlaziz University, Jeddah, Saudi Arabia. Animals were kept for one week acclimatization period under controlled conditions of temperature (23-25 oC), humidity (50-55%) and light/dark cycle (12h L/12h D). Animals had free access to water and standard diet throughout the experiment. Mice were divided randomly and equally into five main groups: - Normal group: healthy animals belonging to this group were ip. injected with 0.2 ml saline, 3 times/week for 2 weeks. - EAC group: animals in this group were ip. inoculated with 2x106 Ehrlich ascites carcinoma cells/mouse and were monitored for 14 days. - Vehicle group: mice were ip. injected with 0.2 ml of the vehicle used to dissolve BBIA-Co complex (DEMSO:H2O, 4:1 v/v) - BBIA-Co group: in this group, healthy animals were ip. injected with 0.2 ml of freshly dissolved Co complex at a final dose equivalent to 40 mg of BBIA-Co/kg body weight (representing 10% of the lethal dose for BBIA-Co), 3 times/week for 2 weeks. - Treated group: animals in this group were i.p. inoculated with EAC cells (2x106 cells/mouse) followed, after 24h, by injection of 40 mg BBIA-Co complex/ Kg body weight dissolved in 0.2 ml vehicle, 3 times/week for 2 weeks. Blood Samples: Animals were monitored regularly for alterations in body weight, for the development of any signs of toxicity and mortality. After the last dose, five mice from each group were left for survival study, while the rest of animals were overnight fasted. Blood samples withdrawn from four mice in equal amount were pooled on a clean tube; resulting in five samples in each group to obtain sufficient quantity of serum for determination of biochemical parameters. Methods: Cytotoxicity parameters: a. Food intake: The amount of food intake/mouse/weak was calculated using the formula described by Anand et al., [6]: Feed consumed by 5 animals/cage/week = Total quantity of feed offered during that week (gm) - Feed left over on last day of week (gm) Feed consumed by individual animal/week = Feed consumed by 5 animals/cage/week ÷ number of mice in the cage b. The percent change in body weight (B.Wt): Body weight was registered for each mouse at the begging of experiment (zero day) and at day 15 (day of animal sacrifice). The Percent of change in body weight was calculated using the formula described by Kuttan et al., [32]: Percentage change in B.Wt = (W2 - W1) / W1 x 100 Where W1 is the body weight of each mouse at the start of experiment and W 2 is the body weight of animal at the end of observation. 162 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 c. Mean Survival time (MST) and Life Span (%LS): Animal survival time, for each group, was recorded and was expressed as mean survival time (days). Both MST and LS (%) were calculated using the equations described by Mazumder et al., [35] and Gupta et al., [23]: MST = (Day of 1st death + Day of last death) / 2 % LS = [(MST of treated group/ MST of control) - 1] × 100 d. Ascetic Fluid Collection: At the end of the experiment and before blood collection, ascetic fluid (if present) was collected and its volume was measured. EAC cells were harvested and cells viability was tested by trypan blue exclusion test using hemocytometer. The total number of cells/ml was calculated using following equation: Cell count = (number of cells x dilution) / (area x thickness of liquid film) Hematological parameters: Blood was obtained from retro-orbital sinus of the mice under light ether anesthesia using heparinized micro-capillaries. Red blood cells (RBCS) and white blood cells (WBCS) were counted by auto-hematology analyzer (BC-2800Vet, Mindray, China). Hemoglobin was measured photometricallly by fully-automated instruments. Biochemical Assays: Separated sera were used for determination of liver and kidney functions by measuring: activities of alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP); in addition to levels of: total proteins, albumin, globulin, urea, creatinine and uric acid. Determination of serum glucose and lipid profile (triglycerides, cholesterol and very low density lipoproteins) was also carried out. All the biochemical parameters were determined using specific kit for each parameter. Statistical Analysis: Statistical analysis was performed using SPSS 24.0 for windows (SPSS Inc, USA). Descriptive statistics were shown as mean ± standard error of the mean. Kruskal-Wallis test was performed for comparing more than two groups of ordinal, non-parametric data to determine if they were statistically different. Independent samples’ Mann-Whitney (U) test was used as a post hoc test to compare the medians of two groups of ordinal, non-parametric data to determine if they were statistically different. Correlation coefficient (r) was used to estimate the effect of size that describe the proportion of total variability attributable to a Variable & used Cohen’s effect size estimates to interpret the meaning of the (r) score. P value < 0.05 was considered statistically significant. Result: Biological activity: Results obtained from testing the potential activity of BBIA-Co against Staphylococcus aureus and Escherichia coli indicated the ability of the complex to inhibit the growth of both bacterial stains, although the antibacterial activity was lower than the antibacterial standards (Fig. 1). In vitro study: Results indicated that BBIA-Co complex exhibited medium cytotoxicity against the tested cell lines. The IC50 value for liver carcinoma cell lines (HepG2) was equivalent to 25 µg/ml, while the IC 50 values for both lung (A549) and prostate (PC3) carcinoma cell lines were 17.5 µg/ml and 21.7 µg/ml, respectively, as indicated in Fig. 2. In vivo study: Cytotoxicity parameters: Food Intake: Results indicated significant increase in food intake by mice bearing EAC tumor, both at 1 st and 2nd week, compared to normal mice. Same trend was noted for BBIA-Co treated EAC inoculated mice (Table 1). 163 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 Fig. 1: Biological activity of BBIA-Co complex Fig. 2: In vitro cytotoxicity of BBIA-Co complex against: liver (A), lung (B) and prostate (C) Carcinoma cell lines. Table 1: The food intake (g/mouse) for the studied groups Groups Normal EAC Vehicle Parameter 1st week 11.5±0.29 14.4±0.19 10.6±0.16 *p value -0.001 0.01 **p value --0.001 2nd week 10.1±0.43 15.8±0.38 9.3±0.27 *p value -0.001 NS **p value --0.001 * p: p value with respect to normal group, **p: p value with respect to EAC group. p value ≤ 0.05 is significant, NS: non-significant. BBIA-Co Treated 12.3±0.54 NS 0.04 13.4±0.27 0.001 0.001 14.4±0.30 0.001 NS 15.0±0.11 0.001 0.001 Change in Body Weight: The percent change in body weight of EAC group, between day zero and day 15, was significantly higher than the matched mean values in control and other groups. No significant difference between the percent change in body weight was noticed between BBIA-Co treated mice and control healthy animals (Fig. 3). It is worth to mention that at the beginning of the experiment all animal groups were matched for body weight. 164 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 Mean Survival Time (MST), Life Span (%LS): MST and % LS were calculated for both drug (BBIA-Co) and treated groups. Normal mice group was used as a control for BBIA- Co group, while EAC group was used as a control for treated group. It is worth noting to indicate that the survival of mice in both normal and vehicle groups were monitored for 120 days and no death in both groups was noted. Treatment with 40 mg Co-complex /kg B.Wt following EAC inoculation improved the mean survival time of mice which in turn resulted in prolonged life span. Normal mice injected with BBIACo complex showed reduction in their MST resulting in 21.5 % decreased life span (Table 2). Fig. 3: The percent change in body weight. p value with respect to normal group. Numbers inside the coulmn indicate standard error. p value ≤ 0.05 is significant; NS: non-significant. Ascetic Fluid Volume and EAC viability: Untreated EAC bearing mice had ascetic fluid volume significantly higher than EAC bearing mice treated with BBIA-Co. Moreover, treatment had significantly reduced the number of viable cells (13 %), resulting in increased percent of non-viable cells (Table 2). Hematological Profile: Results indicated that inoculation of EAC cells to mice resulted in significant reduction in Hb content and RBCs count, with elevated WBCs compared to normal mice group. Significant improvement in hematological parameter, (Hb, RBCs and WBCs) was obvious by treating EAC inoculated mice with Co complex, reaching WBCs mean value comparable to normal (Table 3). Table 2: Mean survival time (MST), life span (LS), ascetic fluid volume and cell viability. Groups Normal EAC Parameters MST (days) 120 26.5 ILS (%) Ascetic fluid volume (ml) 12.8 ±1.70 * p Viable cells 8.6 ± 1.54 (×108 cells/mouse) * p Non-viable cells 1.1 ±0.26 (×108 cells/mouse) * p % of viable cells 88.8 ±0.97 * p % of non-viable cells 11.0 ±0.99 * p - MST and ILS were calculated for BBIA-Co group using normal group as a control group. - MST and ILS were calculated for BBIA-Co treated group using EAC group as a control group. * p: p value with respect to EAC group. p value ≤ 0.05 is significant. Vehicle BBIA-Co Treated 120 - 94 - 21.5 - - - 47 76.6 1.7 ± 0.27 0.0001 2.7 ± 0.47 - - 0.0001 11.8 ± 1.81 - - - - 0.0001 13.3 ± 1.93 0.0001 86.7 ± 1.94 0.0001 165 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 Table 3: The hematological Profile for the studied groups Groups Normal EAC Parameter Hb (g/dl) 14.5 ± 0.22 11.4 ± 0.19 * p p Vehicle BBIA-Co Treated 13.1 ± 0.76 13.1 ±0.31 12.5 ± 0.36 -- 0.001 -- NS 0.01 0.001 0.001 0.01 Ns RBCs (×106cell/mm3) * p 8.6 ± 0.09 6.7 ± 0.13 7.8 ± 0.47 97.6 ± 0.13 7.3 ± 0.2 -- 0.001 NS 0.001 0.001 ** -- -- 0.01 0.001 0.02 7.0 ± 0.4 23.8 ± 3.63 5.2 ± 0.53 7.1 ± 0.31 6.8 ± 0.16 -- 0.001 0.02 NS NS -- 0.001 0.001 0.001 ** p WBCs (×103cell/mm3) * p ** p * p: p value with respect to normal group, **p: p value with respect to EAC group. p value ≤ 0.05 is significant, NS: non-significant. Biochemical Studies: Liver and kidney Functions: Results revealed significant change in almost all markers of liver function by EAC inoculation. Significant increase in the mean values of ALT, AST and reduced total proteins, albumin and globulin was obtained after 15 days of EAC inoculation. Injection of BBIA-Co to EAC bearing mice had significantly adjusted mean values of total proteins, albumin and globulin reaching mean values comparable to healthy animals (Table 4). Concerning kidney function, no significant changes in the mean values of urea and creatinine were noted between normal mice group and EAC bearing tumor, however, increased uric acid mean value was detected in EAC bearing mice. Treatment by 40mg BBIA-Co/kg B.Wt had significantly restored the uric acid to normal value (Table 4). Table 4: Liver and kidney functions for the studied groups Groups Normal EAC Vehicle Parameter ALT (U/L) 29.4±1.83 50.4±2.27 42.8±0.98 * p 0.001 0.001 ** p 0.02 AST (U/L) 57.7±3.22 102.6±10.89 68.6±1.59 * p 0.001 0.01 ** p 0.001 ALP (U/L) 74.6±5.10 84.8±9.07 44.3±2.25 * p NS 0.001 ** p 0.001 Proteins (g/dl) 4.6±0.03 3.6±0.01 4.8±0.05 * p 0.001 0.01 ** p 0.001 Albumin (g/dl) 2.1±0.07 1.7±0.01 2.3±0.02 * p 0.001 0.02 ** p 0.001 Globulin (g/dl) 2.5±0.08 1.9±0.001 2.5±0.05 * p 0.001 NS ** p 0.001 Urea (mg/dl) 72.0±3.24 70.1±1.15 71.8±1.49 * p NS NS ** p NS Creatinine (mg/dl) 0.3±0.01 0.3±0.01 0.3±0.01 * p NS NS ** p NS Uric acid (mg/L) 2.7±0.10 5.9±2.71 3.6±0.36 * p 0.01 NS ** p 0.01 * p: p value with respect to normal group, **p: p value with respect to EAC group. p value ≤ 0.05 is significant, NS: non-significant. BBIA-Co Treated 54.1±4.60 0.001 NS 98.5±2.29 0.001 NS 76.2±4.60 NS NS 4.2±0.04 0.001 0.001 1.9±0.03 NS 0.001 2.3±0.04 NS 0.001 63.3±2.61 0.03 0.02 0.3±0.01 NS NS 3.2±0.12 0.01 0.001 58.2±5.06 0.001 NS 104.7±1.73 0.001 NS 86.2±2.97 0.01 NS 4.3±0.04 0.001 0.001 2.1±0.02 NS 0.001 2.1±0.04 0.01 0.001 59.0±1.68 0.001 0.001 0.3±0.001 0.001 0.001 2.6±0.05 NS 0.001 Serum glucose and lipid profile: The current study demonstrated significant reduction in serum glucose in untreated EAC bearing mice group. Treatment with BBIA-Co had resulted in elevated blood glucose to value greater than normal value. Moreover, compared to normal mice group, significantly elevated triglycerides and VLDL mean values were noted in all animal groups (Table 5). 166 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 Table 5: Serum glucose and lipid profile for the studied groups Groups Normal EAC Vehicle Parameter Glucose (mg/dl) 116.2±4.33 85.2±3.07 98.2±0.63 * p 0.001 0.01 ** p 0.01 Triglycerides (mg/dl) 61.4±3.94 123.6±10.96 95.8±6.09 * p 0.001 0.001 ** p NS Cholesterol (mg/dl) 101.2±3.72 111.2±2.95 105.9±2.51 * p NS NS ** p NS VLDL (mg/dl) 12.2±0.82 28.4±2.83 20.8±1.14 * p 0.001 0.001 ** p NS * p: p value with respect to normal group, **p: p value with respect to EAC group. p value ≤ 0.05 is significant, NS: non-significant. BBIA-Co Treated 145.3±4.92 0.001 0.001 119.3±5.86 0.001 NS 93.1±2.44 NS 0. 01 23.9±1.15 0.001 NS 190.0±4.23 0.001 0.001 131.0±7.68 0.001 NS 120.6±10.67 NS NS 26.2±1.54 0.001 NS Discussion: Cancer is one of the leading causes of mortality worldwide. The failure of conventional chemotherapy to affect major reduction in the mortality indicates that new approaches are critically needed. Currently the curative effects of the exciting chemotherapy drugs are not good enough and they have plentiful side effects. The major focus of research in chemotherapy for cancer includes the identification, characterization and development of new and safe cancer chemo- preventive agents. In the current study, the antibacterial activity of a novel synthetic cobalt complex of bromobenzaldehyde-iminacetophenone (BBIA-Co) was estimated. The in vitro cytotoxicity of the complex against different cell lines was examined. Moreover, in vivo antitumor activities of the complex against EAC inoculated mice; at a dose equivalent to 40 mg/kg body weight was examined. The present data indicated that the metal complex under investigation has the capacity to inhibit the growth of the tested gram-positive (Staphylococcus aureus) and gram-negative (Escherchia coli) bacteria. The activity of this Schiff base complex may arise from the presence of C=N and C=O moieties. The mode of action may involve the formation of hydrogen bonding between these functional groups and the active centers of the cell process [1]. The initial step of evaluating most anticancer agents is cell culture. The in vitro cytotoxicity of the metal complex showed different antitumor activities against different human cancer cell lines. BBIA-Co exhibited medium in vitro cytotoxicity, with IC50 values of equivalent to 25.0 µg/ml, 17.5 µg/ ml and 21.7 µg/ ml against HPG2, A549 and PC3 cell lines, respectively. To establish the validity of the metal aqua complex as anticancer agent, further studies were carried out to demonstrate their in vivo cytotoxic activity against EAC cells inoculated in male albino mice. In this study, the BBIA-Co complex had resulted in life span prolongation of EAC inoculated mice. The mean survival time (MST) of untreated EAC bearing mice was 26.5 days. Because of rapid EAC cell division during the proliferating phase, ascites fluid accumulated, resulting in host animal death due to the pressure exerted by the tumor volume and/or the damage that resulted from the tumor itself [41,5]. Treatment with BBIA-Co complex resulted in increased MST, reaching 47 days, with percentage increase in life span equivalent to ~77 %. This result might point out to effectiveness of the complex as anticancer agent, since one of the reliable criterions for judging the value of anticancer drug is the prolongation of animal's life span by more than 25% [21]. In our study, the increased food intake by mice bearing EAC tumor during the studied experimental period probably may enhance the growth of transplanted EAC tumor cells, by enhancing glycolysis rate of the tumor, Warburg’s effect [57,31,58]. It is well known that dietary energy restriction (DER) often referred to “as under nutrition without malnutrition” is a potent inhibitor of the process of carcinogenesis. It has been hypothesized that DER primarily targets neoplastic cells by reducing net energy status and particularly glucose metabolism, therefore lower the metabolism of transformed cells, since more than 75% of tumor types relay on aerobic glycolysis [57]. This finding was recently supported by Singh et al., [53]. The authors reported that dietary supplementation of the glycolytic inhibitor, 2-diphosphoglycerate, to mice effectively inhibits tumorigenesis same as DER effects. Inoculation of EAC cells to mice, in the current research, resulted in significant increase in body weight after 14 days of inoculation (33.4 %) compared to normal mice (14.4%), which could be attributed to tumor burden. The Ehrlich tumor cells are aggressive rapidly growing cells [48] which can grow in different mice strains [3]. The implantation of EAC cells induces local inflammatory reaction with increasing vascular permeability resulting in edema, cellular migration and a progressive ascetic fluid formation [18]. The ascetic fluid constitutes nutritional source for the cells, therefore, it is essential for tumor cell growth [50]. Treating EAC group with BBIA-Co had prevented the accumulation of ascetic fluid resulting in reduction in weight gain (17.8%); almost comparable to normal mice group; at the end of experiment. 167 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 In cancer chemotherapy, the major problems that are being encountered are myelosuppression and anemia [43,24]. In the current study, EAC bearing mice had significantly decreased RBCs count and hemoglobin contents, indicating the existence of anemia. This may occur either due to iron deficiency or due to hemolytic or myelopathic conditions [19]. After treating mice inoculated with EAC cells with 40 mg of BBIA-Co complex/Kg, three times/week for two weeks, the changes in RBCs and Hemoglobin were reversed, indicating a protective action on the hemopoietic system. Moreover, tumor induction resulted in significant elevation in WBCs count while it was significantly decreased by treatment to values comparable to control mean value. The effect of EAC cells inoculation on hematololgical profile in mice was reported by many authors [11,54]. The reduction in WBCs count in animals bearing tumor after treatment was suggested to be one of the reliable criteria for judging the efficiency of the drug as an anticancer agent [9]. The presence of tumors in experimental animals was found to affect many functions of the vital organs, even when the site of tumor does not interfere directly with organ function (DeWys,1982). In the current study, elevated serum ALT and AST activities were observed in EAC bearing mice. Elevated liver enzymes in EAC bearing mice was previously reported by different researchers [22,12,11]. Our study indicated that treatment by Co- complex did not ameliorate the activities of the liver enzymes. Moreover, injecting the complexes to healthy animals had resulted in significant increase in ALT and AST activities, with AST mean values more than twice that for ALT in all groups receiving BBIA- Co complex. ALT is found predominantly in the liver, with clinically negligible quantities found in the kidneys, heart and skeletal muscles. On the other hand, AST is found in the liver, cardiac muscle, kidneys and red blood cells. In this study as AST activity is higher than ALT activity in the groups of animals receiving the complex under examination, this may reflect the existence of muscle source of these enzymes, probably cardiac muscles, rather than liver inflammation. Furthermore, plasma proteins yield most useful information in chronic liver disease, since the liver is the site of albumin synthesis and also possibly of some α and β –globulins. In this study, EAC bearing mice exhibited decreased total proteins mainly, albumin. This finding is consistent with other studies [30,2]. Treatment of tumor bearing mice with the complexes under investigation had significantly restored the changes in total proteins and albumin toward normal, suggesting restoration of liver function, which in term may support the suggestion of other sources for elevated enzymes rather than the liver. In the present work, no significant differences in serum urea or creatinine levels were noted, indicating unchanged kidney function by EAC inoculation. On the other hand, remarkable increase in serum uric acid mean value was obtained in EAC bearing mice group. Treatment with BBIA-Co had significantly restored uric acid to normal values. Uric acid is the final product of purine metabolism and its circulating concentrations are regulated by the balance in its production and excretion [56]. The elevated serum uric acid in EAC bearing animals could be due to the malignant process itself, resulting from the increased nucleic acid turnover in the rapidly proliferating diseased tissue [49]. The current study demonstrated that EAC inoculation to mice had resulted in a significant decrease in blood glucose compared to normal mice. Most studies had observed lower serum glucose in tumor bearing mice [40,10,26]. One reason for hypoglycemia could be an augmented consumption of glucose by the cells of the tumor [13,39]. As indicated in the current study, treatment had resulted in increased serum glucose, although levels highly exceeded those for normal control. In the current study, EAC induced defects in lipid profile of mice, as manifested by elevated TAG and VLDL. In this regards, several authors have presented evidences indicating that EAC had affected lipid metabolism of the host animals. Highly significant increase in serum TAG, free fatty acids and ketone bodies in tumor bearing mice were previously demonstrated [8,33,14]. Several mechanisms have been proposed by different authors to explore the hypertriglyceridemia and elevated VLDL associated with EAC bearing mice. Lyon et al., [34] attributed the elevated TAG to defective removal of VLDL-TAG. Others [29] reported that fasting lowers plasma TAG in mice, but chronically lowered insulin levels and other hormonal changes that followed reduced food intake might cause inhibition of VLDL-TAG removal from the circulation. The high degree of lipemia in tumor bearing animals could be also due to mobilization of body fat for energy production [42,36,37]. Another possible explanation for hypertriglyceridemia was the suggestion that the presence of tumor leads to secretion of TAG in the form of VLDL; properly from the liver; which enters the peritoneal cavity. TAG and cholesterol contained in VLDL are taken up and utilized by Ehrlich cells, suggesting that one function of VLDL is to transport lipids from host tissue to the growing tumors [34]. In this study, although treatment with BBIA-Co complex had ameliorated some of the abnormalities induced by EAC inoculation, however the complex was unable to improve the changes in lipid profile. The mode of action of the cobalt drug under investigation, [Co(BBIA) 2(H2O)4]2+, might be comparable to that of the established cisplatin drug. Cisplatin, cis-diamminedichloroplatinum (II) [PtCl2(NH3)2], is a wellknown chemotherapeutic drug. Following administration, one of the chloride ligands is slowly displaced by water, in a process termed aquation. The aqua ligand in the resulting [PtCl(H2O)(NH3)2]+ is itself easily displaced, allowing the platinum atom to bind to bases. Of the bases on DNA, guanine is preferred. Subsequent to formation of [PtCl(guanine-DNA)(NH3)2]+, cross-linking can occur via displacement of the other chloride 168 Amani F. H. Noureldeen et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170 ligand, typically by another guanine. Its mode of action has been linked to its ability to cross-link with the purine bases on the DNA; interfering with DNA repair mechanisms, causing DNA damage, and subsequently inducing apoptosis in cancer cells. Cisplatin cross-links DNA in several different ways, interfering with cell division by mitosis. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible [18,15]. Also, various transition metal complexes were synthesized and checked for the possibility to act as antitumor drugs. Our cobalt complex ([Co(BBIA) 2(H2O)4]2+) has proven to have promising in vitro and in vivo cytotoxicity. Therefore, it is suggested that the cobalt complex, [Co(BBIA) 2(H2O)4]2+, could presumably behave the same as cisplatin. Interestingly, the drug already has coordinated water molecules which eliminate the need for the aquation step necessary for the cisplatin. 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