Hindawi Publishing Corporation Journal of Chemistry Volume 2016, Article ID 8570321, 9 pages http://dx.doi.org/10.1155/2016/8570321 Research Article The Coffee Protective Effect on Catalase System in the Preneoplastic Induced Rat Liver Cristiana Schmidt de Magalhães,1 Jéssica Emi Takarada,1 Nathália Costa Carvalho,1 Dayene do C. Carvalho,2 Felipe Lopes de Andrade,1 Eric Batista Ferreira,1 Pedro Orival Luccas,2 and Luciana Azevedo3 1 Exact Sciences Institute, Federal University of Alfenas, Rua Gabriel Monteiro da Silva 700, Centro, 37130-000 Alfenas, MG, Brazil 2Chemistry Institute, Federal University of Alfenas, Rua Gabriel Monteiro da Silva 700, Centro, 37130-000 Alfenas, MG, Brazil 3Nutrition Faculty, Federal University of Alfenas, Rua Gabriel Monteiro da Silva 700, Centro, 37130-000 Alfenas, MG, Brazil Correspondence should be addressed to Cristiana Schmidt de Magalhaes;˜ [email protected] Received 15 October 2015; Revised 2 March 2016; Accepted 28 March 2016 Academic Editor: Philippe Jeandet Copyright © 2016 Cristiana Schmidt de Magalhaes˜ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study aimed to evaluate the effect of organic/conventional coffee in liver tissues in the cancer process, taking into account the level and activities of catalase. The experiments were carried out with 8 groups of rats during 12 weeks. They received two injections of ethylenediaminetetraacetic acid solution 1.5% (v/v) prepared in 0.9% NaCl or 1,2-dimethylhydrazine (DMH) subcutaneous dose −1 −1 of 40 mg⋅kg ⋅bw for 2 weeks. The organic/conventional coffee infusions were at 5, 10, and 20% and were incorporated tofeed −1 (100 mL of infusion⋅kg of diet). The catalase activity showed a decrease for livers which received DMH and DMH plus organic coffee at 5% and 10%. However, an increase was observed for those receiving organic 20% and conventional 10% coffee, slowing down and favoring the reversibility of the carcinogenic process. By SDS-PAGE, we observed an intensity decrease of 59 kDa bands, as the percentage of coffee was increased. The iron concentration (by ET-AAS) confirmed the electrophoretic results, suggesting that the DMH influenced the catalase expression conditions, reducing the activity by the loss of iron ions. Thus, the coffee may restore the catalase system in the liver, exerting its chemopreventive effects. 1. Introduction organic acids, lipids, and aromatic compounds [9]. They form a complex mixture of different concentrations which depend Liver cancer is considered one of the most aggressive ones and corresponds to the third leading cause of cancer deaths on environmental factors, cultivation, crop management, and worldwide [1–3]. Among the new treatment alternatives and grain processing [10]. The differences between conventional clinical procedures, the use of chemopreventive approaches and organic coffee are mainly attributed to fertilization meth- hasbeenresearchedandapplied.Dietarycomponentshave ods. According to Carvalho et al. [10], the concentrations of shown great potential to suppress carcinogenesis in pre- chlorogenic acids, trigonelline, and caffeine in organic arabica clinical neoplastic models as well as prevent or delay the coffee (ORC) were higher than in the conventional coffee occurrence of this disease [4]. (CC) (4.6, 9.6, and 4.4% higher, resp.). The antioxidant com- In this scenario, coffee emerges as one of the alternatives pounds in coffee contribute to the reduction of free radicals for these chemopreventive approaches [5]. Coffee is the third that naturally occur or are chemically induced in the human most popular beverage in the world after water and tea, body[11].Forexample,caffeineisusedinin vitro studies, as with average daily consumption of 150 mL per person [6–8]. an inhibitor in N-demethylation reactions [12, 13]; it is also In addition, roasted coffee infusion has thousands of com- able to reduce liver fibrogenic processes, which regulate the pounds such as phenolic polymers, amino acids, sugars and growth of liver cells [6, 14, 15]. Polyphenols and melanoidins polysaccharides, niacin, chlorogenic acids, minerals, caffeine, also act as protectors, reducing liver chronic diseases [16, 17]. 2 Journal of Chemistry Considering that researches related to the functional Table 1: Experimental design. properties of the bioactive constituents of coffee, as well as the influence and interaction of these compounds to human Group Reagent Coffee health, we decided to compare the protein changes, especially G1/DMH + CD DMH — catalase (CAT), in liver tissues of animals under colon neo- G2/EDTA + CD EDTA — plastic lesions, induced by DMH. In this context, proteomics G3/DMH + ORC 5% DMH Infusion 5% has played an important role in the global monitoring of G4/DMH + ORC 10% DMH Infusion 10% protein changes in tissues or individuals with cancer [18], G5/DMH + ORC 20% DMH Infusion 20% once these modifications are catalyzed by critical enzymes in G6/DMH + CC 5% DMH Infusion 5% numerous cellular functions. G7/DMH + CC 10% DMH Infusion 10% The CAT are tetrameric metalloproteins (heme or linked G8/DMH + CC 20% DMH Infusion 20% to Mn), which act as antioxidants [19] that catalyze the Animals/group = 10, CD: commercial diet, ORC: organic coffee diet, CC: −1 −1 degradation reaction of hydrogen peroxide into oxygen and conventional coffee diet, DMH: 1,2-dimethylhydrazine 40 mg⋅kg ⋅bw ; water. CAT has been found to significantly decrease chro- and EDTA: 1.5% in NaCl 0.9% (DMH vehicle). Infusion 5%,10%,and20%: −1 mosomalaberrationsandalsotodelayorpreventtheonset considering the incorporation of 100 mL of infusion⋅kg of ground feed. of spontaneous neoplastic transformation in mice fibroblasts and epidermal keratinocytes [20]. 2.1. In Vivo Experiment: Sample/Liver Collection. Samples of It is known that liver catalase activity becomes reduced in liver tissues were taken from Wistar rats, males, 21 days old, thepresenceofagrowingtumor.However,afterthetumor with acclimatization period of 2 weeks. Eighty animals were is removed, this activity returns to normal, showing that the used, divided into 8 groups (=10); see Table 1. The proce- tumor tends to suppress it as an antioxidant [21] but there is dures herein were approved by the Animal Ethics Committee no evidence about the food interference in this mechanism in the Federal University of Alfenas, protocol: 235/09, and yet. followed the Ethical Principles in Animal Experimentation The use of 1,2-dimethylhydrazine (DMH) and its metabo- adopted by the Brazilian College of Animal Experimentation lites are an excellent experimental model for studies of new (COBEA). cancer treatments [22]. It is an aliphatic methylating carcino- The experiment was conducted during 12 weeks. In the1st gen, metabolized in the liver to form active intermediates and2ndweeks,theanimalsweretreatedwitheitherethylene- like azomethane, azoxymethane, methylazoxymethanol, and diaminetetraacetic acid (EDTA) solution 1.5% (v/v) prepared ultimate carcinogenic metabolite, diazonium ion (by NAD+- in 0.9% NaCl or 1,2-dimethylhydrazine (DMH) subcutaneous −1 −1 dependent dehydrogenase), which are actively transported doses of 40 mg⋅kg ⋅bw . These were supplied considering subsequently into the colon via bile and blood [23]. Once the proportion of 1.0 mL per 100 g of animal weight. The solu- metabolizedintheliver,theactivemethyldiazoniumions, tions were applied for two weeks, twice per week [25]. capable of methylating DNA, RNA, or protein of colonic epi- To analyze the coffee protective potential, 100 mL of each thelial cells, induce more oxidative stress, resulting in over- coffee infusion was incorporated to 1 kg of commercial diet production of reactive oxygen species (ROS). (CD). Therefore, the organic coffee infusions (BCS-OKO Ga- Therefore, the liver was chosen as the study tissue, and rantie Master Certificates numbers POCO-7569/07.08/14291- catalase activity can be used as excellent preventive chemical BR)at5,10,and20%correspondedtogroupsG3,G4,and marker [24] to investigate cancer occurrence, as well as treat- G5, respectively, whereas conventional coffee infusions at 5, ment efficiency. The objectives were to compare the differ- 10, and 20% corresponded to groups G6, G7, and G8, respec- encesofcatalaseactivityinthistissuecausedbytheinges- tively.G1wasthepositivecontrolgroupandG2wasthe tion of coffee infusion, obtained from organic and conven- negative control group. The animals were euthanized for liver collection in the tional management systems. We also compared the elec- end of the 12th week. The livers were subjected to perfusion trophoretic liver protein band intensities and determined the with NaCl 0.9% (v/v) and stored in Ultrafreezer (U7386S60 iron concentrations in CAT SDS-PAGE bands, relating these ∘ Sanyo MDF, Japan) at −80 C. Samples of liver tissues were differences with chemopreventive mechanisms. taken in triplicate (livers from three animals) from each group. 2. Materials and Methods 2.2. Chemical Analyses. All solutions were prepared employ- To evaluate the coffee protective effect on catalase system, ing deionized water from a purification system Milli-Q® water we performed an in vivo experiment aiming to obtain pre- (Millipore®, Bedford, MA, USA), with 18.2 MΩ⋅cm resistivity. neoplastic induced rat livers. From these livers all analyses The sucrose and phosphoric acid were purchased from Isofar