Vitamins C and E as Effective Protectors against Potassium Bromate-induced Cardiac Injury in Rats

Potassium bromate (KBrO3) is widely used in foods and water, in spite of its well-known oxidative cell and tissue damage. Therefore, vitamins C and E are examined to alleviate its cardiac injury. For this purpose 72 adult male albino rats were categorized into 6 groups. Group1 served as control; group 2 served 30 mg/Kg/ day vitamin C; group 3 served 300 mg/kg/day vitamin E; group 4 was injected intraperitoneally with KBrO3 20 mg/Kg/ dose twice weekly; and groups 5 and 6 received either vitamins C or E with KBrO3 in the same scheme. After 4 weeks, heart and serum were collected for analysis. KBrO3-induced cardiac injury was evidenced by a significant increase in serum asparate transaminase (AST), creatine phosphokinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) activities and cardiac troponin I (cTnI) level. Significant reduction in cardiac collagen synthesis and elevation in matrix metalloproteinase-1 (MMP-1) and tumor necrosis factorα (TNF-α) were noticed in KBrO3-intoxicated rats. These changes were ameliorated in the vitamins C and E-treated groups through their antioxidant and anti-inflammatory properties.


INTRODUCTION
Potassium bromate (KBrO 3 ) is a food additive used primarily as a maturing agent to dough, a conditioner for fish paste and added to beer, cheese or fermented beverages. In addition, KBrO 3 is a constituent in cold-wave hair solutions [1]. It is also generated as a disinfectant in drinking water managed with ozone or chlorine [2].
Several reports on the assessment of KBrO 3 danger show that it is highly toxic as it causes lipid peroxidation and oxidative DNA damage in human and other mammals, which has induced by BrO 3 -in the presence of intracellular sulfhydryl compound such as reduced glutathione or cysteine [1]. Oral doses of 185-385 mg/Kg body weight results in irreversible toxic effects on body systems. The LD50 of KBrO 3 was reported to be 157 mg/Kg body weight, while lower doses are associated with vomiting, diarrhea, nausea and abdominal pain [3][4][5].
These reactive oxygen species (ROS) is believed for the cardiotoxicity generation. Different cardiac oxidases especially xanthine oxidase, monoamine oxidase and NADH oxidase produce H 2 O 2 , which directly and/or indirectly produce reactive hydroxyl radicals responsible for initiation and progression of myocardial injury. However, the myocardium has enzymatic and non-enzymatic system to neutralize free radicals [6]. But, due to the highly oxidative metabolism of KBrO 3 and fewer reactive cardiac antioxidant defenses, that heart is very sensitive to ROS induced damage [7].
Vitamins as vitamin C and E are important components in the human diet. They exert protective effect against diseases such as cancer [8,9], cardiovascular diseases [10,11] and fatty liver [12] which may be attributed to its powerful antioxidant properties. As an antioxidant, they protect against the damaging effects of free radicals by scavenging lipid peroxy radicals and singlet oxygen [13]. Moreover, they stabilize the membrane and biological molecules such as DNA, proteins and lipids [14].
Vitamin C is an effective antioxidant, acting to lessen oxidative stress that it acts as a reducing agent, donating electrons to various enzymatic and a few non-enzymatic reactions. The oneand two-electron oxidized forms of vitamin C, semidehydroascorbic acid and dehydroascorbic acid, respectively, can be reduced in the body by glutathione and NADPH-dependent enzymatic mechanisms. The presence of glutathione in cells and extracellular fluids helps maintain ascorbate in a reduced state. Ascorbic acid performs numerous physiological functions in the human body. These functions include the synthesis of collagen, carnitine, and neurotransmitters; the synthesis and catabolism of tyrosine; and the metabolism of microsome [15].
Vitamin E has many biological functions. As an antioxidant, vitamin E acts as a peroxyl radical scavenger, disabling the production of damaging free radicals in tissues, by reacting with them to form a tocopheryl radical, which will then be reduced by a hydrogen donor (such as vitamin C) and thus return to its reduced state. As it is fat-soluble, it is incorporated into cell membranes, which protects them from oxidative damage [16]. Other functions include enzymatic activities [17], gene expression [18], and neurological functions [19].
Thus, the aim of this work was to ascertain the role of vitamins C and E as antioxidants to inhibit the toxic effects of KBrO 3 on heart.

Chemicals
KBrO 3 used was imported from Alpha Chemica, India. Vitamin C was in the form of Cevarol oral drops from UniPharma Company, Cairo, Egypt. While vitamin E used from vitamin E tablets of Pharco Pharmaceuticals, Alexandria.

Animals and Treatment
Seventy two adult male albino rats "Sprague Dawely" 158-198 g were kept in stainless steel cages in the well-ventilated animal house of the Medical Research Center of the Faculty of Medicine, Ain Shams University from acclimatization (7 days) till the end of the experimental period (4 weeks). They had access to 12 h cycle of light/dark and provided with standard diet prepared by AIN [20] and tap water ad libitum.
KBrO 3 was injected intraperitoneal at a toxic dose of 20 mg/Kg body weight/dose twice weekly prepared as 40 mg/ml distilled water [21]. The vitamin C was given orally in a high dose of 30 mg/Kg body weight/dose daily [14]. The vitamin E was given orally in a dose of 200 mg/Kg body weight/dose daily [12]. The animals were weighed weekly and the change in body weight was calculated at the end of the experimental period.

Sample Collection
At the end of the experimental period, animals were sacrificed under ether anesthesia. Blood was collected from the hepatic portal vein, centrifuged (10 min, 3000 rpm, 4°C) for serum separation. Hearts were excised, washed, dried and weighed for calculating its relative weight.

Serum Biochemical Assays
Creatine kinase isoenzyme-MB (CK-MB), asparate aminotransferase (AST) and lactate dehydrogenase (LDH) activities were determined according to standard methods using diagnostic kits from BioSystems S.A. (Barcelona, Spain) according to Reitman and Frankel [22], Buhl and Jackson [23] and Dawson et al. [24], respectively. Assessment of serum cardiac troponin I (cTnI) level was carried out by enzyme-linked immunosorbent assay using kit purchased from Cloud-Clone Corp., USA according to Apple et al. [25].

Cardiac Biochemical Assays
Cardiac collagen was extracted from 100 mg tissue by using PBS. The homogenized solution (1 ml) was stored overnight at -20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 X g, 2-8°C. The supernatant was removed for assay by the quantitative sandwich immunoassay technique ELISA kit (Cusabio, USA) according to Neuman and Logan [26].
Cardiac matrix metalloproteinase-1 (MMP-1) and tumor necrosis factor-α (TNF-α) were extracted by rinsing the tissue in 5-10 ml PBS with a glass homogenizer on ice. The resulting suspension was subjected to two freeze-thaw cycles to further break the cell membranes. Then the homogenates were centrifuged for 5 minutes at 5000 X g and the supernatants were removed for assay by enzyme-linked immunosorbant assay kits (Cloud-Clone Corp., USA) according to Zhang

Statistical Analysis
Results were expressed as mean ± Standard deviation (S.D) of the mean. Differences among means were tested for statistical significance by one-way analysis of variance using SPSS package version 16. Statistical significance was considered when P ≤ 0.05.

Effect of Vitamins C and E Administration on the Body Weight Gain and Relative Heart Weight in KBrO 3 -toxicated Rats
The results of this study indicate that vitamin C or E administration alone did not affect the body weight gain and relative heart weight of rats as compared to control group (Table 1). KBrO 3 treatment decreased significantly (P ≤ 0.05) body weight gain with no change in relative heart weight. Furthermore, the vitamins treatments of KBrO 3 showed significant (P ≤ 0.05) improvement in body weight gain compared to the toxicated group.

Effect of Vitamins C and E Administration on the Cardiac Enzyme Activities in KBrO 3 -Toxicated Rats
Significant alterations in serum cTnI, LDH, CK-MB, and AST activities were seen in rats treated with KBrO 3 as compared to control group ( Table  2). Supplementations of vitamins C or E alone did not affect serum cTnI level as compared to control group. While their administration to rats treated with KBrO 3 showed significant (P ≤ 0.05) serum reduction compared to KBrO 3 treated group indicating tissue improvement.

Effect of Vitamins C and E Administration on Collagen Synthesis and Inflammation in KBrO 3 -Toxicated Rats
Treatment of rats with KBrO 3 led to marked (P ≤ 0.05) elevation in heart collagen, MMP-1, and TNF-α levels indicating cardiac injury (Table 3). Moreover, vitamins C or E supplementation alone did not have any effect on these parameters which were similar to control. Furthermore, their administration to rats treated with KBrO 3 showed significant (P ≤ 0.05) decrease of collagen, MMP-1 and TNF-α levels compared to the KBrO 3 treated group.

DISCUSSION
Previous studies have reported that oxidative stress plays an important role in the pathophysiology of KBrO 3 -mdiated damage [7,14,29]. The oxidative stress of KBrO 3 promotes oxidative cell damages and injuries in different tissues and organs through the production of ROS which react with protein, lipids and nucleic acids [30]. As there is a negative balance between oxidative stress damage and antioxidants; the heart tissue is very sensitive to ROS damage induced by KBrO 3 because of its high oxidative metabolism and weak antioxidant defense [14]. However antioxidants are molecules that can prevent or reduce the extent of oxidative destruction of biomolecules [31]. El-Deeb and Abd-El-Hafez [14] concluded that there were morphological changes of mitochondria induced by ROS produced from KBrO 3 . These alterations lead to impair the ability of mitochondria to synthesize ATP and to carry out their wide range of metabolic functions. Moreover myofibrillar injury were arised as a secondary event of the mitochondrial dysfunction, which could lead to an imbalance in calcium uptake and loss of ATP production, resulting in disturbance of normal myofibrillar structure and function causing altered myofilament formation and renewal [32].  As a consequence of the ROS generated from KBrO 3 intoxication, that the cardiac structure integrity was greatly affected. This was revealed from the decreased level of cardiac collagen synthesis accompanied with increased activity of MMP-1 and level of TNF-α in the KBrO 3 toxicated rats. The myocardial structural integrity is determined by the fibrillar collagen which is regulated by balance between synthesis and degradation [43]. ROS effect on collagen metabolism in cardiac fibroblasts is taken place by affecting both synthesis and the activity of degradative enzymes. ROS decreased collagenase-sensitive [ 3 H] proline incorporation and the abundance of mRNA for procollagenaseα 1 (I), α 2 (I) and α 1 (III). On the other hand, ROS increased the total activity of extracellular MMP-1 through the transcriptional and posttranscriptional levels. The posttranscriptional is activated by ROS through the latent proenzymes [44]. Moreover El-Deeb and Abd-El-Hafez [14] concluded that KBrO 3 toxicity induced cardiac extracellular matrix (ECM) expansion and remodeling as a result of the increased fibroblasts activity which leads to fibrosis. In addition, fibroblasts produce a number of cytokines, peptides and enzymes among which MMP and their inhibitors directly impact of ECM turnover and homeostasis. However these alterations were arrested from the start in the vitamin C treated rats.

Parameters Groups
The TNF-α is one of the inflammatory cytokines, that is increased as a result of KBrO 3 induced ROS. Oxidative stress is known to activate p-38 mitogen which activates protein kinase and nuclear factor kappa B and thus plays a role in the sequence of signaling events involved in the production of myocardial TNF-α [45]. Previously, it was concluded that TNF-α exerted potent effect on the collagen metabolism resulting in a depletion of fibrillar collagen [46]. Therefore the administration of either of the vitamins C or E parallel to KBrO 3 maintained the regulation between collagen synthesis and degradation and inflammation indices through their antioxidant capacities as previously reported by John et al.

CONCLUSION
The present study indicates that administration of each of the vitamins C or E has a protective effect on cardiac injury induced by KBrO 3 , which is related to their anti-oxidative and antiinflammatory properties.