CHAPTER 10 LACTOBACILLUS PLANTARUM AS1 ISOLATED FROM SOUTH INDIAN FERMENTED FOOD KALLAPPAM SUPPRESS 1, 2-DIMETHYL HYDRAZINE (DMH)

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1 CHAPTER 10 LACTOBACILLUS PLANTARUM AS1 ISOLATED FROM SOUTH INDIAN FERMENTED FOOD KALLAPPAM SUPPRESS 1, 2-DIMETHYL HYDRAZINE (DMH) INDUCED COLORECTAL CANCER IN MALE WISTAR RATS Introduction Probiotic bacteria are increasingly being shown to be capable of influencing gastrointestinal disease and disorders including colorectal cancer (Guarner & Malagelada, 2003; Allan et al., 2006). Colorectal cancer (CRC) is one of the leading causes of cancer death both in men and women (Greenlee et al., 2000; Yamamoto, 2000). Prognosis for advanced CRC is poor (Sant et al., 1995), and hence prevention is required to control the incidence of disease. Epidemiological studies show that diet plays a role in the aetiology of most large bowel cancers, implying that it is potentially preventable disease. Many studies confirm the involvement of the exogenous microflora in the onset of colon cancer. This makes it reasonable to think that changing the intestinal microflora could influence tumor development. 1, 2-Dimethylhydrazine (DMH) is a potent carcinogen that has been reported to induce colon cancer in experimental animals. Humans are exposed to DMH and other hydrazines through environment (Frazier et al., 1991) and diet (Tracy et al., 1998; Toth & Gannett, 1990). DMH is metabolized in the liver, resulting in the production of electrophilic diazonium ion, which is known to elicit oxidative stress (Fiala, 1975; Fiala et al., 1987). Experimental colon cancer induced by DMH in rats is a prolonged multistage process, bearing many of the same cell kinetics, histopathological and molecular characteristics of tumorigenesis that mimics human colon cancer (Halline et al., 1989). 140

2 In the present study, Lactobacillus plantarum AS1 which was isolated previously from South Indian traditional fermented food Kallappam was checked for its efficacy in the suppression of colorectal cancer induced by 1, 2-dimethyl hydrazine in male albino Wistar rats Materials and Methods Animals, housing, and diet Thirty six male albino Wistar rat were obtained from King s Institute (Chennai, TN) and housed in stainless steel wire cages (3 rats/cage). The temperature and relative humidity were maintained at 27 C ± 2 and 50% respectively. Light and dark cycles were 12 h each. Feed and water were provided ad libitum. After a 2 week period of acclimatization, the animals were divided into six groups of six rats each and fed the experimental diets for 26 weeks. During this time weekly body weights and daily feed intake were recorded. Animals were maintained as per the principle and guidelines of the Ethics Committee of Animal care of Pondicherry in accordance with the Indian National Law on animal care and use (Ref. No. PU/SLS/IAEC/15/08-09) Probiotic strain and media Probiotic strain L. plantarum AS1 (Genebank accession no. GQ ) isolated previously from south Indian fermented food Kallappam (Satish et al., 2010) was used for the treatment of colon cancer in rats. MRS agar (HiMedia, Mumbai, India) was used for routine cultivation of L. plantarum AS1. Approximately 10 9 CFU/ml of L. plantarum AS1 was suspended in 0.85% saline and used for the treatment study. 141

3 Experimental Design The rats were divided into six groups and each group consisted of six animals. Colon tumors were induced in groups 3, 4, 5 and 6 with a weekly subcutaneous (s.c.) injection of 1, 2- dimethylhydrazine ( Sigma, USA) in saline at 20 mg/kg body weight starting from +0 week to +6 week. Group 1 control animals were given the normal rat chow. Groups 2, 4 6 animals were treated with L. plantarum AS1 (1 ml containing approximately 10 9 CFU of L. plantarum AS1 suspended in saline /day/oral) for various time periods as indicated below. The time point of DMH administration was taken as zero (0); minus ( ) and plus (+) signs represents the time in weeks before and after DMH administration, respectively. The schedule of treatment in groups 2, 4-6 was as follows: groups 2 and 6, 5 to +21; group 4, 5 to 0; group 5, +0 to +21. The experiment was terminated at the end of 26 weeks (Fig.16). Fig. 16. Treatment Schedule 142

4 Biochemical Estimations Lipid peroxidation was estimated by measurement of thiobarbituric acid reactive substances (TBARS) in plasma and colon tissue (Yagi, 1978). The stock solution contained equal volume of trichloroacetic acid 15% (w/v) in a 0.25 N HCl. One volume of test sample and two volume of stock reagent were mixed in a screw capped centrifuge tube, vortexed and heated for 15 min in a boiling water bath. After cooling on ice the precipitate was removed by centrifugation at 10,000 g for 15 min and the absorbance was measured at 532 nm against the enzyme blank. The values are expressed as μmol of malondialdehyde formed. Total Superoxide dismutase (SOD, EC ) activity was measured by the method of Marklund & Marklund, (1974). Briefly, 100 μl of the tissue homogenate was mixed with 125 μl of ethanol and 625 μl of chloroform in a mechanical shaker for 15 min and centrifuged. The supernatant (0.5 ml) was mixed with 1.0 ml of 0.1 M Tris-HCl, ph 8.2, 750 μl of distilled water and 250 μl of 1 mm pyrogallol in 0.05 M Tris-HCl, ph 7.4; optical density at 420 nm was determined at 0, 1 and 2 min. Control tubes containing 0.5 ml of distilled water were also treated in a similar manner against a buffer blank. The enzyme activity is expressed as unit/mg protein (one unit is the amount of enzyme required to cause 50% inhibition of pyrogallol autooxidation). Catalase (EC ) activity was assayed as described (Sinha, 1972). Briefly, to 100 μl of the tissue homogenate, 0.5 ml of 0.01 M phosphate buffer, ph 7.0, and 0.25 ml of 0.2 M H 2 O 2 were added. After incubation for 10 min at 37 C, the reaction was arrested by the addition of 1.0 ml of diluted (1:5 in water) 5% dichromate-acetic acid (1:3; v/v). Standard H 2 O 2 in the range of 0 to 20 μmol was treated similarly for comparison. The tubes were heated in a boiling water bath for 10 min, and the green color developed was read at 570 nm. Catalase activity is expressed as μmol of H 2 O 2 consumed/min/ mg protein. 143

5 Glutathione-S- transferase (GST, EC ) according to the method of Habig et al., (1974). Briefly, the assay mixture contained 0.1 M potassium phosphate buffer, ph 6.5, 1 mm 1-Chloro- 2,4-dinitrobenzene (Sigma, USA), 1mM EDTA, 1mM glutathione (Sigma, USA). The reaction was started by addition of 100 µl of sample and change in absorbance at 340 nm was studied. A unit of activity is defined as µmol product formed/min under the condition of assay. Alkaline(EC ) (King, 1965) and acid phosphatase (EC ) (Moog, 1946) activities in plasma and colon were measured. Protein content was estimated by the method of Lowry et al., (1951) Histology The colon was processed as follows (Sengottuvelan et al., 2006). The entire colon (from cecum to anus) was removed and washed thoroughly with 0.9% NaCl, cut longitudinally, laid flat on a polystyrene board, and fixed with 10% buffered formaldehyde solution overnight. The colon was then stained with 0.2% methylene blue for 3 5 min in saline in order to identify aberrant crypt foci (ACF), formed by one or more aberrant crypts, which are easily visualized on a background of normal crypts since aberrant crypts have larger, often elongated openings and a thicker lining of epithelial cells compared with normal crypts. These tissues were also processed (dehydrated, embedded in paraffin blocks, sectioned (5 µm), and stained with hematoxylin and eosin) for microscopic examination In vitro antioxidant activity of Lactobacillus plantarum AS1 Antioxidant activity of L. plantarum AS1 performed in vitro to confirm that scavenging of free radicals and anti-peroxidation activities in treated rats were due to this probiotic strain. 144

6 Antioxidative activity The measurement of antioxidative activity of L. plantarum AS1 was performed by the thiobarbituric acid (TBA) method, based on the monitoring of inhibition of linoleic acid peroxidation. Linoleic acid was chosen as the source for unsaturated fatty acid. The TBA method was used for the measurement of lipid peroxidation and a Fe/H 2 O 2 system was used for the catalysis of oxidation (Decker & Faraji, 1990). Linoleic acid emulsion (20 ml) was made up of 1 ml linoleic acid, 0.2 ml Tween 20, and 19.7 ml deionized water. Phosphate buffer solution (0.5 ml, 0.02 M, ph 7.4), 1 ml linoleic acid emulsion, 0.2 ml of FeSO4 (0.01%), 0.2 ml of H 2 O 2 (0.56 mm), and 0.4 ml L. plantarum AS1 (10 9 CFU/ml) were mixed and incubated at 37 C. Blank samples contained either PBS or deionized water. After 12 h of incubation, 2 ml of the reaction solution was mixed with 0.2 ml trichloroacetic acid (TCA; 4%), 2 ml TBA (0.8%), and 0.2 ml butylated hydroxytoluene (BHT; 0.4%). This mixture was incubated at 100 C for 30 min and allowed to cool. Chloroform (2 ml) was then added for extraction. The extract was obtained and the absorbance was measured at 532 nm. The percentage of inhibition of linoleic acid peroxidation was defined as follows: [1 A532 (sample)/a532 (blank)] 100%. Determination of α, α-diphenyl-β-picrylhydrazyl (DPPH) Radical The scavenging of DPPH by L. plantarum AS1 was analyzed by a modification of method utilized by Shimada et al., (1992). Eight tenths of a milliliter of L. plantarum AS1 cells and 1 ml of freshly prepared DPPH solution (0.2 mm in methanol) were mixed and allowed to react for 30 min. Blank samples contained either PBS or deionized water. The scavenged DPPH was then monitored by measuring the decrease in absorbance at 517 nm. The scavenging ability was defined as follows: [1 A517 (sample)/a517 (blank)] 100%. 145

7 Statistical Analysis All data were statistically analysed (analysis of variance) using the SPSS 16 Statistical program Results Average body weight and feed intake by Wistar rats Figures 17 and 18 shows the final body weight and food intake in control and experimental rats. We found significantly increased weight gain among the rats fed with L. plantarum AS1 (group 2; 254 ± 10.25) compared to DMH control (Group 3; 225 ± 14.71). There was also a 9.89% increase in the feed intake by L. plantarum AS1 treated rats (group 2) compared to DMH control (group 3). Fig. 17. Average weight increase/month in albino Wistar rats Groups were treated as mentioned in Materials and methods section. 146

8 Fig 18. Average feed intake by albino Wistar rats Groups were treated as mentioned in Materials and methods section. *The mean difference is significant at the 0.05 level Incidence of colon tumors, number of tumors per tumor bearing rats and average tumour size in experimental groups Tumor count was performed in the DMH and DMH+AS1-treated rats (Table 15). A total of 13 tumors were found in DMH (group 3) control rats with 100% incidence and average tumor count/rat of L. plantarum AS1 treated groups showed significant reduction in both incidence and number of tumors compared to the DMH control group. L. plantarum AS1 pretreated rats (group 4) and L. plantarum AS1 post-treated rats (group 5) had 1.8 tumor/rat (total count = 9) and 1.6 tumor/rat (total tumor = 8) with 83.33% incidences. L. plantarum AS1 pre and post treated rats (group 6) showed a marked decrease in the incidence of colon cancer, with only four rats being affected (incidence = 66.66%) with average tumor number of 1.25 per rat (total 147

9 count = 5). As anticipated L. plantarum AS1 and control groups did not develop any tumors. Mean tumor sizes also differed significantly among DMH control and DMH+AS1 treated groups. DMH control group had mean tumor size of 0.36 ± cm 2 whereas L. plantarum AS1 pre and post treated rats had 0.23 ± 0.15 cm 2. Similar reductions in mean tumor sizes were observed in L. plantarum AS1 pre-treated rats (0.27 ± cm 2 ) and L. plantarum AS1 posttreated rats (0.26 ± cm 2 ) Level of Lipid peroxidation, activities of antioxidant enzymes and marker enzymes in colon and plasma of different experimental groups Level of lipid peroxidation (TBARS) and activities of catalase, GST, SOD, alkaline phosphatase and acid phosphatase in colon and plasma of control and experimental animals were shown in Tables 16 and 17. In the present study a significantly increased level of lipid peroxidation and antioxidant and marker enzyme activities in DMH (group 3) treated rats when compared to control (group 1) rats has been observed. An elevated level of lipid peroxidation parallel with the antioxidant enzyme activities has been found and the possible explanation for this contradictory finding could be that increased antioxidant enzyme activities may not able to cope-up with elevated level of lipid peroxidation in DMH treated animal. However similar findings were observed in plasma of humans suffering from colon cancers (Gul et al., 1998; Krishna & Venkata ramana, 2006). In pre- and post-treatment (group 6) rats, there was significant reduction in the level of peroxidation and antioxidant enzymes compared to DMH (group 3) control (Table 16 & 17). Similar reductions in enzyme activities and peroxidation level were observed in plasma of group 6 animals compared to DMH control (group 3) rats. Pre- (group 4) and posttreated (group 5) animals also show reduction in TBARS level and antioxidant and marker enzyme activities in colon and plasma when compared to DMH-treated (group 3) rats. 148

10 Table 15. Incidence of colon tumors, number of tumors per tumor bearing rats and average tumour size diameter (cm 2 ) Groups No. of Rats No. of tumor bearing rats Group 1 (Control) Tumor Total tumor No. of tumor/ incidence *(%) number tumor bearing rats Nil Nil Nil Mean tumor size (cm 2 ) Group 2 (AS1) Group 3 (DMH) Group 4 (Pre-TG) Group 5 (Post-TG) Nil Nil Nil a 13 a 2.16 a 0.36±0.115 a b 9 b 1.8 b 0.27±0.068 b b 8 b 1.6 c 0.26±0.057 b Group c 5 c 1.25 d 0.23± 0.15 c (Pre & Post- TG) Groups were treated as mentioned in materials and methods section. Values are mean ± SD from 6 rats in each group. Values not sharing a common superscript letter (a d) differ significantly at P < *(Number of tumor-bearing rats/total number of rats in each group) x

11 Table 16. Level Lipid peroxidation, activities of some antioxidant enzymes and some marker enzymes in colon of different experimental groups (n=6). Particulars Lipid Peroxides SOD GST Catalase ALP ACP Group 1 (Control) Group 2 (AS1) Group 3 (DMH) Group 4 (Pre-TG) Group 5 (Post-TG) Group 6 (Pre & Post TG) 0.105±0.08 a 2.01±0.05 a 0.35±0.012 a 0.427±0.014 a 23.33±2.08 a 49.6±6.5 a 0.075±0.01 a 1.99±0.11 a 0.38±0.049 a 0.567±0.02 b 20.33±2.51 a 36.0±6.5 a 1.63±0.284 b 3.76±0.12 b 1.82±0.02 b 3.33±0.038 c 112.0±10.1 b 213±7.0 b 1.08±0.068 c 2.71±0.06 c 1.04±0.027 c 1.34±0.028 d 67.6±2.5 c 122±9.16 c 1.27±0.074 c 2.81±0.07 c 1.51±0.02 d 1.56±0.024 e 70.0±7.0 c 148±14.0 d 0.488±0.04 d 2.35±0.05 d 0.803±0.02 e 0.97±0.012 f 42.3±2.5 d 121±11.59 c Groups were treated as mentioned in Materials and methods section. Values are mean ± SD from six rats in each group. Values not sharing a common superscript letter (a f) differ significantly at P < Values are expressed as follows: Lipid peroxidation, TBARS formed/min/mg of protein; SOD, Units/mg of protein; GST, μmoles of CDNB utilized /min/mg of protein; Catalase, μmoles of H utilized/min/mg of protein; ALP & ACP, μmoles phenol liberated/min/ mg of protein. 150

12 Table 17. Level lipid peroxidation, activities of some antioxidant enzymes and some marker enzymes in plasma of different experimental groups (n=6). Particulars Lipid Peroxides SOD GST Catalase ALP ACP Group 1 (Control) Group 2 (AS1) Group 3 (DMH) Group 4 (Pre-TG) Group 5 (Post-TG) Group 6 (Pre & Post TG) 0.74±0.022 a 1.22±0.02 a 1.34±0.05 a 0.733±0.06 a 760±8.71 a 522.3±23.02 a 0.67±0.11 a 1.2±0.015 a 1.18±0.06 b 1.11±0.074 b 743±25.1 a 476.3±24.1 a 3.54±0.033 b 3.07±0.057 b 3.28±0.04 c 7.55±0.221 c 967±23.64 b 1034±31.4 b 1.75±0.04 c 2.75±0.013 c 2.13±0.06 d 2.31±0.086 d 855±31.99 c 680±23.0 c 2.96±0.081 d 2.87±0.023 d 2.16±0.05 d 2.35±0.052 d 842.6±25.1 c 792.3±26.5 d 1.53±0.024 e 1.45±0.033 e 1.83±0.04 e 1.77±0.059 e 794±5.56 a 506±25.05 a Groups were treated as mentioned in Materials and methods section. Values are mean ± SD from six rats in each group. Values not sharing a common superscript letter (a f) differ significantly at P < Values are expressed as follows: Lipid peroxidation, TBARS formed/min/mg of protein; SOD, Units/mg of protein; GST, μmoles of CDNB utilized /min/mg of protein; Catalase, μmoles of H utilized/min/mg of protein; ALP & ACP, μmoles phenol liberated/min/mg of protein. 151

13 Histological examination of colon tissue sections Methylene blue stained sections of DMH induced colon tissue revealed the presence of tumor (Fig. 19). Moreover, there were notable differences in the epithelial cells of DMH affected colon tissues: affected cells were abnormal in their apprearances with very thick cell membrane as compared to normal ones. Hematoxylin and eosin stained colon sections of control and experimental animals were observed under the light microscope at 20 magnification field. Control rats and L. plantarum AS1 alone treated rats colon tissues were normal and lesion-free. Except for the control groups (groups 1 and 2), all experimental animals showed the presence of lesions, but their count varied (Fig. 20) In vitro antioxidant activity of L. plantarum AS1 Table 18 shows the in vitro antioxidant and free radical scavenging activity of L. plantarum AS1. An inhibition of ± 2.47% linoleic acid peroxidation by L. plantarum AS1 strain was found. The free radical scavenging effect was measured by measuring the absorbance decrease of DPPH at 517 nm, and it was observed to be ± 3.84%. Hence, L. plantarum AS1 had an antioxidant effect which could be one of the reasons for significant reduction in cancerous tumors and related symptoms in L. plantarum AS1 treated group rats. 152

14 Fig. 19. Methylene blue stained sections of colon tissue A- Tumor in colon tissue of DMH induced rat B- Cell morphology of cancer bearing colon tissue Fig.19 represents methylene blue stained sections of DMH induced cancer bearing colon tissue. (A) Longitudinally dissected section of DMH induced rat colon reveals (arrow) the cancerous tumor over its epithelial surface. (B) Microscopic (20 ) examination of colon tissue from DMH induced rat shows (arrow) abnormal thickening of epithelial cell walls typical of colon cancer 153

15 Fig. 20. H & E stained sections of colon tissue A- Control rat (group 1) colon section B- DMH control rat (group 3) colon section C- Pre-& post-treatment rat (group 6) colon section Fig. 20 Histological appearance of Control and DMH injected rat sections. (A) Control rat colon section. All Cells have normal appearance. (B) DMH injected (group 3) rat colon tissue section, arrows indicate crypts exhibiting slit- like lumen. (C) Pre- & posttreatment rat colon section. This group of rats received L. plantarum AS1 throughout the experimental period and received DMH for 7 weeks. There is a reduced number of aberrant crypts in the tissue section. 154

16 Table 18. In vitro antioxidant activity of L. plantarum AS1 Antioxidant Assay L. plantarum AS1 Intact cell Antioxidant activity ± 2.47 (% inhibition) DPPH Radical ± 3.84 (% Radical Scavenged) Discussion L. plantarum AS1 isolated from South Indian traditional fermented food Kallappam was evaluated for probiotic properties earlier (Satish et al., 2010). This property makes it a suitable organism to be tested for its disease controlling attributes. The present study was designed to investigate the chemopreventive role of L. plantarum AS1, by considering its antioxidant property with respect to lipid peroxidation (LPO) and the antioxidant system against DMHinduced multistage colon carcinogenesis. Male albino rats were employed in the present study as they were used for similar studies earlier (Manju & Nalini, 2005; Tracy et al., 1998; Daniel et al., 1999). DMH, which was used to induce colorectal cancer is a powerful carcinogen for several rodent species with a very high degree of specificity for the colon. DMH is first oxidized, perhaps non-enzymatically, to azomethane (AM), a poisonous gas soluble in both organic and 155

17 aqueous solvents. AM is easily oxidized with microsomal N-oxygenase to azoxymethane (AOM). AOM may be hydroxylated by the microsomal monoxygenase system in colon mucosa to methylazoxymethanol (MAM). MAM non-enzymatically methylates nucleic acids and produce colon tumors (Fiala, 1975). L. plantarum AS1 treated animals showed increased weight gain when compared to DMH alone treated rats. This weight increase may be due to enhanced uptake (about 9.89%) of feed. Probiotic bacteria improve absorption and digestion of food particles in the intestine (David, & Glenn, 1999).A maximum of 42.13% decrease in the mean tumor number per rat was observed when they were administered with probiotics L. plantarum AS1. There was also 36.12% decrease in mean tumor size when compared with DMH control. The precise mechanisms by which L. plantarum AS1 may inhibit colon cancer are presently unknown. However, the mechanism may include the following: binding and degrading potential carcinogen; production of antitumorigenic or antimutagenic compounds; enhancing the host s immune response and effects on physiology of the host (Rafter, 2002). Free radicals and lipid peroxides have been considered to be very important in carcinogenesis (Capel & Thornley, 1983; Goldstein & Witz, 1990). Moreover, free radicals formed during the activation of DMH have been considered to be an important factor in colon carcinogenesis. Numerous studies have reported elevated lipid peroxidation in experimental (Deschner, & Zedeck, 1986) and human (Keshavarzian, 1992; Otamiri, & Sjodahl, 1989) colorectal cancer. We observed increased level of lipid peroxide and increased antioxidant enzyme (SOD, CAT, and GST) activities in DMH injected control rats. Similar findings were reported in experimental and human cancer patients (Gul et al., 1998; Krishna & Venkata ramana, 2006). These alterations reverted to normal upon L. plantarum AS1 administration; the enzyme activities decreased with the increase in time of L. plantarum AS1 administration. Antioxidant enzymes usually become 156

18 enhanced in the system when there is increased ROS (Priscilla & Healther, 2000). From the above result, we presumed that L. plantarum AS1 may possess antioxidant activity by which it may inhibit peroxidation and scavenge free radicals. We examined this by performing in vitro antioxidant assays. Peroxidation inhibition was performed using the 2-thiobarbituric acid (TBA) method and free radical scavenging assay was done by DPPH scavenging method. L. plantarum AS1 inhibited peroxidation of linoleic acid by ± 2.47% and scavenged DPPH by ± 3.84%. Thus, the results revealed the antioxidant property of L. plantarum AS1 which might be one of the factors for its anti-carcinogenic activity. L. plantarum AS1 might have scavenged the free radicals generated by DMH and reduced their ill effects. In conclusion, this study has supported earlier observations that certain probiotic bacteria, such as the L. plantarum strain, are capable of diminishing colon tumor. L. plantarum AS1 exerts its anticancer action through its antioxidant properties. Long-term administration of L. plantarum AS1 is necessary to achieve maximum inhibitory effect. Further studies are needed to elucidate the exact mechanism for L. plantarum AS1 acting as an anticarcinogen in multistage carcinogenesis. 157