|Year : 2018 | Volume
| Issue : 6 | Page : 1379-1388
Orientin mitigates 1, 2-dimethylhydrazine induced lipid peroxidation, antioxidant and biotransforming bacterial enzyme alterations in experimental rats
Kalaiyarasu Thangaraj, Karthi Natesan, Kandakumar Settu, Mariyappan Palani, Mydhili Govindarasu, Vanitha Subborayan, Manju Vaiyapuri
Department of Biochemistry, Periyar University, Salem, Tamil Nadu, India
|Date of Web Publication||28-Nov-2018|
Department of Biochemistry, Periyar University, Periyar Palkalai Nagar, Salem - 636 011, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Colorectal cancer (CRC) is the second most diagnosed cancer often identified during the later stages of carcinogenesis. Orientin, a C-glycoside of luteolin, is well known for its versatile therapeutic action toward oxidative stress-induced cellular response may exert chemoprevention against CRC.
Materials and Methods: In our study, we investigated the modulatory effect of orientin on lipid peroxidation, antioxidant defense, and biotransforming bacterial enzymes in 1, 2-dimethylhydrazine (DMH)-induced male albino Wistar rats in a dose-dependent manner. Animals were induced with DMH (20 mg/kg b.wt) for 15 weeks and administered with orientin in three different doses (5 mg/kg, 10 mg/kg, and 20 mg/kg b. wt) daily under distinct phases (initiation, postinitiation, and the entire) for a total treatment period of 30 weeks.
Results: Orientin reinstates the alterations induced by DMH on lipid peroxidation and enzymatic antioxidants through its rich-free radical scavenging properties. In addition, orientin curtails the DMH-induced augmentation of biotransforming bacterial enzymes to inhibit the colon cancer progression. Overall, experimental findings suggest that orientin significantly inhibits the DMH induced colon cancer in all the three different doses, however, maximum inhibition was observed on supplementation of 10 mg/kg b.wt for the entire period of the study.
Conclusion: Hence, the intraperitoneal administration of 10 mg/kg b.wt orientin for the entire period is recommended for further molecular investigation to elucidate the precise mechanism of inhibition and so orientin can be used as a novel chemotherapeutic agent for CRC.
Keywords: 1, 2-dimethylhydrazine, antioxidants, bacterial enzymes, colorectal cancer, lipid peroxidation, orientin
|How to cite this article:|
Thangaraj K, Natesan K, Settu K, Palani M, Govindarasu M, Subborayan V, Vaiyapuri M. Orientin mitigates 1, 2-dimethylhydrazine induced lipid peroxidation, antioxidant and biotransforming bacterial enzyme alterations in experimental rats. J Can Res Ther 2018;14:1379-88
|How to cite this URL:|
Thangaraj K, Natesan K, Settu K, Palani M, Govindarasu M, Subborayan V, Vaiyapuri M. Orientin mitigates 1, 2-dimethylhydrazine induced lipid peroxidation, antioxidant and biotransforming bacterial enzyme alterations in experimental rats. J Can Res Ther [serial online] 2018 [cited 2019 Sep 17];14:1379-88. Available from: http://www.cancerjournal.net/text.asp?2018/14/6/1379/228634
| > Introduction|| |
Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths (8% of all cancer death) and the third most commonly diagnosed cancer worldwide, and has poor prognosis when metastasized to lymph nodes or distant organs. Increase in incidence associated with race, ethnicity, lifestyle habits, and dietary patterns, especially the intake of the typical Western diet with a high quantity of animal (saturated) fat and/or red meat and a low amount of fiber and/or vegetables and fruits. The alkylating agent, 1, 2-dimethylhydrazine (DMH), is a colon-specific procarcinogen used widely to induce tumors in experimental rodent models., The DMH-induced colon carcinogenesis mimics human colon carcinoma in epithelial origin, anatomy of colonic mucosa, morphological, histological, and tumorigenic characteristics, and therefore serve as an ideal experimental model for chemoprevention studies., The subcutaneous administration of DMH undergoes metabolic activation in the liver by dehydrogenation to form metabolic intermediates such as azoxymethane and methylazoxymethanol. The intermediate metabolized further to form active electrophilic methyldiazonium glucuronide by NAD+ dependent dehydrogenase in the liver and excretes through bile and blood to reach the colonic lumen. The bacterial glucuronidases in the mucosal cells hydrolyze the glucuronides and generate active carbonium ion which methylates nucleic acids and proteins to induce oxidative stress and triggers tumorigenicity., Apart from colon specificity, DMH also alkylates the hepatocellular DNA and acts as a hepatic necrogenic agent.
Colon cancer is often linked with persistent oxidative stress, and the reactive oxygen species (ROS) produced during DMH metabolism were highly reactive, damages cellular macromolecules such as nucleic acids, fat, carbohydrate, proteins and also interacts with lipid bilayers, which ultimately results in cytotoxicity, mutagenicity, and carcinogenicity. ROS possess the ability to oxidize PUFAs and initiate lipid peroxidation, an endogenous pathway that produces free radicals and substances such as malondialdehyde (MDA), conjugated dienes, hydroperoxides. Oxidative stress in colonic mucosa was counteracted by the endogenous antioxidant defense portal systems, including superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH)-dependent enzymes. Antioxidants work together and if there is any alteration in the function of any of these enzymes would result in loss of equilibrium and cellular damage leading to malignancies. The intestinal lumen enriched with bacterial enzymes generates toxins and carcinogenic metabolites from procarcinogens which may influence the risk of colon cancer.
The chemoprevention of CRC is of immense interest throughout the world which can be influenced by phytochemicals with rich-free radical scavenging properties. Flavone C-glycosides, an important subclass of flavonoids, have shown to act as effective scavengers owing their free hydroxyl groups on the B ring. Orientin (luteolin-8-C-glucoside, ORI), a bioactive C-glycosyl flavone profusely found in basil herb, passion fruit, bamboo leaf, dayflower, and pigeon pea leaves. Earlier studies have reported that orientin-induced apoptosis through the caspase-dependent and mitochondria-mediated pathways in HeLa cervical cancer cell lines and human esophageal carcinoma EC109 cells.
None of the information is available regarding the protective role of orientin against colorectal carcinogenesis. Therefore, in the light of above inferences we designed this study to evaluate the dose-dependent chemoprotective efficacy of orientin against DMH-induced colorectal carcinogenesis in Wistar rats.
| > Materials and Methods|| |
Orientin (ORI), 1, 2-dimethylhydrazine (DMH) hydrochloride, nitroblue tetrazolium (NBT), reduced GSH, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and 1′-chloro-2,4-dinitrobenzene (CDNB) were purchased from Sigma-Aldrich Chemical Company, Saint-Louis, MO, USA. All other chemicals and solvents employed were of analytical grade.
Orientin dissolution and administration
Orientin was dissolved in 0.01% of dimethyl sulfoxide just before use and administered intraperitoneally at a daily dose of 5 mg/kg, 10 mg/kg, and 20 mg/kg body weight.
Colorectal tumor induction
DMH was dissolved in 1 mM ethylenediaminetetraacetic acid just before use and the pH adjusted to 6.5 with 1 mM NaOH to make sure the stability of carcinogen. Animals were given subcutaneous injections of DMH in the groin at a dose of 20 mg/kg body weight for 15 consecutive weeks of the total experimental period.
Healthy male albino Wistar rats of 100–120 g were obtained from Vinayaka Mission College of Pharmacy, Salem, Tamil Nadu, India. All rats were acclimatized 1 week and housed in polypropylene plastic cages with six rats per each and maintained in specific pathogen-free environmental conditions set to 25°C ± 2°C room temperature on a 12 h dark/light cycle with 50% ±10% relative humidity. Animals were fed with modified high fat where commercial pellet diet containing 4.2% fat (Sri Venkateshwara Enterprises, Bengaluru, Karnataka, India) was pulverized and mixed with 15.8% peanut oil, making a total of 20% fat in the diet and tap water ad libitum. The animals were cared for in compliance with the principles and guidelines of the institutional animal ethical committee in accordance with the Indian National Law on Animal Care and Use.
Experimental design and treatment schedule
Animals were randomly segregated into 14 groups of six rats and fed with modified high-fat pellet throughout the experimental period of 30 weeks. Group 1 rats served as untreated controls. Group 2 rats were induced colorectal tumor through subcutaneous administration of 20 mg/kg body weight DMH once a week for 15 consecutive weeks. Group 3–5 rats received orientin intraperitoneally at a daily dose of 5 mg/kg, 10 mg/kg, and 20 mg/kg body weight, respectively throughout the experimental period. Group 6–14 rats administered DMH (20 mg/kg body weight), as in Group 2, in addition, rats were treated with orientin in three different phases as follows. Group 6–8 rats (Initiation) received orientin intraperitoneally at a daily dose of 5 mg/kg, 10 mg/kg and 20 mg/kg body weight 1 week prior to 1st DMH injection till the end of the final DMH administration. Group 9–11 (postinitiation) rats received orientin intraperitoneally a daily dose of 5 mg/kg, 10 mg/kg, and 20 mg/kg body weight 1 week after the final DMH administration till the end of the experimental period. Group 12–14 rats received orientin intraperitoneally at a daily dose of 5 mg/kg, 10 mg/kg, and 20 mg/kg body weight throughout the entire period. The detailed experimental protocol is shown in [Figure 1]. The body weight of all rats was recorded at the beginning and weekly once till the sacrifice period. After euthanization, blood hemolysate and tissue homogenate were prepared and stored as aliquots for further biochemical analysis. The mucosal scrapings and fresh fecal pellets were homogenized in phosphate-buffered saline and used for colonic bacterial enzyme analysis.
The level of thiobarbituric acid reactive substances (TBARS) was measured by the formation of pink chromogen complex with MDA, and absorbance was recorded at 535 nm in tissues and plasma. Formation of conjugated dienes through rearrangement of the double bonds in the polyunsaturated fatty acids was measured by the absorbance at 233 nm. Lipid hydroperoxides (LOOH) are determined by their ability to oxidize ferrous iron leading to the formation of a chromophore with an absorbance maximum at 560 nm.
Superoxide dismutase and catalase
SOD (EC.184.108.40.206) was quantified based on 50% inhibition of the formation of NADH-phenazine methosulfate-NBT formazan complex at 560 nm. CAT (EC.1.11.16) was determined by measuring the amount of chromium acetate produced while reducing dichromate in acetic acid to chromium acetate.
Glutathione and glutathione-dependent enzymes
Reduced GSH content is determined through Ellman method based on the formation of yellow color when DTNB is added to the compounds with sulfhydryl groups. Glutathione peroxidase (GPx) (EC.220.127.116.11) was assayed by incubating known amount of the enzyme preparation with H2O2 in the presence of GSH for 5 min. Glutathione reductase (GR) (EC 18.104.22.168) was assessed based on the oxidation of NADPH; the absorbance was measured spectrophotometrically at 340 nm. Glutathione-S-transferase (GST) was quantified by measuring the formation GSH conjugate using CDNB as substrate and the increase in absorbance was recorded at 340 nm.
Colonic mucosal and fecal content of bacterial enzymes
β-Glucuronidase (EC 22.214.171.124) activity was assessed by the hydrolysis of p-nitrophenyl-β-D glucopyranoside. β-Glucosidase (EC 126.96.36.199) and β-galactosidase (EC 188.8.131.52) activity was measured using p-nitrophenyl-β-D-glucopyranoside and p-nitrophenyl β-D-galactopyranoside as substrates, respectively. Mucinase (184.108.40.206) was quantified using porcine gastric mucin as substrate. Nitroreductase (EC 220.127.116.11) activity was determined using p- nitrobenzoic acid substrate. Sulfatase (EC 18.104.22.168) activity was measured by assessing the amount of p-nitrocatechol liberated from p-nitrocatechol sulfate.
Values are given as the mean ± standard deviation. The significant difference between the mean of the 14 groups was significantly analyzed by one way analysis of variance and Duncan's Multiple Range Test (DMRT). The results were considered statistically significant at P < 0.05. Statistical analysis was performed using SPSS 16.0 software package (SPSS, IBM product, Chicago, IL, USA).
| > Results|| |
Effect of orientin on body weight and growth rate
The body weight gained by the rats in control and treatment groups was recorded and growth rate was determined by calculating the difference between the final and initial body weight divided the total experimental days. As shown in [Figure 2], body weight gained by rats in control (Group 1) > orientin (Group 3–5) > DMH + orientin (entire period) (Group 12–14) > DMH + orientin (post-initiation) (Group 9–11) >DMH + orientin (initiation) (Group 6–8) > DMH (Group 2). Orientin supplemented rats (Group 3–14) showed a (P < 0.05) significant increase in growth rate when compared with DMH alone-treated rats as shown in [Figure 3].
|Figure 2: Effect of orientin on body weight in control and treated rats. Ori 5 mg/kg - Orientin 5 mg/kg b.wt, Ori 10 mg/kg - Orientin 10 mg/kg b.wt, Ori 20 mg/kg - Orientin 20 mg/kg b.wt|
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|Figure 3: Effect of orientin on growth rate in control and treated rats. Ori 5 mg/kg - Orientin 5 mg/kg b.wt, Ori 10 mg/kg - Orientin 10 mg/kg b.wt, Ori 20 mg/kg - Orientin 20 mg/kg b.wt. (i)- Initiation, (PI) - Post initiation, (EP) - Entire period. The values are presented as the mean ± standard deviation of six rats per each group. a–g P < 0.05; values not sharing a common superscript letter are significantly different from the 1, 2-dimethylhydrazine-treated groups (analysis of variance followed by Duncan's Multiple Range Test)|
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Changes in circulating and tissue lipid peroxidation
[Table 1] and [Table 2] shows the effect of orientin on lipid peroxidation in control and experimental rats. Our results showed that the circulation and hepatic levels of lipid peroxidation were significantly increased in DMH administered rats (Group 2) as compared to control rats (Group 1) whereas the colonic and intestinal levels were significantly decreased. Orientin supplementation (Group 6–14) significantly decreased the circulatory and hepatic levels of lipid peroxidation to near normal as compared to DMH alone administered rats whereas in colonic and intestinal tissues significantly increased. The effect of orientin in restoring the lipid peroxidation status was more pronounced in the rats supplemented with 10 mg/kg body weight orientin for the entire period (Group 13) as compared to other DMH administered groups treated with orientin (Group 6–12, 14). Orientin alone-treated rats (Group 3–5) did not show much significant effect when compared to control rats (Group 1).
|Table 1: Effect of orientin on circulatory and hepatic level of lipid peroxidation in control and experimental rats|
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|Table 2: Effect of orientin on colonic lipid peroxidation in control and experimental rats|
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Changes in circulating and tissue endogenous antioxidants
[Table 3] and [Table 4] shows the effect of orientin on colonic lipid peroxidation in control and experimental rats. Our results showed that both the circulation and tissue levels of SOD and CAT were significantly decreased in DMH-treated rats (Group 2) as compared to control rats (Group 1). Orientin supplementation (Group 6–14) significantly increased the levels of SOD and CAT to near normal as compared to DMH alone administered rats. A more pronounced effect was observed in the rats supplemented with orientin at 10 mg/kg body weight for the entire period (Group 13) as compared to other DMH administered groups treated with orientin (Group 6–12, 14). Orientin administration (Group 3–5) enhanced the activities of SOD and CAT when compared to control rats (Group 1).
|Table 3: Effect of orientin on circulatory and hepatic level of superoxide dismutase and catalase in control and experimental rats|
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|Table 4: Effect of orientin on colonic level of superoxide dismutase and catalase in control and experimental rats|
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Changes in circulatory and tissue glutathione and glutathione-dependent enzymes
[Table 5], [Table 6], [Table 7], [Table 8] show the circulatory and tissue levels of GSH- and GSH-dependent antioxidant enzymes (GPx, GST, and GR) in control and experimental rats. Our results showed that both the circulation and tissue levels of GSH- and GSH-dependent enzymes were significantly decreased in DMH-treated rats (Group 2) as compared to control rats (Group 1). Orientin administration (Group 6–14) significantly increased the levels of GSH- and GSH-dependent enzymes to near normal as compared to DMH alone administered-treated rats. A more pronounced effect was in the rats administered with orientin at 10 mg/kg body weight for the entire period (Group 13) as compared to other orientin supplemented DMH-treated groups (Group 6–12, 14). Orientin administration (Group 3–5) enhanced the activities of GSH- and GSH-dependent enzymes when compared to control rats (Group 1).
|Table 5: Effect of orientin on circulatory and hepatic level of reduced glutathione and glutathione peroxidase in control and experimental rats|
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|Table 6: Effect of orientin on circulatory and hepatic level of glutathione-S-transferase and glutathione reductase in control and experimental rats|
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|Table 7: Effect of orientin on colonic level of reduced glutathione and glutathione peroxidase in control and experimental rats|
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|Table 8: Effect of orientin on colonic level of glutathione-S-transferase and glutathione reductase in control and experimental rats|
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Effect of orientin on bacterial enzymes
[Table 9] and [Table 10] show the effect of orientin on the activities of bacterial enzymes in the fecal and colonic mucosal contents of the control and experimental rats. The bacterial enzymes were found to be significantly increased in DMH-treated rats (Group 2) as compared with the control rats (Group 1). Orientin supplementation (Group 6–14) significantly decreased the activities of these enzymes in the fecal and colon mucosal content as compared with the DMH alone administered rats (Group 2). A more pronounced effect was in the rats administered with orientin at 10 mg/kg body weight for the entire period (Group 13) as compared to other orientin supplemented DMH-treated groups (Group 6–12, 14).
|Table 9: Effect of orientin on fecal and colonic mucosal β-glucuronidase, β-glucosidase and β-galactosidase enzymes|
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|Table 10: Effect of orientin on fecal and colonic mucosal mucinase, nitroreductase and fecal sulfatase enzymes|
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| > Discussion|| |
CRC is often a pathophysiological consequence of the persistent oxidative stress with the increased influx of reactive oxygen species. In the present investigation, orientin was supplemented through intraperitoneally in three different doses under three different regimens to determine the effective dose of orientin against DMH-induced colorectal carcinogenesis. The decrease in body weight and growth rate of DMH-treated rats fed with high-fat diet may be due to the increased tumor burden, loss of appetite accompanied by increase in polyps driven-cachexia and anorexia. Orientin-treated rats have shown a significant increase in body weight despite the metabolic changes induced by DMH owing their ability to reinstate the cellular metabolic dysfunctions.
DMH enhances the production of ROS, which in turn induce oxidative stress in the hepatic and colon tissues. In our study, the oxidative stress induced was exemplified by the increased circulatory lipid peroxidation in DMH-treated rats. This may be due to excessive ROS generation with reduced host antioxidant defense, which attacks the cell membrane-associated polyunsaturated fatty acids and thereby disintegrates cell membranes, leading to epithelial cell transformation. The lipid peroxidation increases in premalignant cells immediately after DMH injection, but in case of malignant neoplasms, the lipid peroxidation starts decreasing, as the tumor progresses, which could be due to the decreased expression of cytokines by tumor cells. The inverse relationship between the lipid peroxidation and the extent of cellular proliferation and dedifferentiation of the malignant cells was clearly evident by the increased proliferation of cancerous cells while TBARS, LOOH, and CD levels were reduced in colonic tissues. In our study, orientin supplementation restored the level of both circulatory and tissue lipid peroxidation to the near normal level despite the changes induced by DMH which may be due to the antiproliferative efficacy of orientin. The present finding provides ample evidence for the restitution of oxidative ability by orientin in the colonic tumor cells by donating hydrogen from the hydroxyl group of (−OH) on the ring structure to free radicals to make them unreactive.
In the present study, the circulatory and hepatic SOD and CAT were found to be significantly decreased in DMH-treated rats as compared to the control rats due to the sequestration of antioxidants in circulation and the increased level of hepatic lipid peroxidation. DMH administration significantly decreased the colonic level of SOD and CAT activities when compared with untreated control rats in line with the previous experimental findings. The decreased SOD and CAT activities could be due to the anomalous increase in ROS and the oxidative stress with the increased proliferation of malignant colon cells. Orientin elevated SOD and CAT activities similar to our previous laboratory reports with umbelliferone. The present findings demonstrated that orientin promotes the dismutation of superoxide anions to H2O2 and the catalysis of H2O2 into H2O and O2; thereby protecting the cellular constituents from the hydroxyl radicals-induced oxidative damage.
DMH-treated rats showed decreased levels of GSH and dependent enzymes in the circulation and liver due to the increased utilization of GSH to counteract the circulatory lipid peroxidation and detoxification of carcinogenic metabolites of DMH which is in corroboration with the earlier findings. The decreased colonic level of GSH and dependent enzymes in DMH-treated rats compared with control might be due to the reduced synthesis and availability of sufficient GSH to neutralize H2O2. Orientin revert back the changes in the level of GSH and GSH-dependent enzymes induced by DMH metabolite due to the powerful quenching ability of orientin toward the free radicals by donating electrons to the unstable oxidizing molecules.
Changes in the intestinal microflora
Colonic microflora plays a predominant role in the etiology of CRC. The bacterial enzymes in the colonic epithelium transform endogenous and exogenous xenobiotics into active carcinogens and activate the metabolites detoxified by the liver. The increased level of bacterial enzymes could be attributed to altered intestinal microflora by high-fat diet and DMH administration. The change in mucinase secretion and degradation influences the transformation of normal mucosa into colon cancer cells. The carcinogenic metabolites that are released by β-glucuronidase activity induce microbial mucinase to interact with the colonocytes resulting in DNA damage and cell proliferation. Nitroreductase and sulfatases aids in the metabolic activation and release of carcinogens in the gastrointestinal tract. Orientin revert back the changes induced by DMH by retarding the action of β-glucuronidase followed by the protection of mucin layer and degradation of desulfated toxins. [Figure 4] represents an overall mechanism of orientin on lipid peroxidation, antioxidants, and bacterial enzymes in 1,2 DMH-induced CRC.
|Figure 4: Overall possible mechanism of orientin against 1, 2-dimethylhydrazine induced alterations|
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Orientin exhibits dose-dependent protection against DMH in different phases; however, the entire treatment period showed more profound inhibitory effect. The overall experimental findings suggest that the higher dose (20 mg/kg b.w) was effective, but not as much as medium dose (10 mg/kg b.w) of orientin which is corroborated with the earlier findings. This may be because of the formation of obnoxious secondary metabolites by increased concentration of orientin that impedes with its antioxidant mechanism. Furthermore, investigations are required to elucidate the precise molecular mechanism of orientin protection to establish it as a potential chemotherapeutic agent for CRC.
The authors thank Dr. R. Kothai and Dr. B. Arul, Vinayaka Mission's College of Pharmacy, Salem, Tamil Nadu, India, for their generous support and guidance during this study.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]