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Mate tea attenuatesDNA damage and carcinogenesis induced by

diethylnitrosamine and thermal injury in the rat esophagus

Juliana Ferreira da Silva1, Lucas Tadeu Bidinotto2, Kelly Silva Furtado2, Daisy Maria Fávero Salvadori2_{, Maria Aparecida Marchesan Rodrigues}2_{, Luis Fernando Barbisan}1,2_*

1_{UNESP São Paulo State University, Institute of Biosciences, Department of Morphology,} 18618-000 Botucatu, SP, Brazil.

2_{UNESP São Paulo State University, School of Medicine, Department of Pathology, 18618-000} Botucatu, SP, Brazil.

Running title: mate tea and DNA damage and esophageal carcinogenesis

*Address correspondence to: Luis Fernando Barbisan, Ph.D.

Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), 18618-000 Botucatu, SP, Brazil.

Abstract

Drinking hot mate has been associated with risk for esophageal squamous cell carcinoma in South America. Thus, the modifying effects of mate tea intake on DNA damage and esophageal carcinogenesis induced by diethylnitrosamine (DEN) plus thermal injury were evaluated in male Wistar rats. In the initiation phase, rats were treated with DEN injections (8 x 80 mg/Kg b.w.) plus esophageal thermal injury and concomitantly received mate tea (2.0% w/v, test group) or green tea (2.0% w/v, positive control group) as the sole source of drinking fluid for 8 weeks. Any additional treatment was introduced at post-initiation until week 20. Peripheral blood was collected 4 hr after the last DEN application for comet assay at week 8 and samples from esophagus and liver were collected at weeks 8 and 20. At week 8, mate or green tea intake itself were non-genotoxic and significantly decreased DNA damage levels in peripheral blood leucocytes from DEN-treated animals. Also, a significant reduction of cell proliferation rates in both esophageal epithelium and liver parenchyma and on the number of putative preneoplastic liver lesions were observed in mate or green tea-treated animals at week 8. A significant lower incidence of esophageal and liver neoplasms and tumor multiplicity was observed in the groups previously treated with mate or green tea when compared to the DEN initiated/thermal injury group at week 20. These data indicate that mate tea presented protective effects against DNA damage and esophageal and liver carcinogenesis induced by DEN.

Key words: Mate tea, Green tea, DNA damage, diethylnitrosamine-induced esophageal and liver lesions

Introduction

Esophageal cancer is the eight most common human cancer worldwide and ranks sixth as a cause of cancer mortality (Parkin et al., 2001). Squamous cell carcinoma (SCC) is the predominant histological type with a variable geographic distribution and occurs at very high incidence in certain parts of China, Central Asia, Iran, South Africa, South America, France and Italy (Stoner and Gupta, 2001; Parkin et al., 2001). In Brazil, the highest incidence of esophageal SCC occurs in the most Southern region, Rio Grande do Sul, with age-adjusted mortality rates reported as 20.4/100,000/year for men and 6.5/100,000/year for women (Fagundes et al., 2006). The markedvariations in geographical distribution of esophageal SCC indicate thatenvironmental factors are involved in its pathogenesis (Stoner and Gupta, 2001). Thus, there is a need to develop effective strategies for the prevention of this malignancy and the detection of potential risk factors and chemoprevention are potentially viable approaches.

Tobacco and alcohol are the main agents involved in the etiology of esophageal SCC in Europe and North America, where over 90% of cases can be attributed to these causes (Parkin et al., 2001). Besides, drinking a local hot beverage has been shown to increase the risk for development of this malignancy in Uruguay, Paraguay, northeastern Argentina and southern Brazil (Victora et al., 1990; Pintos et al., 1994; Castelletto et al., 1994; Rolón et al., 1995;; Castellasagué et al., 2000; De Barros et al., 2000; Sewran et al., 2003; De Stefani et al., 2003; Goldenberg et al., 2003). Populations from these high risk areas share the habit of drinking a local tea known by the folk name of “mate”, “yerba mate” or “erva-mate”, an infusion of dried leaves of the perennial tree Ilex paraguariensis St. Hil., a native species of South America (Heck and Mejia, 2007). In Brazil, the leaves and stems of mate are usedto prepare different beverages, such as chimarrão (greendried leaves brewed with hot water in a vessel called ‘cuia’ through a metal straw),tererê (green dried leaves brewed with cold water in thesame kind of vessel and straw) and the mate tea (roasted leaves brewedwith hot water and drank as any other herbal tea) (De Barros et al., 2000; Alves et al., 2008; Miranda et al 2008,). In southeastern South America where the “gaucho” culture is widespread, large amounts of mate are drunk at high temperatures (65 to 71 0C), placing the oropharynx and esophagus with very hot fluids (de Barros et al., 2000). This might partially explain the high- incidence rates of SCC cancer in this region (Castellsagué et al., 2000; Pütz et al., 2002).

The potential mutagenic/genotoxic of mate aqueous extracts is still controversial. Mate solutions showed mutagenic activity in different Salmonella typhimurium strains, at

concentrations of 20 to 50 mg/plate, and genotoxic activity in the inductest (WP2s (lambda) strain), with a maximal phage induction at concentrations of 10 to 20 mg/plate (Leitão and Braga, 1994). Additionally, mate aqueous extracts (range doses 100–750 g/ml) increased the frequency of chromosomal aberrations in cultured human peripheral lymphocytes, but was not clastogenic to bone marrow cells of male Wistar rats (at oral doses up to 2 g/kg) (Fonseca et al., 2000). Recently, Alves et al. (2008) demonstrated that mate tea was not a clastogenic or/and aneugenic agent (range doses of 175–1400 g/ml) for culture of human peripheral lymphocytes using a cytokinesis-block micronucleus assay in the absence of exogenous metabolic activation. Also, mate tea intake (0.5, 1.0 or 2.0 g/kg, for 60 days) was non genotoxic to the liver, kidney and bladder cells from male Swiss mice. Besides, the regular ingestion of mate tea increased the DNA resistance to H2O2-induced DNA strand breaks and improved DNA repair after H2O2 challenge in liver cells from male Swiss mice (Miranda et al., 2008).

Various studies have detected contamination with polycyclic aromatic hydrocarbons (PAH) in different samples of commercial mate leaves and hot and cold mate infusions (Zuin et al., 2005; Kamangar et al., 2008). Also, an increase in urine 1-hydroxypyrene glucuronide (1-OHPG) levels was detected in healthy subjects with mate drinking habits from Rio Grande do Sul (Fagundes et al., 2006). Besides the association with upper aerial-digestive cancer, mate drinking has been associated with cancer development in other organs like the bladder, which is not influenced by thermal injury (Bates et al., 2007; De Stefani et al., 2007). Therefore, it is unclear whether the increased risk of esophageal SCC previously attributed to hot mate consumption is related to thermal injury at which mate is often drunk, or due to carcinogenic contaminantion, or both (Castellsagué et al., 2000; Pütz et al., 2002; Zuin et al., 2005; Kamangar et al., 2008).

The purpose of this study was evaluate the modifying influence of mate tea intake on DNA-damage, assessed by the Comet assay on peripheral blood leukocytes, and the development of esophageal preneoplastic and neoplastic lesions in male Wistar rats treated with the carcinogen diethylnitrosamine (DEN) plus esophageal thermal injury by instillation with hot water 650C. Additionally, positive control groups treated with green tea were included in this study due to be one of the most common beverages worldwide with remarkable chemopreventive properties (Yang et al., 2002; Chung et al., 2003).

Materials and Methods

Animals and treatments

The animals were handled in accordance with the Ethical Principles for Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA). Four-week- old male Wistar rats were obtained from CEMIB (UNICAMP Campinas, SP, Brazil). The animals were housed in polypropylene cages (four animals/cage) covered with metallic grids under standard conditions (22 ± 2 0C, 55 ± 10% humidity under a 12-hr light-dark cycle). They were fed commercial NUVILAB-CR-1 chow (NUVITAL, Curitiba, PR, Brazil) and water ad libitum for a 2-week acclimation period before beginning the experiment. Samples of leavesof Ilex paraguariensis St. Hil (mate) and Camelia sinensis (green tea) were generously supplied by the CentroFlora Group (Botucatu-SP, Brazil).

The animals were randomly allocated into seven groups, consisting of 20 rats in Groups G1 to G3 and 05 rats in Groups G4 to G7 (Figure 1). Groups 1 to 3 were treated once a week with intraperitoneal (i.p.) injections of diethylnitrosamine (DEN, Sigma-Aldrich Co., St. Louis Mo, USA) (8 x 80 mg/kg of body weight) and received hot water 650C (1ml/rat, instilled into the esophagus) twice a week for 8 consecutive weeks. This esophageal DEN initiation and thermal injury protocol was adopted with minor modifications from previously described protocols (Balansky et al., 1994; Li et al., 2003). Group 4 was treated with DEN and received water 25 0_{C (1ml/rat, instilled into the esophagus) twice a week for 8 consecutive} weeks. Groups 5, 6 and 7 received i.p. injections of NaCl 0.9% (DEN vehicle) once a week and water 250C (1ml/rat) twice a week for 8 consecutive weeks. Concomitantly, the groups received drinking water (groups G1, G4 and G7) or mate tea (2.0% w/v, groups G2 and G5) or green tea (2.0% w/v, groups G3 and G6) as the sole source of drinking fluid for 8 weeks. After the initiation period, animals from groups G1 to G3 received drinking water and food ad

libitumuntil at week 20. The groups were sacrificed at weeks 8 and 20 by a CO2 atmosphere.

Body weight and food/liquid consumption were measured twice a week during the entire experimental period. Tea solutions (2.0% w/v) were prepared by adding 20 g of minced leaves of Ilex paraguariensis or Camelia Sinensis to 1000 ml hot water (70 0C) for 20 min, filtering and allowed to cool down at room temperature. Tea solutions were prepared daily and offered

Comet assay

Samples of peripheral blood from the periorbital vein plexus were collected from all groups at 4 hr after the last DEN treatment at week 8 to perform the Comet assay. The single- cell gel electrophoresis assay or Comet assay was performed under alkaline conditions according to a previously described protocol (Tice et al., 2000). Briefly, 5 μl of each blood sample were mixed with 120 μl of 0.5% low-melting-point agarose (Invitrogen, Carlsbad, CA) at 37ºC and layered onto conventional microscope slides, precoated with 1.5% normal- melting-point agarose (Invitrogen). The slides were placed overnight in cold freshly-prepared lysing solution (1% Triton X-100, 2.5 mM NaCl, 0.1 mM Na2EDTA, 10 mM Tris with 10% dimethylsufoxide, pH 10.0) and then into a horizontal electrophoresis apparatus containing alkaline electrophoresis buffer (0.3 M NaOH, 1 mM Na2EDTA, pH>13) at 4ºC for 20 min. Using the same buffer, electrophoresis then was performed at 25 V and 300 mA for 20 min. After electrophoresis, the slides were washed twice for 5 min in neutralizing buffer (0.4 M Tris-HCl, pH 7.5), fixed for 5 min in absolute alcohol, air-dried, and stored at room temperature.

Immediately before analysis, the DNA was stained with 50 μl of 20 μg/ml ethidium bromide. The slides were examined with a 40X objective using an epi-illumined fluorescence microscope (Olympus-Bx60) attached to a color CCD video camera, and connected to an image analysis system (Comet II; Perspective Instruments, Suffolk, UK) (Figure 2 A). Coded slides were scored blindly and 50 blood cells were randomly analyzed for each animal (25 cells per slide from two slides per animal)

Tail intensity (the quantity of DNA in the tail of the comet) and tail moment (product of DNA density in the tail and the mean distance of DNA migration in the tail) were used to measure the extent of DNA damage. The comet images with a “cloudy” appearance or a very small head and a tail like a balloon (necrotic/apoptotic cells) were excluded from the evaluation under the assumption that they represent dead cells (Hartmann and Speit, 1997).

Tissue processing, histology and immunohistochemical procedures.

At necropsy, the esophagus was excised, opened longitudinally and fixed flat in 4% phosphate-buffered formalin during 24 hours for paraffin embedding. Serial sections of esophagus (at least 4-6 strips per animal) were routinely processed. The paraffin blocks were cut into 5-m-thick sections and stained with hematoxylin-eosin (HE) for histophatological analysis or for immunohistochemical analysis of epithelial cell proliferation using proliferating cell nuclear antigen (PCNA) (Figure 2 B) (Siglin et al., 1995).

The liver was excised, weighted, and representative samples were fixed in 4% phosphate-buffered formalin during 24 hours for paraffin embedding. The paraffin blocks were cut into 5-m-thick sections and stained with HE for histophatological analysis or for immunohistochemical analysis of preneoplastic hepatocellular lesions positive for glutathione S-transferase P form (GST-P) and PCNA (Figure 2 C and D) (Ito et al., 1988; Eldridge et al., 1993).

At week 20, macroscopically visible lesions on the esophageal mucosa and in the liver were mapped and registered. The classification scheme for esophageal and liver preneoplastic and neoplastic lesions was based on criteria previously described (Goodman et al., 1994; Whiteley et al., 1996). Preneoplastic esophageal and liver lesions and tumor yield within each experimental group were expressed in terms of both incidence and multiplicity.

GST-P and PCNA expression were detected by the avidin-biotin peroxidase complex (ABC) method. Briefly, deparaffinated 5-Pm tissue sections on poly-L-lysine-coated slides were treated sequentially with 3% H2O2 in phosphate-buffered saline (PBS) for 10 min, nonfat milk for 60 min, anti-GSTP antibody (clone 311, 1:1000 dilution; MBL laboratory, Tokyo, Japan) or anti-PCNA antibody (clone PC10, 1:200 dilution; Dako, Glostrup, Denmark) overnight, biotinylated horse anti-rabbit or anti-mouse IgG (1:300 dilution) for 60 min, and avidin-biotin-peroxidase solution (1:50 dilution) for 45 min (Elite ABC kit; Vector Laboratory, Burlingame, CA). Chromogen color development was accomplished with 3,3´-diaminobenzidine tetrahydrochroride (Sigma). The slides were counterstained with Harris haematoxylin.

GST-P positive lesions and PCNA labeling analysis.

Morphometric analyses of putative preneoplastic hepatocellular lesions (PNL) positive for GST-P were measured using a Nikon photomicroscope (Microphot-FXA, Tokyo, Japan) connected to a KS-300 apparatus (Kontron Electronic, Munich, Germany). Liver area was measured in a special Macro-Stand device (supported by Canon TV zoom lens V6x16/16-100 mm plus a Canon 58 mm close-up 240 lens connected to a CCD black-and-white video camera module with a Sony DC-777 camera unit) connected to the KS-300. GST-P-positive PNL were also classified according to two different classes: AFH and hepatic nodules  3 or > 3 mm in diameter. Data were expressed as number of GST-P-positive PNL per liver area (lesions/cm2).

The PCNA-labeling indices, expressed asa percentage, were calculated by dividing the number of squamous epithelial cells or hepatocytes with PCNA-labelednuclei (S phase) by the total number of cells countedin the each tissue sample (1000 to 2000 cells for esophagus and 5,000 to 10,000 cells for liver).

Statistical analysis.

Statistical analysis was performed using the Jandel Sigma Stat Software (Jandel Corporation, San Rafael, CA, USA). The body weight, body weight-gain food and liquids intake, relative liver weight, PCNA-labeling indices, GST-P-positive data and tumor multiplicity were analyzed by ANOVA test or the Kruskal-Wallis test. The incidence of different types of preneoplastic or neoplastic lesions was examined using F2 test or the Fischer exact test. Significance was set at P< 0.05.

Results

General findings

Seven animals were found dead during the course of the experiment: three rats from the DEN/TI-treated group (G1), two rats from the DEN/IT/mate tea-treated group (G2) and one rat from the DEN/TI/green tea-treated group (G3). A complete necropsy was not performed due to advanced postmortem changes and/or cannibalism.

Table I shows the mean values of the final body weight, body-weight gain, and, mate tea, green tea, water and food consumption during the first 8 week. Values of the final body weight, body-weight-gain and liquid intake were higher for the DEN/TI plus mate or green tea-treated groups (G2 and G3) than to the DEN/TI and DEN-treated groups (G1 and G4) (Table I). No differences in relative liver weight were observed among the DEN-treated groups but the values for alanine aminotransferase (ALT) were lower in mate (G2) and green tea-treated groups (G3) (P= 0.062 and P < 0.05, respectively). At week 8, the final body weight, body-weight-gain, ALT levels were significantly different in DEN-treated groups (G1 to G4) when compared to the non-DEN-treated groups (G5 to G7) (P < 0.001).

During the post-initiation phase, food and water consumption did not differ between the groups (G1 to G3). No differences in final body weight and body-weight gain values were observed at week 20 (data not shown). Moreover, an increase in relative liver weight values was detected in DEN/TI-treated group (G1, 5.3 ± 0.7) when compared to the DEN/TI plus mate or green tea-treated groups (G2 and G3, 4.2 ± 0.5 and 4.1 ± 0.6) (P < 0.001).

Genotoxicity data and esophagus and liver analysis at week 8

Figure 3A shows the values obtained in the Comet assay analyses (tail moment and tail Intensity parameters) on peripheral blood leukocytes evaluated 4 hours after the last DEN injection. The levels of DNA damage were significantly higher in the DEN-treated groups (G1 to G4) when compared to the non-initiated groups (G5 to G7) (P < 0.001). Mate or green tea intake itself was non genotoxic (G5 and G6 vs. G7). Significantly decreased levels of DNA damage were detected in the peripheral blood leucocytes, when these teas were administered simultaneously with DEN treatment (G2 and G3 vs. G1 and G4, P < 0.001).

Thermal injury regimen induced mild hyperkeratosis and hyperplasia in the squamous epithelial of esophagus from DEN-treated animals (G1 vs. G4). A significant reduction of incidence of animals with hyperkeratosis/hyperplasia (40 and 50%) and in cell proliferation rates in esophageal epithelium were observed in mate or green tea-treated animals (G2 and G3 vs. G1) at the end of DEN treatment and thermal injury regimens (Figure 3B).

GST-P positive hepatocellular lesions were observed only in DEN-initiated groups (G1 to G4). The number of preneoplastic lesions (PNL > 3 mm) was significantly lower in mate or green tea-treated groups (G2 and G3) when compared to the DEN/TI group (G1).

Mate or green tea treatments per si did not cause any histological changes in the esophageal epithelium or in the liver parenchyma in the non-initiated groups (G5 and G6 vs. G7).

Esophagus and liver histopathological analysis at week 20

Table II summarizes the incidence data of histologically-diagnosed esophagus and liver preneoplastic and neoplastic lesions and tumor multiplicity. Specially, there was a reduction on the incidence of esophageal dysplastic lesions in DEN/TI plus green tea group (G3) (Figure 4A) and esophageal papilloma (Figure 4B) and hepatic adenoma in DEN/TI plus mate or green tea-treated groups (G2 and G3) when compared to the DEN/TI-treated group (G1) (P = 0.054; P < 0.04 and P < 0.09, respectively). The morphology of the hepatocellular carcinomas (Figure 4C) was more undifferentiated in DEN/TI group (G1) that in the groups receiving mate or or green tea (G2 and G3). Cholangiocellular neoplasms (Figure 4D) were detected only in DEN/TI group. Also, tumor multiplicity for both esophagus and liver neoplasms was lower among the groups receiving mate or green tea (G2 and G3) when compared to the DEN/TI-treated group (G1) (P < 0.05).

Discussion

The results described herein indicate that mate tea intake reduced the levels of DNA damage in peripheral blood leukocytes and esophageal carcinogenesis induced by DEN/thermal injury protocol in male Wistar rats. Besides, liver carcinogenesis was also inhibited in the DEN-initiated group receiving concomitantly mate tea. The protective effects of mate tea on chemically-induced DNA damage and carcinogenesis were similar to the observed in the positive control group treated with green tea. In fact, green tea or their specific catechins have been proved to inhibit DNA damage and carcinogenesis in rodents and human (Yang et al., 2002; Chung et al., 2003).

Experimental and epidemiological studies have supported the role of thermal injury on the promotion of esophageal carcinogenesis (Yioris et al., 1983; Kinjo et al., 1998; Castellsagué et al., 2000; Li et al., 2003; Sewram et al., 2003). The consumption of hot beverages can provoke changes in esophageal epithelium that could increase the risk of damage from contact with refluxed gastric/duodenal contents or others potential carcinogens (Tobey et al., 1999; Yang et al., 2002; Pütz et al., 2002; Li et al., 2003). Besides, chronic exposure to high luminal temperature can result in an inflammatory process that may led to the formation of nitrosamines and free radicals able to initiate and/or promote the esophageal carcinogenesis process (Pütz et al., 2002). The thermal injury protocol used in this study resulted in an increase in the rates of cell proliferation, a well established parameter for cancer risk development on esophageal epithelium from DEN-treated animals (group G1 vs. group G4). Moreover, Li et al. (2003) showed that thermal injury abolished the inhibitory effects of epillocatechin-3 gallate from green tea against esophageal carcinogenesis process induced by N-nitrosomethylbenzylamine in male Fisher 344 rats. In contrast, using a more suitable thermal injury protocol, our findings indicate that the morphological and proliferative changes induced by thermal injury were attenuated by treatment with mate or green tea.

Various chemicals with antioxidant properties have been found to inhibit chemically- induced mutagenesis and carcinogenesis in different animal models (Kelloff, 2000; De Flora et al., 2001). In the present study, we have observed that the treatment with mate or green tea, during the initiation phase of carcinogenesis, significantly reduced the levels of DNA damage induced by DEN in peripheral blood cells. Since mate has demonstrated