Isolation, characterization and identification of antigenotoxic and anticancerous indigenous probiotics and their prophylactic potential in experimental colon carcinogenesis

Colorectal cancer, the third most commonly diagnosed cancer, is a lifestyle disease where diet and gut microbiome contribute intricately in its initiation and progression. Prophylactic bio-interventions mainly probiotics offer an alternate approach towards reducing or delaying its progression. Therefore, the present study was designed wherein a robust protocol for the isolation, characterization, and identification of indigenous probiotics having antigenotoxic and anticancerous activity was followed along with their prophylactic potential assessment in early experimental colorectal carcinogenesis. Among forty-six isolated lactic acid bacterial strains, only three were selected on the basis of antigenotoxicity against N,N-Dimethyl dihydrazine dihydrochloride and 4-Nitroquinoline 1-oxide and probiotic attributes. All three selected probiotic strains exhibited anticancerous potential as is evident by the reduced Aberrant Crypt Foci, reduced fecal pH, enhanced fecal lactic acid bacteria and altered fecal enzymes (β-glucuronidase, nitroreductase, β-glucosidase) that modulated gut microbiota and microenvironment resulting into restored histoarchitecture of the colon. The results are a clear indicator of the prophylactic potential of selected indigenous probiotics which may be used as an alternative prophylactic biological therapy against colon carcinogenesis particularly in highly susceptible individuals.


Secondary screening of isolated LAB for anticancerous activity in vitro.
To observe the anticancerous effect of isolated LAB in vitro, Caco 2 and HT-29 cells were employed. Isolated LAB viable cell culture as well as their cell-free supernatants (CFS) inhibited the proliferation of both Caco 2 and HT-29 cells. Specifically, antiproliferative potential against Caco 2 cells was most significant (p < 0.01) with LAB isolate #14 (56%) followed by LAB isolate #17A, 22, 14A, 12, 1A, 20 and 19 respectively (Fig. 3a). Although, it was also observed that CFS of all selected isolated LAB reduced the proliferation of Caco 2 cells but CFS of LAB isolate #17A had maximum reduction in proliferation of Caco 2 cells (69%) followed by CFS of LAB isolate #14, 22, 14A 12, 1A, 19 and 20 respectively (Fig. 3a). Thus it was observed that most of the selected isolated LAB cultures and their supernatants had antiproliferative effect on Caco-2 cells but viable cells of isolated LAB #14 had a maximum reduction in proliferation whereas CFS of isolated LAB #17A exhibited maximum antiproliferative activity.
Similarly, with HT-29 cells, all selected isolated LAB viable cell cultures and their CFS significantly (p < 0.01) reduced proliferation (Fig. 3b). It was observed that viable cells of LAB isolate #22 showed a marked reduction in the viability of HT 29 cells (60%) followed by LAB isolate #14, 17A, 12, 1A, 19, 14A and 20 respectively. Though, In vivo study. The selected and identified isolated probiotics i.e. L. rhamnosus MD14, L. plantarum GMD and P. pentosaceus GMD17A were employed to assess their anticancerous potential in early experimental colon carcinogenesis.
Body mass and growth rate. It was observed that one-week prior supplementation of isolated probiotics before the initiation of DMH induced CRC, led to a gradual increase in body mass of animals belonging to all probiotic-fed animals (Fig. 4a). More specifically, it was observed that animals belonging to L. rhamnosus MD14+ DMH-treated (Group III) showed significant (p < 0.05) increase in both body mass and growth rate followed by L. plantarum GMD+ DMH-treated (Group IV) and P. pentosaceus GMD17A+ DMH-treated (Group V) compared with DMH-treated animals (Group II; Fig. 4a,b), which had least increase in body mass and lowest growth rate.
Fecal lactobacilli count. The recovery of lactobacilli in feces indicates the survival, colonizing and transiting ability of probiotics thereby modifying the gut microbiota and microenvironment. It was observed that though prior supplementation of different probiotics led to a gradual increase in lactobacilli count in all probiotic-fed animals but L. rhamnosus MD14+ DMH-treated (Group III) had significantly (p < 0.05) higher lactobacilli count, followed by L. plantarum GMD+ DMH-treated (Group IV), P. pentosaceus GMD17A+ DMH-treated (Group V) compared with DMH-treated (Group II) animals (Fig. 4c).
Fecal pH. It has been hypothesized that alkaline feces increases the risk of colon cancer therefore, the fecal pH of animals belonging to different groups was assessed weekly. It was observed that fecal pH was comparable among animals belonging to all the groups with no significant difference at the beginning of experiment. However, fecal acidification was observed from the second week onwards with probiotic supplementation. It was interesting to observe that at the end of experiment, animals belonging to L. rhamnosus MD14+ DMH-treated (Group III) had maximum acidification of feces (pH-5.4), followed by L. plantarum GMD+ DMH-treated (Group IV; pH-5.8) and P. pentosaceus GMD17A+ DMH-treated (Group V; pH-5.9) compared with DMH-treated animals (Group II; pH 8.1) and control animals (Group I; pH 7.6; Fig. 4d).
Liver function test. The plasma levels of serum bilirubin, ALT, AST and Alkaline phosphatase (AP) were measured as an indicator of liver function in order to assess the effect of LAB supplementation on DMH-treated animals. More specifically, it was observed that animals fed with different isolated probiotic led to a significant decrease (p < 0.05) in all the liver biomarkers in spite of DMH treatment compared with DMH-treated animals (Group II) that had elevated levels of serum bilirubin, ALT, AST and AP (Fig. 5).
Aberrant crypt foci. Aberrant crypt foci (ACF), the earliest and reliable hallmark of colon carcinogenesis at the early stage of experimental carcinogenesis, were found to be significantly decreased (p < 0.05) in all probiotic treated animals compared with DMH-treated animals (Supplementary Table 1 Fecal enzymes assay. Fecal enzymes i.e. β-glucuronidase, nitroreductase, β-glucosidase have been implicated in converting pro-carcinogens into carcinogens thus the activity of these enzymes were assessed to deduce the modulating potential of isolated probiotic in the colonic environment. It was observed that animals supplemented with probiotics along with DMH-treatment had reduced β-glucuronidase and nitroreductase activity with increased β-glucosidase activity (Fig. 6). Specifically, it was observed that animals belonging to L. rhamnosus MD14+ DMH-treated (Group III) had significant (p < 0.05) reduction in the activity of β-glucuronidase and nitroreductase and increased activity of β-glucosidase followed by P. pentosaceus GMD17A+ DMH-treated (Group V) and L. plantarum GMD+ DMH-treated (Group IV) compared with DMH-treated animals (Fig. 6).
Histopathological study. Histology of the colon of control animals (Group I) exhibited normal histo-architecture and cellular morphology of mucosa, submucosa, and muscularis propria with intact epithelium lining (Fig. 7a) compared with disrupted crypts and mucous glands with moderate degree of dysplasia in the form of glandular dilation along with markedly dense inflammatory infiltrate comprising of inflammatory cells, lymphocytes and plasma cells forming a lymphoid nodule along with pseudostratification and hyperchromasia in DMH-treated (Group II) animals (Fig. 7b). Interestingly, the colon of L. rhamnosus MD14+ DMH-treated (Group III) showed intact epithelium lining and closely packed mucus glands with minimal inflammatory infiltrate comprising of eosinophil, plasma cells, neutrophils (Fig. 7c) compared with moderate inflammatory infiltrate forming lymphoid nodule in the submucosal layer in L. plantarum GMD+ DMH-treated (Group IV) animals (Fig. 7d). Similarly, the colonic tissue of animals supplemented with P. pentosaceus GMD17A+ DMH-treated (Group V) had dense inflammatory cells along with edema in lamina propria (Fig. 7e).

Discussion
Colorectal cancer, a lifestyle disease, remains a leading cause of morbidity and mortality worldwide. Among other factors such as genetics, age, exposure to carcinogens, lifestyle, smoking, and alcohol use, diet plays an important contribution to its health risk 3 . Ample evidence suggests that colonic microflora is greatly involved in the etiology of CRC, therefore, dietary interventions and natural bioactive supplements such as probiotics have been studied experimentally extensively to reduce the risk of CRC 13,17,20 . In our earlier studies, we found that prior supplementation of either established probiotics (L. rhamnosus GG, L. acidophilus) alone or in combination with www.nature.com/scientificreports www.nature.com/scientificreports/ Celecoxib exhibited anti-cancerous activity as evident by reduced ACF and procarcinogenic biomarkers: NFκB, COX-2, β-catenin, K-ras [9][10][11][12] . Since the response of probiotics is species and strain specific, the present study was aimed at isolating indigenous probiotics exhibiting antigenotoxic and anticancerous potential vis-à-vis assessing prophylactic potential in experimental CRC.
We observed that isolated LAB from infant fecal samples and fruit peels possessed antigenotoxic and anticancerous activities as these are regarded as the functional properties for characterizing probiotic microorganisms to be used either as prophylactic agents or to reduce the occurrence of CRC in highly susceptible individuals 21,23,24 . In earlier studies, it has been reported that LAB and bifidobacteria inhibited the genotoxic effect of 4-NQO  www.nature.com/scientificreports www.nature.com/scientificreports/ in SOS-Chromotest 23,25,26 . Interestingly, in present study as well, it was observed that both isolated LAB cultures and their respective CFS inhibited the growth of Caco-2 and HT-29 cell lines indicative of antiproliferative potential and corroborates with earlier studies which have also reported that probiotics, both live cultures, and heat-killed cells, and their respective metabolites inhibit the growth of HT-29, HeLa Caco-2 and PANC-1cancer cell lines 7,24,27 .
To further validate the anti-cancerous potential of selected indigenous probiotics, DMH model of experimental colon carcinogenesis was employed as it is a well established, preferred and most widely used model that mimics human sporadic CRC 28 . It was observed that prior supplementation of selected indigenous probiotic resulted in a gradual increase in body mass and growth rate in spite of DMH treatment and is in accordance with earlier [9][10][11][12]15,16 . In these studies, it was observed that prior supplementation of L. acidophilus and L. rhamnosus GG alone or in combination with celecoxib (NSAID) led to an increase in both body mass and growth rate of animals.
Interestingly, in the present study, we observed that continued supplementation of indigenous probiotics, prior to initiation of CRC with DMH, enhanced the fecal lactic acid bacterial count indicating the survival and transiting ability of probiotics thereby modifying the gut microbiota and microenvironment and corroborates with earlier observations 12,15 . Epidemiologically, it has been shown that populations with alkaline fecal pH are at greater risk for colon cancer than populations with acid fecal pH 29 . The observed acidification of fecal pH in the present study may be due to the generation of Short Chain Fatty Acids (butyrate and conjugated linoleic acids) as scientists have also observed that acidification of feces occurred due to probiotic L. acidophilus administration in a DMH induced carcinogenic rat model 30 .
The prophylactic potential of indigenous probiotics was also observed by assessing the hepatophysiology, as the liver is the pivotal organ playing an important role in the normal physiology of human beings. A remarkable reduction in the liver biomarkers (ALT, AST, Alkaline phosphatase and Serum Bilirubin) were observed in probiotic-supplemented animals despite DMH treatment compared with DMH treated animals, where severe liver damage was observed. The results are suggestive of attenuation of cellular leakage and restoration of functional integrity of cell membrane in the liver due to probiotic supplementation. Earlier reports have also documented that prior administration of different probiotics (Lactobacilli and Bifidobacterium) in an acute liver injury rat model reduced hepatocellular damage as well as attenuated liver injury as observed by ALT, bilirubin, and glutathione levels 31,32 .
Aberrant crypt foci (ACF), the earliest preneoplastic marker of CRC, were found to be reduced in animals administered with probiotics. However, the percentage reduction in ACF was variable among probiotics but is comparable with the observation of Verma and Shukla 12 . They have also reported that different probiotics have different percentage of ACF reduction and was maximum with L.GG followed by L. acidophilus and L. plantarum treated animals respectively. The possible mechanism of reduced ACF in probiotics + DMH treated animals may be due to the difference in the interaction of isolated LAB with DMH metabolites that may have altered the intestinal microenvironment mainly by alteration of gut pH and metabolites produced, thus preventing DNA damage in the colon. Moreover, the observed different percentage of ACF reduction by different probiotics may also be due to the fact that probiotic response is very much species and strain specific 22 .
Fecal bacterial enzyme activities were monitored to assess the effect of selected probiotics on the colonic microenvironment as these are implicated in converting pro-carcinogens into carcinogens. It was observed that prior supplementation of selected indigenous probiotic led to decreased activity of β-glucuronidase and nitroreductase suggesting their role in preventing chemical carcinogenesis as β-glucuronidase is responsible for converting DMH to its ultimate carcinogen, methylazoxymethanol 33 . All selected probiotic-supplemented animals had high levels of β-glucosidase activity which may be due to the higher β-glucosidase activity in lactobacilli as it is implemented in carbohydrate metabolism through the catabolism of cellobiose and other β-glucosides 34 . The prophylactic potential of selected probiotics was also validated by in the histopathological observations where normal histoarchitecture of colons of probiotic-supplemented animals was observed. Moreover, lymphocytic infiltration was found to be reduced along with normal mucus glands in probiotic-supplemented animals despite DMH treatment and corroborates with our earlier observations [9][10][11][12] .
Taking into consideration the findings of the present study, it can be stated that probiotics confer protection against CRC mainly by improving homeostasis in the colon which lowers fecal pH and modulates metabolism by altering preneoplastic fecal enzymes resulting in the elimination of toxins and carcinogens. Additionally, probiotics ameliorate liver physiology vis-a-vis attenuate colonic damage. However, a further detailed study is underway to assess the role of selected indigenous probiotic in the modulation of various carcinogenic molecular markers in experimental colon carcinogenesis.

Isolation of lactic acid bacteria (LAB).
Lactic acid bacteria (LAB) were isolated from fruit peels (apple, orange) and fresh neonatal fecal samples from human infants collected in sterile containers. A total of 20 fecal samples from healthy breastfed infants (1-6-month-old) were collected only after obtaining informed consent from the parents in a congenial environment. Briefly, fecal sample (1 g) was serially diluted and inoculated on MRS agar, incubated at 37 °C for 48 h, 3 to 5 single colonies of each sample with different morphologies were randomly selected and again inoculated to MRS broth and streaked on MRS agar several times to get pure culture. The pure isolates were Gram stained for the preliminary identification. Purified cultures were maintained in MRS broth for daily use and preserved in 50% (v/v) sterile glycerol for long term storage at −80 °C.

Preliminary screening for antigenotoxicity of isolated LAB. Bacterial strain. Tester strain E. coli
PQ37 used for SOS Chromotest was gifted by Prof. Marina Isidori, Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Italy. It was maintained by inoculating nutrient agar plates with 10% ampicillin and incubating at 37 °C for 24 h and preserved at −80 °C. SOS chromotest. Antigenotoxicity of isolated LAB was assessed by SOS Chromotest using DMH and 4-NQO as genotoxicant as described by Quillardet et al. 35 . Briefly, the isolated LAB were incubated in MRS broth at 37 °C overnight, cold centrifuged at 4000 g for 10 minutes, washed twice and resuspended at a concentration of 10 9 CFU/mL. An equal volume (20 µL) of LAB suspension and genotoxicant [either DMH or 4-NQO] were incubated at 37 °C for 2 h, cold centrifuged at 4000 g for 10 minutes and the supernatant collected. E. coli PQ37 (600 µL) was added to supernatant of genotoxicant co-incubated with isolated LAB (20 µL). After 2 hr incubation at 37 °C with shaking, β-galactosidase and alkaline phosphatase activities were assayed. Results were expressed in terms of reduction in induction factor (IF).
where IF1 is the induction factor of the test compound, IF2 is the induction factor of positive control (with genotoxicant alone), IF0 the induction factor of the negative control.
Secondary screening for the anticancerous activity of isolated LAB in-vitro. After successful primary screening selected isolates were assessed for their antiproliferative activity against human colon cancer cells in-vitro.
Cell lines. Colon cancer cell lines Caco2 and HT-29 were procured from the Cell Repository, National Center for Cell Sciences, Pune, India, and maintained in Minimum Essential Medium and RPMI-1640 supplemented with penicillin 10 U/mL, streptomycin 100 µg/mL and 20% and 10% heat-inactivated FBS respectively, by incubating at 37 °C in a humidified atmosphere with 5% v/v CO 2 in carbon dioxide incubator. www.nature.com/scientificreports www.nature.com/scientificreports/ Bacterial strains and culture conditions. The isolated LAB were inoculated in MRS broth and incubated at 37 °C overnight; cold centrifuged at 4000 g and the pellet was collected, washed twice with PBS (pH 7.4) and re-suspended at a concentration of 10 9 CFU/mL.

Preparation of cell-free supernatants.
Cell-free supernatant (CFS) of isolated LAB was obtained by cold centrifugation of overnight grown LAB cultures at 4000 g for 10 minutes and filtered through a 0.2 µm membrane filter 36 .
Cell proliferation assay. Effect of isolated LAB and its supernatant on Caco2 and HT 29 colon cancer cell lines was assessed by MTT assay 37 . Caco2 and HT 29 cells (2 × 10 4 cells/mL) were seeded in 96 well tissue culture plates and incubated for 24 h. Thereafter, the media was changed with fresh antibiotic free media and lactobacilli suspension (10 9 CFU/ml; 20 µL) or CFS of LAB (20 µL) was added followed by incubation at 37 °C for 24 h. 20 µL of MTT (5 mg/mL) was then added to each well and incubated for 4 h. At the end of the incubation period, the medium was removed followed by the addition of DMSO (150 µL) to solubilize the formazan crystals. Absorbance was measured at 570 nm using ELISA reader (TECAN Infinite M200). Results were expressed in terms of % survival of cells after treatment and were calculated as (OD of test/OD of control) × 100. All tests were performed in triplicate and repeated thrice. For viable cells, PBS (pH 7) served as control and for CFS MRS broth served as control.
Probiotic characterization of selected LAB. Acid and bile tolerance. To assess the growth of isolates under acidic condition, MRS broth was adjusted to pH 2.5. Overnight LAB cultures were inoculated to the acidic MRS broth and incubated at 37 °C for 1-2 h. Cultures were sampled hourly for viable cell count. Standard MRS broth (pH 6.5) was used as a control.
MRS broth supplemented with 0.3% and 1% oxgall (B3883, Sigma) (w/v) was prepared to assess bile-salt tolerance. Overnight LAB cultures were inoculated to the modified MRS broth and incubated at 37 °C for 2-4 h. Cultures were sampled at 2 h and 4 h for viable cell count. Standard MRS broth served as control.
Cell surface hydrophobicity. The method described by Rosenberg et al. 38 was used to assess bacterial adhesion to hydrocarbons. Briefly, LAB suspension (3 mL) and 1 mL hexadecane or Xylene were mixed by vortexing for 2 min followed by incubation at 37 °C. Samples were withdrawn at 2 h and absorbance was measured at 600 nm. The percentage cell surface hydrophobicity was calculated as per Valeriano et al. 39 .
where A0 = absorbance of the control; A1 = absorbance of aqueous phase Identification of the selected isolates. The screened LAB isolates were identified both phenotypically and phylogenetically mainly by colony characteristics, Grams staining, catalase reaction as well as by partial sequencing of 16S rRNA. The standard method for bacterial DNA isolation was used to extract genomic DNA from LAB isolates. 16S rRNA gene was amplified using universal primers (UNI 27F-5′ AGAGTTTGATCCTGGCTGAG 3′, UNI 1492R-5′ GGTTACCTTGTTACGACTT 3′). The conditions for PCR were 94 °C for 4 min, 94 °C for 30 secs, 49 °C for 40 secs, 72 °C for 100 secs, and 72 °C for 5 min. Amplicon obtained was purified and sequenced. The sequence obtained was subjected to nucleotide Blast at NCBI database for species-level identification of bacteria. Sequences obtained were submitted to the GenBank with the following accession numbers MH656799, MH656803, and MH889142.
In vivo assessment. Based  Housing and husbandry. The animals were housed in polypropylene cages (n = 3 per cage) with a wire mesh top and a hygienic bed of husk (changed regularly) in a well ventilated, temperature and humidity controlled, rat room with 12 h light/dark cycle. The Animals were acclimatized for 7-10 days and were given water and standard pellet diet (Hindustan Lever Products, Kolkata, India) ad libitum.
Induction of colon carcinogenesis. N,N-Dimethyl hydrazine dihydrochloride (DMH) was prepared in 1 mM EDTA saline and adjusted to pH 7.0 with 1 mM NaOH. Single dose of DMH (20 mg/kg body weight) was given intraperitoneally (i.p.) to animals once a week and the treatment was continued for 6 weeks 11 .
Preparation of probiotic dose. For experimental inoculation, 18-h old isolated LAB culture was cold centrifuged at 4000 g for 10 min, washed, and suspended in phosphate buffered saline (PBS, pH 7.2) to contain 1 × 10 9 lactobacilli/0.1 ml 11 . Experimental design. Animals were randomly divided into five groups. Each group comprised of 6 animals and treated as follows Follow up of the animals. During the treatment, body mass, lactobacilli count, fecal pH was monitored weekly. A day before sacrificing the animals, feces of animals was collected, for enzyme (nitroreductase, βglucosidase and βglucuronidase) analysis. Animals were sacrificed after six weeks of DMH treatment under anesthesia (ketamine) and cervical dislocation. Aberrant crypt foci count, liver function test and histopathological analysis of the colon was then performed.
Estimation of body mass and growth rate. The body mass of all animals was recorded weekly on ordinary balance (SD-300, S.D fine chemicals Ltd, Chandigarh, India). The growth rate was calculated as described by Verma and Shukla 11 .
Fecal lactic acid bacterial count. In order to assess the effect of DMH treatment on the beneficial lactic acid bacterial count in the colon, freshly voided fecal material (0.5 g/rat) from each group was homogenized in normal saline, serially diluted, plated on MRS agar and incubated at 37 °C for 24 h and colony forming units (CFU) were recorded 11 .
Fecal pH. In order to evaluate the fecal pH, freshly voided fecal material (0.1 g/rat) from each group once in a week was diluted in 2 ml of saline, homogenized with a glass Teflon homogenizer and immediately checked for pH with a pH meter (Deluxe pH meter, model 101E) as per Giovanna et al. 40 .

Enumeration of aberrant crypt foci (ACF).
The entire colon was removed and cut into small sections (2 × 5 cm) and was processed immediately for ACF counts. The sections were stained with 0.2% methylene blue and ACF were counted using a light microscope. The total number of ACF/rat was calculated as the sum of the small, medium and large ACF as described by Bird 41 . Liver function test. 0.5 ml blood/ rat was collected in a microcentrifuge tube through cardiac puncture and serum was prepared and analyzed for Bilirubin, SGOT, SGPT and Alkaline phosphatase using autoanalyzer, Sysmex XP -100.

Histopathological analysis.
A part of distal colonic tissue was used for histopathological studies. The formalin-fixed colonic tissue was dehydrated in different grades of alcohol. The tissue was dipped in molten paraffin wax and was cooled quickly to prevent crystallization. Thin sections of tissue were cut, and embedded tissue sections were kept in a water bath at 50 °C to remove the wax. Sections were mounted on separate clean