Salubrious Therapeutic efficacy of Myrtenal on Colon Carcinoma induced by 1, 2-Dimethylhydrazine studied in experimental albino rats.

Sathishkumar Venkatachalam, Lokeshkumar Boobathi, Maruthaiveeran Periyasamy Balasubramanian*

Department of Pharmacology and Environmental Toxicology, Dr. A.L. Mudhaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, 600113, Tamilnadu, India

*Corresponding Author E-mail: sathish.vmr@gmail.com

ABSTRACT:

Background: LPO-derived DNA adducts in the mammalian cells induce point mutations, chromosomal aberrations, and recombination which are expressed by inﬂammatory mediators, such as cyclooxygenase-2 and lipoxygenases that are found to be increased during colon cancer development. Since the risk of being diagnosed with cancer, it increases with age, most cases occur in adults who are middle aged or older. Objective: About 77% of all cancers are diagnosed in persons 55 years of age and older. Cancer researchers use the word “risk” in different ways, most commonly expressing risk as lifetime risk or relative risk. DMH acts as a potent site and organ specific carcinogen by generating various reactive metabolic intermediates leading to oxidative stress. Experimental design: Male Wistar albino rats were divided into four groups and each group consisting of six animals. Group I and group IV were vector and drug control. The group II and group III animals were treated with DMH 20 mg/kg bodyweight to induce colon carcinoma. Rats received cancer bearing Group III animals were treated with Myrtenal at the concentration of 230 mg/kg bodyweight for 15 weeks . At the end of the experimental period all the rats were sacrificed. DNA adducts where investigated in the experimental animal model with a potent procarcinogen DMH, an alkylating agent that targets DNA and induces the formation of methyl adducts with DNA bases, point mutations, micronuclei, and sister chromatid exchanges yielding macroscopically visible neoplasm in a dose-dependent manner. Results: The colon and liver tissues levels of the enzymic and non-enzymic antioxidants were significantly decreased in cancer bearing animals when compared to the control animals. Lipid peroxide levels (LPO) were estimated. Conclusion: From our results, we conclude that Myrtenal is a potent antioxidant and play a protective role against DMH induced colon cancer.

KEYWORDS: Colon cancer, Myrtenal, 1, 2-Dimethylhydrazine , Antioxidants, Phase I Enzyme

INTRODUCTION:

Colorectal cancer (CRC) is the third most common cause of cancer mortality in women and fourth in men [1]. It is fitting therefore that The Cancer Genome Atlas (TCGA) published a comprehensive characterization of the genetics

of CRC as their third publication [2]. Because CRC is often diagnosed at a late stage, and the early detection of cancer dramatically increases survival, the identification of genetic risk factors in CRC is of the utmost importance. The most widely recognized pathway describing CRC progression is the adenomacarcinoma sequence [3] (Figure 1): beginning as benign polyps/dysplastic lesions before progressing to advanced adenoma, and finally to invasive carcinoma. A variety of molecular pathways and genes are involved in the progression, which typically occurs over years or decades [4]. Carcinomas that remain confined to the colon wall are curable with surgery, while most (73%) of those that progress to stage III tumors (metastasize to regional lymph nodes) are treatable with a combination of surgery, chemotherapy, and radiotherapy [5,6]. Further, the colorectal cancer is characterized as one of the most prevalent cancers in many developed countries, following westernized food practice [7].Various epidemiological studies suggest that, imbalanced diet and obesity as a major risk factor for colorectal cancer[8,9]. Furthermore, environmental carcinogens which include genotoxic and non-genotoxic agents, triggers free radical mediated damage to the cell membrane associated polyunsaturated fatty acids causing lipid peroxidation [10], thereby releasing lipid per oxidation byproducts such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA) that are generated from docosahexaenic acid, arachidonic acid and linoleic acid, results in covalent modifications that can interfere with protein function and stability, enhancing the carcinogenesis process [11]. These intermediates readily react with DNA bases at specific sites to form exocyclic DNA adduct of which several has been characterized as propano and etheno DNA base adducts [12]. LPO-derived DNA adducts in the mammalian cells induce point mutations, chromosomal aberrations, and recombination which are expressed by inﬂammatory mediators, such as cyclooxygenase-2 and lipoxygenases that are found to be increased during colon cancer development [13]. As reported by Miller, DNA adduct formation in cells is critical and if not repaired, can lead to the development of cancer. DNA lesions in human study subjects offer new tools in cancer etiology research and are useful in verifying the efficacy of chemopreventive agents in reducing endogenous DNA damage and cancer risk [14]. DNA adducts where investigated in the xperimental animal model with a potent procarcinogen DMH, an alkylating agent that targets DNA and induces the formation of methyl adducts with DNA bases, point mutations, micronuclei, and sister chromatid exchanges yielding macroscopically visible neoplasm in a dose-dependent manner [15] which produces free radicals in circulation after metabolisation of DMH in the liver yielding electrophilic diazonium ions [16]. Favored by high fat consumption, increased levels of bile acids in the colon can modify the metabolic activity of the intestinal microflora, with the conversion of bile acids and neutral sterols to reactive carcinogens leading to oxidative stress [17]. Human body is equipped with various antioxidants viz. superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), ascorbic acid (Vitamin C), α-tocopherol (Vitamin E), etc., which can counteract the deleterious action of ROS and RNS and protect cellular and molecular distortion [18]. Provided with the family of drug metabolizing phase I enzymes cytochrome P450s (CYPs) Cytochrome b5 and; phase II enzymes including detoxifying and antioxidant enzymes such as glutathioneS-transferases, -glutamylcysteine synthetase, NADP (H): quino oxidoreductase 1 (NQO1), and UDP:glucuronosyl transferases[19] functions by regulating toxic, oxidative damaging, mutagenic and neoplastic effect of chemical carcinogen [20]. They catalyze oxidative or reductive reactions of endogenous lipophilic (steroids, bile acids, fatty acids, prostaglandins) and exogenous compounds (drugs) into more polar (hydrophilic) products, allowing their elimination in the urine.[21] Supporting the antioxidant defense mechanism the pioneering efforts of Wattenberg worked on the exploration of chemopreventive drugs, as a novel approach to control the incidence of colon cancer [22] thereby inducing cell death in cancer cases was experienced with monoterpenes a promising compound composed of two isoprene units which induces apoptosis in cancer cells by modulation in cell signaling pathway with remarkable biological activities such as antioxidant, chemotherapeutic and chemopreventive effects in different models of cancer [23]. Myrtenal a member of monoterpenes is found predominantly in cumin, pepper, mint, eucalyptus has been postulated to possess various biological activities such as anti-malarial, anti-plasmodial, anti-radicular, cyclooxygenase-inhibitor, gonadotrophic, hypocholesterolemic and immunostimulant effects [24] but there is a paucity of information regarding its role as a chemotherapeutic agent, especially in DMH induced colon carcinogenesis. Hence, the present investigation was undertaken to evaluate the anticancer potential of myrtenal against experimental Colon carcinoma (CRC) in Wistar albino rats.

MATERIALS AND METHODS:

Reagents

1, 2-Dimethylhydrazine (DMH) ,Myrtenal was purchased from Sigma Chemical Company, St. Louis, MO, USA. All the other chemicals used in this study were of analytical grade available commercially.

Experimental animals

Experiments were carried out with 5 weeks old male Wistar rats procured from central animal house facility, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai–600113. They were maintained in the controlled environmental conditions of temperature and humidity on alternative 12 h light/dark cycle, noise level maintained below 85 db and had unrestricted access to standard diet consisting of 24% protein, 4.5% fat and 4% fibre. The experiment was sanctioned and approved by the Institutional Animal Ethical Committee (IAEC No.01/13/2013).

Experimental Design

The experimental animals were divided into four groups, each group comprising six animals.

Group 1: Control animals fed with standard diet and pure drinking water.

Group 2: Animals were administered with 20 mg/kg body weight of DMH, in 1 mM EDTA, pH adjusted to 6.5 with 1 mM NaOH and subcutaneously injected once in a week for 15 weeks.

Group 3: Animals were treated with Myrtenal (230 mg/Kg b.wt.) with corn oil as vehicle for 15 weeks by intragastric administration. Myrtenal treatment was started 1 week prior to the first dose of 20 mg/kg body weight of DMH (as in group 2) and continued till end of the experimental period.

Group 4: Animals were treated with Myrtenal (230 mg/Kg b.wt.) for 15 weeks by intragastric administration to assess the cytotoxicity if any, induced by Myrtenal, and rats were referred as drug control.

After the end of the experimental period, the rats were fasted overnight and anesthetized using diethyl ether and sacrificed by cervical decapitation. A portion of colon was used for histopathological studies and remaining tissue was homogenized in 0.1 M Tris–HCl buffer pH 7.4 and centrifuged, the supernatant was used for biochemical studies.

Colon analysis

Colons were excised from experimental rats, and were blotted dry and opened longitudinally, with the inner surface examined for visible macroscopic lesions. Tumor weight, Tumor incidence (percentage of animals with tumors) and multiplicity (mean counted tumors per animals) were determined for the colons. Immediately following sacrifice, colons were removed and washed with ice-cold saline, and colon homogenates (10%) were prepared in ice cold TBS (Tris 50 mM and NaCl 150 mM; pH 7.2) then centrifuged at 10,000g for 10 min at 4^●C and were stored as aliquots at or below -20^●C for subsequent assays.

Biochemical analysis

The protein content was estimated by the method of Lowry et al (1951) [25] using bovine serum albumin as standard. The nucleic acids were extracted by the method of Schneider (1957) [26].DNA was estimated by the method of Burton (1956) [27].RNA was estimated by the method of Rawal et al (1977) [28].

Lipid peroxidation and antioxidant Assay

The macromolecular damage such as LPO was estimated by the method of Ohkawa et al,(1979) [29] For lipid peroxidation (MDA) analysis, butylated hydroxytoluene (BHT) was added to colon homogenates at 1% final concentration to prevent further oxidation during sample storage (a week at ±20^●C). MDA production in the colon was measured according to the method of Yagi, [30] in which MDA forms a pink coloured complex with thiobarbituric acid with maximum absorbance at 535 nm. CAT activity was measured by determining the decomposition of H₂O₂ as described by Sinha, (1972) SOD activity was estimated by the method of Marklund and Marklund(1974)[31]. GPx activity was measured according to the method of Rotruck et al. (1973) .GSH level in the colon was determined by the method of Moron et al., (1979) . GST was estimated by the method of Habig (1981) [32].GR was estimated by the method of staal et al. (1969) . Vitamin E was estimated by the method of Desai (1984) . The levels of phase I enzymes (CytochromeP450, Cytochrome b5, NADPH Cytochrome ‘C’ reductase) and phase II enzymes [33] (Glutathione-S-Transferase(GST) and UDP-Glucuronyl transferase) in liver tissue homogenate were determined.

Histopathology

Fresh colon tissue specimens were fixed in buffered formalin for 48 h, followed by dehydration in ascending grades of alcohol, cleared in benzene and was embedded in paraffin wax. Paraffin block 4μm thick sections were double stained with haematoxylin and eosin, and were analysed using an optical microscope.

Statistical analysis

Values are expressed as mean±S.D. The results were statistically evaluated using one-way analysis of variance (ANOVA) by SPSS 10.0 student version followed by Turkey’s multiple comparison method to compare means of different groups. The mean difference is significant at the 0.05 levels.

RESULTS:

The effect of Myrtenal on body weight of control and experimental animals are presented in Table 1. The body weights were found to be significantly decreased in group 2 cancer bearing animals when compared with group 1 control animals. On the contrary, the administration of Myrtenal increased the body weight in group 3 animals when compared to group 2 animals. However, no significant changes were observed in Myrtenal alone treated group 4 animals when compared to group 1 control animals.

Colon tumor analysis

Tumor incidence

DMH- induced colon tumor development had a tumor incidence of 100%. Myrtenal treatment resulted in decrease of tumor incidence compared to DMH-induced tumor bearing animals in group 2. On the other hand group 1 control and group 4 myrtenal alone treated rats showed no difference.

Table 1: Incidence of colon tumor and the number of tumors/tumor bearing rats

Groups	No. Of Rats	No. Of Tumor Bearing Rats	Tumor Incidence (%)^*	Total No. Of Tumors	No Of Tumors /Tumor Bearing Rats
Control	6	0	0	NIL	NIL
Dmh	6	6	100	17	3.33
Treatment	6	2	33.11	5	2.3
Drug control	6	0	0	NIL	NIL

*(Number of tumor bearing rats/total number of rats in each group) x 100

Fig.1 Effect of Myrtenal on Lipid peroxidation in Colon of Control and Experimental Animals

Results are expressed as mean ± S.D for six rats in each group. Statistical significance p < 0.05 compared with ^agroup 1, ^bgroup 2, and ^cgroup 3 based on Duncan’s multiple range test. Units: n moles of MDA liberated μg/mg tissue.

ROS generation

ROS clearly possess the capacity to behave in a sporadic and destructive fashion [34]. The high rates of lipid peroxidation and the different forms of DNA base lesions that result in genomic instability, such as strand breaks, base modiﬁcations and DNA–protein cross linkages, have been found in the majority of neoplastic tissues. ROS-mediated DNA damage plays an essential role in the initiation of carcinogenesis, as well as in malignant transformation [35]. In this study the ROS level was seen increased in DMH treated group 2 rats compared to group 1 control. Myrtenal treated group 3 rats showed significant decrease in ROS levels Compared to group 2 rats. Group 4 myrtenal alone treated rats showed no changes. DNA-reactive aldehyde can damage DNA either by reacting directly with DNA bases or by generating more reactive bifunctional intermediates, which form exocyclic DNA adducts. Of these, 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA), acrolein, and crotonaldehyde have been most intensely studied with respect to their chemical and biological interactions with nucleic acid bases. The ability of these reactive electrophiles to modify DNA bases, yielding promutagenic lesions, is considered to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO, HNE and MDA which are increasingly been implicated in carcinogenesis [36]. LPO was found to be significantly increased in group 2 cancer bearing animals compared to group 1 control animals. On administration of Myrtenal significantly reduced the peroxidation reaction in group 3 Myrtenal treated animals compared with group 2 cancer bearing animals. However, no significant changes were observed in group 4 Myrtenal alone treated animals. The results show a significant reduction of SOD and CAT activities in DMH alone-treated group 2 rats as compared to the control. But on Myrtenal supplementation in group 3 the SOD and CAT activities were significantly elevated as compared to the unsupplemented DMH-treated group 2.The levels of GSH and GR activity were significantly lowered in animals that were treated with DMH in group 2 as compared to group 1control rats. The levels were significantly increased on supplementation with Myrtenal. GPX and GST levels were seen decreased upon myrtenal supplementation compared to group 2 DMH induced rats. The concentrations of α-tocopherol were higher on DMH treated group 2 as compared to group 1 control animals. But on Myrtenal supplementation, the concentration of α-tocopherol was significantly decreased (P<0.05) as compared to the unsupplemented DMH-treated group. The total protein level in colon of control and experimental animals were studied. A significant decrease in the total protein level was seen in group 2 colon cancer bearing animals compared to group 1 control animals. Upon myrtenal supplementation total protein level was elevated compared to the group2 cancer bearing animals, there were no significant changes observed in the group 4 myrtenal alone treated animals compared to group 1 control animals. In group 2 colon cancer bearing animals Phase I enzyme levels were elevated compared to group 1 control. The activities of Cytochrome P450 and Cytochrome b5 were significantly decreased in myrtenal treated group 3 animals compared to group 2.No significant changes were observed between group 4 myrtenal alone treated animals and group 1 control animals. Phase II biotransformation enzymes projected decreased levels in group 2 cancer bearing animals compared to group 1 control animals. Group 3 myrtenal treated animals showed elevated levels of GST and UDP-GT compared to group 2 animals. Group 4 myrtenal alone treated animals and group 1 control animals showed no significant difference.

DISCUSSION:

Colon cancer incidence with few to no symptoms is often diagnosed in the later stages of cancer with the conventional cancer therapy being surgery followed by administration of anticancer drugs [37], which may lead to side effects hence Chemopreventive and dietary care strategy for colon cancer treatment is extensively studied for its antioxidant ability to prevent cancer in humans [38]. Research on dietary products has so far showed that monoterpenes such as myrtenal, present in spices are well known to cure digestive ailments [39] and resolve cellular abnormality in cancer condition by counteracting Reactive oxygen species [40]. The sources of cellular ROS include leakage from the mitochondrial electron transport chain located in oxidative phosphorylation complexes of mitochondria as well as a number of ROS-generating plasma membrane and cytosolic enzymes [41]. Elevated ROS levels play an essential role in the proliferation of colon carcinoma of epithelial cells, as well as the ability of certain tumors to enhance angiogenesis [42], which alter redox regulation of cellular signaling pathways and induce cellular redox imbalance and shut off immune functions leading to lipid peroxidation which serves as a biomarker of carcinogenesis [43] Increased lipid peroxidation alters membrane fluidity and membrane potential of the colon mucosa leading to loss of cellular function and induces cell death.Elevated levels of MDA an end product of LPO during carcinogenesis was seen as a biomarker which [44] may be due to DMH, a methylating agent which stimulate cell division and induce colorectal tumor formation by releasing ROS which interferes with DNA, in a manner similar to that which occurs in humans [45]. Antioxidants provided by Myrtenal in colon cancer treated animals showed decreased MDA levels thereby stabilizing the cellular integrity of the cancer cells by preventing from further Lipid peroxidation.ROS produced by NADPH oxidases(NOXs) function as signaling molecules in most eukaryotes[55] but over production of ROS by the activity of adenosine on the epithelial cells of colon disrupts the membrane potential and lead to cell death[46] Hence an immediate recovery of metabolic pathways via enzymatic reactors include the (SOD) family members (Mn, Cu and Zn SOD) that catalyze the dismutation of superoxide anion O₂ to form hydrogen peroxide (H₂O₂), which is further detoxiﬁed to water by glutathione peroxidase [47] a selenium-dependent antioxidant enzyme that reduces H₂O₂ and lipid peroxides/hydroperoxides by oxidizing glutathione. [48] The scavenging of H₂O₂ and inhibiting of the inactivation of SOD by GPx or CAT play an important role in the preservation of its antioxidant ability and the balance between the production and destruction of ROS in organisms. On contrary, GPx and CAT could also be inactivated by superoxide radical and this inactivation can be completely prevented by SOD. So the optimal protection of cells could be achieved only when an appropriate balance between the activities of these enzymes is maintained [49].Glutathione S-transferases (GSTs) are known to catalyze the conjugation of glutathione (GSH) with different species of electrophilic compound to detoxify and protect cells against reactive oxygen metabolites . Electrophilic diazonium ions produced by DMH are detoxified by GST dependent enzymes, by the oxidation of myrtenal with catalytic Cytochrome P₄₅₀ analogues which involve oxometallic species such as the metalloporphyrins [50] which are widely and intensely investigated in the area of catalysis and also mimics enzymes like catalase, peroxidase, and P₄₅₀ cytochromes or as transmembrane electron transport agents . Cytochrome P-450 the most important mono oxygenase reacts with molecular 0₂ in such a way that one of the 0-atom is reduced to water and the other is introduced into the organic substrate [51] as a result enhancing the activity of SOD, CAT, GPx, GST, GSH and GR. Results of Myrtenal supplementation exhibits the increased levels of Enzymic antioxidants as a factor of its antioxidant property. Interactive effects between vitamins C and E in preventing lipid peroxidation has been evidenced. Histopathological analysis shows that morphological changes in the tumor dysplasia characterizing the antitumor activity by restoring the distorted cancer cell to near normal.

CONCLUSION:

In conclusion, the present study clearly provides information about the chemo preventive activity of myrtenal in colon cancer induced by DMH. However, studies are in progress in exploring the cellular mechanism by which myrtenal prevents and/or inhibits experimental colon carcinogenesis.

ACKNOWLEDGEMENT:

The authors extremely grateful to Dr. R. Venkatakrishna Murali, M.D., Ph.D., Professor and Head, Department of Pharmacology and Environmental Toxicology, Dr. A.L. Mudhaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai–600113 for providing the laboratory facility.

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S.NO	Particulars	Group I	Group II	Group III	Group IV
S.NO	Particulars	Control	DMH	DMH + Myrtenal	Myrtenal
1	SOD	23.2±0.41	19.41±0.64	21.34±0.5	23.9±0.35
2	CAT	2.16±0.02	1.63±0.06	2.02±0.02	2.47±0.04
2	GPx	26.23±0.43	15.18±0.36	19.21±0.45	25.85±0.31
4	GR	11.42±0.4	6.3±0.30	8.45±0.40	10.61±0.49
5	Vit C	1.27±0.06	2.69±0.11	1.63±0.09	1.16±0.05
6	Vit E	1.11±0.08	1.97±0.06	1.43±0.06	1.29±0.06