Mol Biol Rep (2010) 37:1373–1376 DOI 10.1007/s11033-009-9519-1
Inhibition of 1,2-dimethylhydrazine induced colon genotoxicity in rats by the administration of probiotic curd Arvind Kumar Æ Nikhlesh Kumar Singh Æ Pushpalata Rabindra Sinha
Received: 30 November 2008 / Accepted: 17 March 2009 / Published online: 29 March 2009 Ó Springer Science+Business Media B.V. 2009
Abstract Epidemiologic and experimental studies suggest that the probiotic organisms are effective in preventing colon carcinogenesis, which is the major cause of mortality and morbidity in western countries. Keeping this in view, a curd (a common Indian fermented milk product) was prepared by the addition of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and curd culture Lactococcus lactis biovar. diacetylactis. In present study, we have evaluated the anti tumor effect of probiotic curd by monitoring the DNA damage through comet assay. The rats were allocated to four groups, first group was DMH control group, second group was probiotic curd group in which probiotic curd was given along with DMH (1,2-dimethylhydrazine) injection, third group was normal curd group in which normal curd was given along with DMH injection and fourth group was normal control group. Animals received subcutaneous injection of DMH dissolved in normal saline at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. The rats were dissected at 40th week of experiment and comet assay was done in colonic cells to assess the DNA damage. A significant reduction in DNA damage (54.7%) was observed in probiotic curd group as compared to DMH control group (88.1%). The probiotic curd was effective to significantly reduce the L:W ratio in comparison to DMH control group and normal curd. The results of present study show the protective effects
A. Kumar (&) N. K. Singh P. R. Sinha Animal Biochemistry Division, National Dairy Research Institute, Karnal, Haryana 132001, India e-mail:
[email protected] A. Kumar 4984, Street No.1, Near Old Poultry Farm, Shiv Colony, Karnal, Haryana 132001, India
of probiotic curd against DMH induced genotoxicity in colonic cells. Keywords 1,2-Dimethylhydrazine (DMH) Comet assay Probiotic curd Genotoxicity DNA damage Length:width (L:W)
Introduction Abundant evidence implies that specific bacterial species used for fermentation of dairy products such as yogurt and selected from healthy gut microflora have powerful anti pathogenic and anti inflammatory properties [1]. Epidemiologic and experimental studies suggest that the consumption of fermented milk products and Lactic acid bacteria (LAB) decrease the incidence of certain types of cancer. Probiotic organisms are effective in preventing colon carcinogenesis, which is the major cause of mortality and morbidity in western countries. 1,2-Dimethylhydrazine (DMH) is a common colon carcinogen used in animal studies. It is a pro carcinogen that requires activation through a series of in vivo chemical transformation in liver by oxidation through microsomal oxidases to the carcinogen azomethane, which on further oxidation, produces azoxymethane (AOM), also a common intestinal carcinogen. Cytochrome P450IIE1 metabolizes AOM to MAM, which degrades to formaldehyde and methyl diazonium ion. This ion breaks down to nitrogen and the ultimate carcinogen, the methyl carbonium ion, an alkylating species that forms adduct with nucleic acids [2]. Thus, genotoxic stress may contribute towards the progression of carcinogenesis. Comet assay is the common technique to assess the genotoxicity. Comet assay can be used to investigate the
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genotoxicity of industrial chemicals, biocides, rays and pharmaceuticals. Comet assay can be applied to any tissue, provided that a single cell/nucleus suspension can be obtained. Kanna et al. [3] reported protective effect of vanadium against DMH induced colon carcinogenesis. They reported 86.6% DNA damage in vanadium ? DMH group rats as compared to 93.3% DMH control rats. The length:width (L:W) ratio of comet was also significantly higher in DMH control group (2.218) as compared to DMH ? Vanadium group (1.508). In the present study we assess the inhibition of DMH induced genotoxicity in rat’s colonic cells by feeding probiotic curd containing mixture of probiotic microorganisms.
Materials and methods Preparation of probiotic curd Probiotic curd was prepared by inoculating the probiotic cultures (Lactobacillus acidophilus and Lactobacillus casei; National Collection of Dairy Culture, National Dairy Research Institute (NDRI), Karnal) and curd culture (Lactococcus lactis biovar. diacetylactis DRC-1) in milk procured from the Experimental Dairy, NDRI, Karnal. The milk was boiled at 90°C for 30 min with constant stirring and then allowed to cool up to 30°C. The culture was inoculated and kept for 12 h on 37°C. Normal curd was prepared by inoculating the curd culture and incubated at 30°C for 12 h. Grouping of animals Hundred rats were allocated to four experimental groups consisting of 25 animals in each group: (1) DMH control group: animals were fed with control diet along with DMH injection; (2) Probiotic curd group: probiotic curd was given along with control diet and DMH injection (3) Normal curd group: normal curd was given along with control diet and DMH injection; (4) Normal diet group: control diet was given without any DMH injection. Diets of animals A standard diet containing 45% starch, 25% Bengal gram, 10% casein, 5% sucrose, 10% soybean oil, 1% vitamin mix, 4% mineral mixture, 0.2% choline chloride and 0.35% methionine was given to experimental rats along with supplement diet viz. curd and milk. Vitamin and mineral mixture prepared and mixed according to AOAC [4]. Curd was given as 30% of total diet while control group (DMH control and normal diet) rats received equal amount of milk.
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Induction of colon carcinogenesis and comet assay At 10 weeks of age, all animals received subcutaneous injection of DMH dissolved in normal saline at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. Rats in normal diet group received saline at same rate. At 40th week, Experiment rats from all groups were sacrificed. The colon of rats were excised and washed with phosphate buffer saline (PBS) and processed for comet assay. Colonic epithelial cells were isolated by following the protocol of Tavan et al. [5] and comet assay was performed by following the protocol of Ahuja and Saran [6] with some modification in lysis of cell membrane in which Proteinase K was added. The jar containing this lysis buffer and slides was incubated at 37°C for 2 h and then b-mercapto ethanol (1%) was added and kept in refrigerator for 12 h. Silver staining [7, 8] was done to stain the comet slides. The slides were observed under the inverted microscope (CK40, Olympus) fitted with ocular micrometer. Two slides were prepared from each animal and at least 100 comets were observed from each slide. The ratio of DNA length to width and percentage of damage DNA per group was calculated. Further to estimate the damage, the damaged DNA was scored by following the damage scoring no. in which the damage DNA had given some number from 0 to 4 having no damage to maximum damage. Then the observed DNA was matched with that scoring number and scoring number was assigned to that comet. For scoring the comets at least 1,000 comets were scored from each group. Statistical differences between treatment groups were evaluated by applying ANOVA (Analysis of variation) test in SYSTAT software. Differences were considered significant at P \ 0.05.
Results and discussion Administration of DMH injections resulted in extensive liver damage and colon carcinogenesis. Table 1 represents the incidence, multiplicity, size and number of tumors in all experimental groups which clearly indicates the protective potential of probiotic curd by showing significantly lower (P \ 0.05) tumor incidence, multiplicity and size as compared to DMH control group. Upon histological analysis (Table 1), DMH control and Normal curd group rats showed 12 and 5.88% carcinoma, respectively while no carcinoma was observed in probiotic curd group. Administration of DMH caused DNA damage in all experimental animals. Figure 1 show damaged and undamaged DNA in colonic cells of experimental rats. The comet assay results are shown in (Table 2). The mean L:W
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Table 1 Effect of Ac Dahi on incidence, multiplicity, size and volume of tumors in colon of experimental rats Groups
Tumor incidence
Tumor multiplicity
DMH control
13/15 (86.67%)a b
1.92 ± 0.26a
Probiotic curd group
6/15 (40%)
Normal curd group
10/15 (66.67%)b
1.7 ± 0.15c
Normal diet group
0/15 (0%)
0
1.33 ± 0.20
b
Total number of tumors
Average tumor size (cm)
Adenoma (%)
Adeno carcinoma (%)
Carcinoma (%)
25
1.46 ± 0.069a
56
32
12
8
b
0.51 ± 0.048
75
25
0
17
0.87 ± 0.044c
58.82
35.29
5.88
0
0
0
0
0
Values other than incidence are mean ± standard error mean (SEM) Values with different superscript letters are significantly different (P \ 0.05) Data for tumor multiplicity and tumor size were compared by ANOVA Tumor incidences were statistically compared by z-test
Fig. 1 Various types of comets observed in rats colonic cells. A Undamaged, B Damaged
Table 2 Effect of probiotic curd feeding on DMH induced DNA damage Group
No. of rats
Tailed cells (%)
DMH control
6
88.1 ± 1.1a
3.1 ± 0.13a
6
b
1.4 ± 0.03b
c
2.2 ± 0.04c 1.02 ± 0.05d
Probiotic curd group Normal curd group Normal diet group
6 6
54.7 ± 1.5
70.6 ± 2.9 9.2 ± 1.1d
L:W
Each value represents the mean ± SE of mean of data from six animals DNA damage percentage; L:W ratio of comet DNA mass Different superscript letters are significantly different (P \ 0.05) Statistical differences were evaluated by applying ANOVA
ratio of the DNA mass, which indicates the extent of DNA damage, for DMH control group was significantly higher (P \ 0.05) among all groups. Similarly, probiotic curd significantly reduced the DNA damage (P \ 0.05) in comparison to DMH control group and Normal curd group. In DNA damage scoring (Fig. 2), it was observed that DMH control group rats had more comets in scoring number as high as 3 and 4 in comparison to other groups, while in probiotic curd group, maximum number of comets scored a value of only one. The scoring result of normal dahi group was similar to DMH control group.
Several experimental observations have indicated a potential protective effect of LAB against the development of colon tumors [9]. Lactic acid bacteria plays an important role in retarding colon carcinogenesis by possibly influencing metabolic, immunologic and protective function in the colon [10]. In present study, the probiotic microorganisms were able to inhibit colon carcinogenesis by showing lower DNA damage in comparison to DMH control and Normal dahi group. The probiotic microorganisms present in the probiotic curd are able to reduce the free radicals generated by the DMH metabolism and prevent adduct formation. This may be one of the reasons for less DNA damage in curd treated groups. Tavan et al. [5] evaluated the effect of some LAB on heterocyclic aromatic amines induced colon carcinogenesis in rats. They divided the sixty rats in four groups with supplementation of tap water, 30% non fermented skimmed milk, with 30% B. animalis DN-173 010 fermented milk and 30% Streptococcus thermophilus DN-001 158 fermented milk. Comet assay and 3D test was done to assess the genotoxicity. These workers reported the significant decrease in DNA damage only for rats fed with fermented milk. Similarly two short term studies [11, 12] using 1,2-dimethylhydrazine as a genotoxic agent in rats, showed that various LAB (lactobacilli, bifidobacteria and Streptococcus thermophilus) were able to decrease colon DNA damage. All the
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Fig. 2 Scoring of damaged DNA in groups. Each value represents the percentage of comets observed in each scoring no. of each group (1,000 comets in each group)
100
0
1
2
3
4
90
Scoring No.(percentage)
80 70 60 50 40 30 20 10 0 DMH Control
Probiotic dahi group
Normal dahi group
Normal diet group
Groups
strains of lactobacilli and bifidobacteria prevented DNA damage when given at a dose of 1010 cells/kg body wt, 8 h before the carcinogen administration. In most of the cases the DNA damage was reduced to a level similar to that in untreated rats. Streptococcus thermophilus was not as effective as the other LAB strains. Previous studied conducted in our laboratory showed the antioxidant [13] and free radical scavenging activity [14] of probiotic curd. In present study, the mechanism of less genotoxic effect may be due to enhancement of antioxidant system resulting in the scavenging of free radicals and toxic carcinogenic electrophiles. Reduction in generation of active toxic carcinogen electrophile may cause reduction in formation of DNA adducts with ultimate carcinogen, finally decreasing the mutation and DNA damage. Thus probiotic curd may exert its antigenotoxic effects in protecting DNA by preventing the formation of ultimate carcinogen which forms adduct with DNA and causes genotoxic damage.
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