Protective Effects of Probiotic Lactobacillus rhamnosus IMC501 in Mice Treated with PhIP
Protective Effects of Probiotic Lactobacillus rhamnosus IMC501 in Mice Treated with PhIP
Journal of Microbiology and Biotechnology. 2014. Mar, 24(3): 371-378
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : September 26, 2013
  • Accepted : December 17, 2013
  • Published : March 28, 2014
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About the Authors
Luca, Dominici
Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, Via del Giochetto, 06122 Perugia, Italy
Milena, Villarini
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Perugia, Via del Giochetto, 06122 Perugia, Italy
Francesca, Trotta
Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, Via del Giochetto, 06122 Perugia, Italy
Ermanno, Federici
Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, Via del Giochetto, 06122 Perugia, Italy
Giovanni, Cenci
Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, Via del Giochetto, 06122 Perugia, Italy
Massimo, Moretti
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Perugia, Via del Giochetto, 06122 Perugia, Italy

The aim of the present study was to investigate the antigenotoxic properties of the probiotic Lactobacillus rhamnosus IMC501; DNA damage was induced by one representative food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Mice were treated orally with suspensions of lactobacilli for 10 days before administration of food mutagen. During the treatment, the abundance of lactobacilli in feces, as assessed by qPCR analysis, increased, whereas β-glucuronidase and N -acetyl-β-glucosaminidase activities decreased. The extent of DNA damage was measured in colon and liver cells by comet assay. In colonocytes, diet supplementation with IMC501 resulted in a significant inhibition of DNA damage induced by PhIP. The results obtained in this in vitro study suggest that Lactobacillus rhamnosus IMC501 used as a dietary supplement can provide a useful integration of antimutagen food components of the normal diet, which are generally lower than the protective level.
Consumer interest in functional foods and dietary supplements and their potential health benefits has been increasing in recent years. The scientific attention is mainly focused on products containing probiotic bacteria that can be used either in prophylaxis or therapy of gastrointestinal tract diseases [21] . A particular aspect of this issue concerns the potential role of probiotics in reducing the risk of intestinal carcinogenesis. Antigenotoxicity and antimutagenicity are therefore considered new tools for probiotics and functional foods [45] , even if the evidence from human clinical trials remains quite limited [5 , 16] .
The human gastrointestinal tract is colonized by microorganisms (up to 1,200 species of bacteria) with peak numbers in the colon (about 10 12 bacteria per gram) [13] . Carbohydrates and proteins are the main fermentative substrates for bacteria in the large intestine. The gut microbiota is either able to degrade undigested polysaccharides to short-chain fatty acids, and synthesize biotin, folate, and vitamin K, as well as ferment indigestible dietary residues, and cooperate in calcium, magnesium, and iron absorption [2] . Whereas products of carbohydrates fermentation are usually beneficial, protein catabolites are rather toxic [34] .
Colonic microbiota can generate a wide variety of mutagens, carcinogens, and tumor promoters from dietary and endogenous precursors. Fecal bacterial enzymes such as β-glucuronidase, β-glucosidase, nitroreductase, 7-α-dehydroxylase, etc ., mainly produced by anaerobic colon bacteria ( i.e ., Bacteroides , Clostridium ) are also involved in the mutagenic activation processes [19 , 41] . The expression of fecal bacterial enzymes in the colonic environment can be largely modulated by probiotics [1] . It is well known that foodborne genotoxic compounds ( e.g ., mycotoxins and plant glycosides) or genotoxins created during food processing ( e.g ., polycyclic aromatic hydrocarbons and heterocyclic amines) are capable of expressing risk in the gut [47] . It has been shown that some probiotics are capable of counteracting genotoxic and/or mutagenic effects induced by several chemical compounds [40] .
At present, epidemiologic data support a role of probiotic intake in the reduction of mutagenicity/cancerogenicity in the intestinal tract. Case-control studies have shown an inverse association between yoghurt or fermented milk intake and the incidence of colon cancer, thus indicating a positive role of lactobacilli and bifidobacteria [42 , 44] . Some clinical studies have demonstrated that diet supplementation with probiotic is associated with a reduction in the levels of urinary and fecal mutagens and a decrease in proliferative activity of colonic crypts in patients with an elevated activity before the treatment [40] .
By in vitro approaches, antigenotoxic activities of probiotic bacteria such as lactobacilli, bifidobacteria, and streptococci have resulted in inhibiting or reducing the activity of genotoxic xenobiotics, such as nitrosamines, heterocyclic amines, alkylating agents, and phenolic compounds [3 , 42 , 45 , 48 , 49] . The results of our previous studies have demonstrated the ability of lactobacilli, bacilli, and yeasts to counteract in vitro various food mutagens, among which include 4-nitroquinoline- N -oxide, N -methyl- N ’-nitro- N -nitrosoguanidine, heterocyclic amines, and aflatoxin B 1 [4 , 8 , 51] . Carcinogenesis studies conducted in laboratory animals have shown that some probiotics ( i.e ., lactobacilli and bifidobacteria) seem capable of counteracting the DNA damage activity exerted by potent food mutagens (2-amino-3-methylimidazo[4,5-f]quinoline, DMH, aflatoxin B 1 , azoxymethane) with consequent reduction of a pre-neoplastic state and protection against tumors induced by these compounds [19 , 26 , 39 , 54] . For these reasons, every strategy reducing mutagen presence may be effective in carcinogenesis prevention. However, the presence of compounds originating from food processing such as roasting and frying is difficult to control.
It is known that high temperatures generate various kinds of toxic substances, such as heterocyclic amines. Heterocyclic amines are formed when meat and fish are cooked at 150–300℃ [15] . A number of studies have reported positive associations between higher consumption of well-done meat and the risk of colon, breast, lung, and gastric cancers [35] . PhIP, the most frequent heterocyclic amine in the human diet [10] , is included in IARC Group 2B as a possible carcinogen to humans [23] and is used as a reference colon carcinogen in murine models [32] . The aim of the present study was to verify in a short-term murine model the ability of Lactobacillus rhamnosus IMC501, a probiotic strain already characterized for its in vitro antigenotoxic capabilities [53] , to modulate the activity of the gut microflora and to inhibit the activity of genotoxic xenobiotics.
The experiments were carried out using a secondary exogenous food mutagen, namely PhIP. The lactobacilli protective effects were studied in liver and colon cells, targets for tumor induction by the considered compound [6 , 36] . The strain efficacy was evaluated by the single-cell microgel-electrophoresis (comet) assay [50] .
Materials and Methods
- Chemicals
Genotoxin : 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP; CAS No. 105650-23-5) was acquired from Maxchemco Chemical Industry Ltd. (Shanghai, China).
Reagents : Dimethyl sulfoxide (DMSO), ethanol, ethylenediamine tetraacetic acid disodium (Na 2 EDTA) and tetrasodium (Na 4 EDTA) salts, hydrochloric acid (HCl), sodium chloride (NaCl), and sodium hydroxide (NaOH) were of analytical grade and were purchased from Carlo Erba Reagenti Srl (Milan, Italy). Ethidium bromide (EtBr), low- and normal-melting-point agarose (LMPA and NMPA, respectively), 4-methylumbelliferone (4-MU), 4-MU-β-Dglucuronide, 4-MU-β-D-glucoside, 4-MU-N-acetyl-β-D-glucosaminide, tris(hydroxymethyl)aminomethane (Tris), and Triton X-100 were obtained from Sigma-Aldrich Srl (Milan, Italy). The Bradford protein assay kit was purchased from Bio-Rad Laboratories (Milan, Italy).
- Probiotic Microorganism
Lactobacillus rhamnosus IMC501, a probiotic strain used as ingredient of functional food and dietary supplement [52 , 53] , was kindly supplied by Synbiotech Srl, a spin-off of University of Camerino (Camerino, Italy).
- Animals and Experimental Procedures
Male CD-1 mice, aged 4–6 weeks, weight 25–30 g, were purchased from Harlan Italy (Milan, Italy) and quarantined for 1 week before the experiments started. Mice were housed individually in plastic cages with wood chip bedding, maintained at 20℃ under a 12 h light/dark cycle. Each animal was allowed free access to water and a standard pellet diet (Altromin-R, A. Rieper SpA, Vandoies, Italy). A total of 20 mice were allocated randomly to four groups (five animals per group) as follows:
  • Group 1: untreated (negative) control, mice given physiological saline (0.9% NaCl).
  • Group 2: IMC501, mice supplemented with the probioticLactobacillus rhamnosusstrain.
  • Group 3: PhIP positive control, mice given physiological saline and treated with PhIP.
  • Group 4: IMC501 + PhIP, mice supplemented with probiotics and treated with PhIP.
To reduce noise in the data and ensure repeatability, a blocked design approach was used; the animal population was stratified into four homogeneous subgroups (blocks). In each repeated experimental set, a block was composed of 8 animals, with two animals randomly assigned to each of the four experimental groups. For the experimental procedure, groups 1 and 3 received physiological saline for 10 consecutive days, whereas groups 2 and 4 received the Lactobacillus suspension (10 9 cells/ml, 10ml/kg b.w.); physiological saline and probiotics were administered by gavage (at 9:00 a.m. of every day). On the 10 th day, 3 h after physiological saline or lactobacilli administration (12:00 a.m.), the mice of groups 3 and 4 were treated by gavage with PhIP (100 mg/kg b.w.). The genotoxin dose was derived from the literature [43] , where PhIP was dissolved in corn oil at a concentration of 20 mg/ml. After a further 3 h (3:00 p.m.), the animals were killed by cervical dislocation and the colons and livers rapidly excised.
The experimental protocol was approved by the Ethical Committee for Animal Experimentation at the University of Perugia, and by the Commission for Animal Experimentation of the Ministry of Health, Rome, Italy.
- Fecal Microbiota: Quantitative Analysis
Fecal samples were collected at the beginning of the experimentation (day 0), after 5 days (day 5), and on the last day, before administration of food mutagens (day 10); feces were stored at -20℃ until analyses. Samples were pooled, thoroughly homogenized and analyzed in triplicate. Total community DNA was extracted from 200 mg of homogenized samples using the MoBio UltraClean Fecal DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) following the manufacturer’s instructions.
The abundance of total bacteria and of the Lactobacillus genus was determined by quantitative polymerase chain reaction (qPCR) analyses of 16S rRNA gene fragments. qPCR was performed on an Mx 3000P qPCR System (Stratagene, La Jolla, CA, USA) using the GoTaq qPCR Master Mix (Promega, Madison, WI, USA) following the manufacturer’s instruction. PCR primers and cycling conditions used for total bacteria [14] and Lactobacillus group [29] were reported elsewhere. The absolute quantification of the target gene copy number was performed as previously reported [14] . Standard curves for the calibration of qPCR assays for total bacteria and Lactobacillus genus were constructed with serial dilutions of linearized plasmids where the full 16S rRNA genes from an Escherichia coli and a Lactobacillus rhamnosus strain, respectively, were cloned.
- Fecal Enzyme Activities
The activities of three microbial enzymes (β-glucuronidase, β-glucosidase, and N -acetyl-β-glucosaminidase) were quantified in fecal samples of mice administered with Lactobacillus rhamnosus IMC501 or physiological saline. The assays were performed according to fluorimetric methods by measuring 4-MU released from 4-MUconjugated substrates. Briefly, 50 mg of fecal sample was homogenized in 5 ml of phosphate buffer at pH 7.0, sonicated (5 cycles, 30 sec, 0℃, 1 min interval), and centrifuged (8,000 ×g, 5 min). Analyses were made on supernatants of cell-free extracts [7] . Fecal proteins were assayed according to the Bradford method. Results were expressed as specific activities (μmol of 4-MU/h/mg protein) and reported as the group mean ± standard error (SE).
- Isolation of Colon and Liver Cells
The explanted portions of colon were emptied of feces and then washed with ice-cold homogenization buffer (75 mM NaCl, 24 mM Na 2 EDTA, pH 7.5). Each colon segment was cut lengthwise and the mucosa was pulled away using a scraper, put in 1 ml of ice-cold homogenization buffer, and homogenized gently using a Potter-Elvehjem on ice to obtain a suspension of isolated cells. The explanted livers were washed with ice-cold homogenization buffer. Each liver was chopped, washed again, and resuspended in 1 ml of the homogenization buffer; an aliquot of liver was gently homogenized using a Potter-Elvehjem on ice to obtain a suspension of isolated cells [30] . Colonocyte and hepatocyte viability was checked by measuring the percentage of the cytosolic enzyme lactate dehydrogenase (LDH) activity released in the extracellular medium [55] . The activity of LDH was determined using a LDH cytotoxicity detection kit (Takara Bio Inc., Shiga, Japan), according to the manufacturer’s instructions. Absorbance was recorded using a Tecan Sunrise microplate reader (Tecan Italia Srl, Milan, Italy).
- Single-Cell Microgel-Electrophoresis Assay
Primary DNA damage in liver and colon cells was evaluated by the single-cell microgel-electrophoresis (comet) assay performed under alkaline conditions according to the original procedure [46 , 50] . The standard alkaline procedure (lysis at pH 10, unwinding and electrophoresis at pH >13) allows the detection of both singleand double-strand DNA breaks as well as apurinic/apyrimidinic sites ( i.e . alkali labile lesions) that are expressed as frank strand breaks in the DNA under the alkaline conditions of the assay. For genotoxicity testing, suspensions of isolated cells were embedded into an agarose microgel onto a microscope slide and subsequently lysed by detergents at high salt concentration. The liberated DNA was then exposed to alkali for unwinding from the strand breakage sites and electrophoresed under alkaline conditions. Processing of cells for ex vivo evaluation of primary DNA damage was carried out according to the method published elsewhere [12] .
After staining with EtBr, DNA results for structures resembling comets with the tail length or tail fluorescence content reflecting the frequency of DNA strand breaks and hence DNA damage [11] . The comets in each microgel were analyzed using a computer-based semi-automated image analysis system (Comet Assay III, Perceptive Instruments Ltd., Suffolk, UK). Computerized imaging, described in detail elsewhere [31] , was performed on coded slides and the percent of DNA in the comet tail ( i.e ., tail intensity; TI) was used to measure the level of DNA damage.
Each result was expressed as the group mean ± standard error (SE). Remaining PhIP-induced genotoxic activity (RGA%) was calculated according to the following formula:
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whereas the genotoxic inhibition rate (GIR%) was expressed as
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- Statistical Analysis
Statistical analysis was performed with the SPSS statistical package (SPSS, Chicago, IL, USA). Comet tail intensity distributions were checked for normality by Kolmogorov-Smirnov goodnessof-fit test. Differences among groups were then evaluated by oneway analysis of variance (ANOVA) followed by Bonferroni post hoc analysis for multiple comparisons. Fecal microbiota differences (qPCR and enzyme activities) were evaluated by Student’s t -test. Two-sided p values <0.05 were regarded as statistical significance.
- Effects on Fecal Microbiota
After IMC501 diet supplementation, the qPCR assay revealed a progressive increase of lactobacilli 16S rRNA with a slight, but significant difference with respect to the control group. The effect was already evident after 5 days administration of IMC501 ( Fig. 1 ).
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Abundance of lactobacilli in mice feces during Lactobacillus rhamnosus IMC501 diet supplementation. Data shown are mean ± SE (n = 3). White bars, control group; grey bars, probiotic-treated group. Values with one different superscript letter are significantly different from each other (p < 0.05, Student’s t-test).
- Effects on Fecal Enzyme Activities
Administration of IMC501 for 10 days was effective in modulating enzyme activities of mice fecal microflora ( Fig. 2 ). In particular, a marked decrease was observed for both β-glucuronidase and N -acetyl-β-glucosaminidase specific activities ( p < 0.05) with fold-reduction in enzyme activities, as compared with the control group, being 63% and 26%, respectively. Any significant reduction of β-glucosidase activity was however observed following IMC501 diet supplementation.
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Effect of Lactobacillus rhamnosus IMC501 diet supplementation for 10 days on fecal bacterial enzyme activities. (A) β-glucuronidase; (B) N-acetyl-β-glucosaminidase; (C) β-glucosidase. White bars, control group; grey bars, probiotic-treated group. Values shown are the mean ± SE (n = 5). ap < 0.05 vs. control, Student’s t-test.
- PhIP-Induced DNA Damage in Colon and Liver Cells
Percent viability observed in colon and liver cells isolated from animals administred with IMC501 and/or treated with PhIP was comparable to that observed in control (untreated) animals. Cell viability group mean values for colon cells were always around 95% (data not shown), and for hepatocytes cell viability was never found to be below 99% (data not shown).
The extent of primary ( i.e ., basal or induced) DNA damage in colon cells, as evaluated by the alkaline comet assay, is summarized in Fig. 3 . Dietary supplementation with L. rhamnosus IMC501 strain (group 2) did not affect primary DNA damage in mice colon cells, with tail intensity values overlapping those of control animals (group 1). DNA strand breakage observed in colon cells from mice receiving PhIP (group 3) was significantly greater than in cells from negative controls (group 1). In the animals treated with IMC501+PhIP (group 4), the extent of primary DNA damage induced by the model mutagen was significantly decreased by the administration of probiotic bacteria, as compared with animals receiving PhIP only (group 3), with a GIR of 69.6%.
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Effect of 10 days of Lactobacillus rhamnosus IMC501 diet supplementation on PhIP-induced DNA damage in mice colon cells. Data are summarized as the group mean value ± SE (n = 5). White bars, negative/positive (PhIP) control groups; grey bars, groups supplemented with probiotics. a,bValues with one different superscript letter are significantly different from each other (p < 0.05) by ANOVA.
Dietary supplementation with IMC501 strain (group 2), as well as treatment with PhIP (group 3) did not cause any increase in the extent of primary DNA damage in mice liver cells, with tail intensity values being similar to those of control animals (group 1); consequently, any effect could be observed in mice supplemented with the Lactobacillus rhamnosus IMC501 strain and treated with IMC501+PhIP (group 4) (data not shown).
The present study was aimed at investigating in vivo the possible protective effects exerted in mice by diet supplementation with probiotic Lactobacillus rhamnosus IMC501 against dietary mutagen-induced injury in colon and liver cells.
Lactobacilli abundance assessed by qPCR confirmed the capability of IMC501 to integrate on the fecal microbial ecosystem of the murine animal model used in this study. The lactobacilli increase in feces, observed already after 5 days of diet supplementation with IMC501, was associated with a reduced activity of fecal enzymes, in particular β-glucuronidase and β- N -acetyl-glucosaminidase that are peculiar of some components of autochthonous fecal microflora. Indeed, β-glucuronidase has the ability to hydrolyze the bond formed during glucuronidation of xenobiotics by the enzyme UDP-glucuronosyltransferase, thus reversing phase II detoxifying reactions in which noxious chemicals ( e.g ., dietary carcinogens) undergo conjugation in the liver; as a consequence, conjugated carcinogens are not excreted and parent genotoxic xenobiotics are released in the colon lumen [22] . As a high activity of bacterial β-glucuronidase in human colon was observed in patients with colorectal cancer [24] , our results highlight the IMC501-related favorable modulation of biomarkers of colorectal cancer risk indicating a harmful activity of colonic microbiota. The decrease of deconjugation activities induced by IMC501 is in line with the findings reported for humans administered with Lactobacillus casei and Lactobacillus paracasei probiotic strains [34] ; more generally, species belonging to Bifidobacterium or Lactobacillus genus are reported to display a low activity of enzymes involved in carcinogens formation, as compared with other major colonic anaerobes ( e.g ., Bacteroides , Clostridium , and Eubacterium ) [1] .
Furthermore, β- N -acetyl-glucosaminidase catalyzes the random hydrolysis of β-1,4 glycosidic bonds between N -acetyl-β-D-glucosamine and D-glucuronic acid residues in hyaluronate. The beneficial and/or detrimental effects of degradation of the gut mucus gel in the intestinal mucosa still need to be defined [17 , 34] . However, as an increase in some mucolytic bacteria ( e.g ., Ruminococcus gnavus , Ruminococcus torques ) was found in subjects with diagnosis of Crohn’s disease or ulcerative colitis [38] , a decreased mucolytic activity could be indicative of a reduced pathogenic risk, with mucus probably protecting colonocytes from biological/chemical noxae.
Supplementation of diet with IMC501 significantly reduced the extent of DNA damage induced by PhIP in colonocytes. PhIP did not show any genotoxic effect in liver cells and, consequently, it was not possible to evaluate potential antigenotoxic properties of the tested Lactobacillus rhamnosus strain in this organ. The beneficial effects reported in the present paper for the challenge lactobacilli/ PhIP are in agreement with previous reports indicating that oral administration of probiotics effectively reduces the extent of induced DNA damage in model animals [12 , 33 , 49 , 54] .
In vitro and in vivo data suggest that lactobacilli may exert their antigenotoxic effects by both direct and indirect mechanisms. Diet supplementation with lactobacilli can modify gut microbiota metabolism, leading to the inhibition of enzyme activities involved in the re-toxification of detoxified xenobiotics [18 , 20] . Direct binding of dietary carcinogens to the cell walls of probiotic bacteria has been reported and is currently considered as the most effective detoxification mechanism preventing absorption of genotoxic xenobiotics through the intestinal mucous. Several food mutagens, including PhIP, were reported to be very effectively bound to the cell walls of Lactobacillus acidophilus and Bifidobacterium longum [25 , 37] . It has been also hypothesized that probiotics can prevent the absorption of genotoxic xenobiotics in the intestine [56] or reduce their bioavailability [27] . It has been also reported that probiotics adhere to colonocytes, both in vitro and in vivo , a feature that seems to play a role in limiting the uptake of xenobiotics through the intestinal barrier [28] . Moreover, probiotics can inactivate food mutagens, and/or their precursors, by several other mechanisms, such as chemical or enzymatic inactivation of mutagens ( e.g ., scavenging) [4 , 9] . However, among mechanisms by which probiotics may exert their beneficial effects, significant differences exist between different probiotic strains [33] .
In this study, the protective effects of Lactobacillus rhamnosus IMC501 strain against PhIP-induced DNA damage were observed in colon cells, where direct positive effects of dietary probiotics could be expected, but not in liver cells, where a systemic chemopreventive activity probably involves resorbed protective agents.
In conclusion, our results confirm that the Lactobacillus rhamnosus IMC501 strain used in this study possesses beneficial properties. Diet supplementation with IMC501 was associated with a reduced activity of fecal enzymes associated with a harmful activity of colonic microbiota and used as biomarkers of colorectal cancer risk. Moreover, the tested probiotic bacteria were capable of reducing significantly the extent of PhIP-induced DNA damage in colon cells. The possibility of using probiotic strains as dietary supplements to counteract the activity of food mutagens in the human gut might be of interest for the production of functional foods, especially dairy products, such as yoghurts and fermented milks containing Lactobacillus and Bifidobacterium stains. With regard to the microorganism used in this in vivo approach, our data suggest that the incorporations of Lactobacillus rhamnosus IMC501 in the diet might contribute to suppress or reduce the genotoxic activity of potentially harmful compounds.
Allsopp P , Rowland I 2009 Potential protective effects of probiotics and prebiotics against colorectal cancer, p. 997-1048. In Charalampopoulos D, Rastall RA (eds.). Prebiotics and Probiotics Science and Technology. Springer New York.
Blaut M , Clavel T 2007 Metabolic diversity of the intestinal microbiota: implications for health and disease. J. Nutr. 137 751S - 755S
Burns AJ , Rowland IR 2004 Antigenotoxicity of probiotics and prebiotics on faecal water-induced DNA damage in human colon adenocarcinoma cells. Mutat. Res. 551 233 - 243    DOI : 10.1016/j.mrfmmm.2004.03.010
Caldini G , Trotta F , Villarini M , Moretti M , Pasquini R , Scassellati-Sforzolini G , Cenci G 2005 Screening of potential lactobacilli antigenotoxicity by microbial and mammalian cell-based tests. Int. J. Food Microbiol. 102 37 - 47    DOI : 10.1016/j.ijfoodmicro.2004.11.015
Capurso G , Marignani M , Delle Fave G 2006 Probiotics and the incidence of colorectal cancer: when evidence is not evident. Dig. Liver Dis. 38 S277 - S282    DOI : 10.1016/S1590-8658(07)60010-3
Carere A 2006 Genotoxicity and carcinogenicity of acrylamide: a critical review. Ann. Ist. Super. Sanita 42 144 - 155
Cenci G , Caldini G , Mastrandrea V , Votoni MR 1993 Carbohydrate enriched diets and bacterial glycosidases in rat faeces. Microbios 76 143 - 151
Cenci G , Caldini G , Trotta F , Bosi P 2008 In vitro inhibitory activity of probiotic spore-forming bacilli against genotoxins. Lett. Appl. Microbiol. 46 331 - 337    DOI : 10.1111/j.1472-765X.2007.02314.x
Chalova VI , Lingbeck JM , Kwon YM , Ricke SC 2008 Extracellular antimutagenic activities of selected probiotic Bifidobacterium and Lactobacillus spp. as a function of growth phase. J. Environ. Sci. Health B 43 193 - 198    DOI : 10.1080/03601230701795262
Dingley KH , Curtis KD , Nowell S , Felton JS , Lang NP , Turteltaub KW 1999 DNA and protein adduct formation in the colon and blood of humans after exposure to a dietaryrelevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Epidemiol. Biomarkers Prev. 8 507 - 512
Dominici L , Cerbone B , Villarini M , Pagiotti R , Moretti M 2013 In vitro genotoxicity testing of Lawsonia inermis and henna extracts in HepG2 cells by the comet assay. Int. J. Nat. Prod. Sci. 3 12 - 21
Dominici L , Moretti M , Villarini M , Vannini S , Cenci G , Zampino C , Traina G 2011 In vivo antigenotoxic properties of a commercial probiotic supplement containing bifidobacteria. Int. J. Probiotics Prebiotics 6 179 - 196
Egert M , de Graaf AA , Smidt H , de Vos WM , Venema K 2006 Beyond diversity: functional microbiomics of the human colon. Trends Microbiol. 14 86 - 91    DOI : 10.1016/j.tim.2005.12.007
Federici E , Giubilei MA , Cajthaml T , Petruccioli M , D’Annibale A 2011 Lentinus (Panus) tigrinus augmentation of a historically contaminated soil: matrix decontamination and structure and function of the resident bacterial community. J. Hazard. Mater. 186 1263 - 1270    DOI : 10.1016/j.jhazmat.2010.11.128
Felton JS , Knize MG , Wu RW , Colvin ME , Hatch FT , Malfatti MA 2007 Mutagenic potency of food-derived heterocyclic amines. Mutat. Res. 616 90 - 94    DOI : 10.1016/j.mrfmmm.2006.11.010
Fotiadis CI , Stoidis CN , Spyropoulos BG , Zografos ED 2008 Role of probiotics, prebiotics and synbiotics in chemoprevention for colorectal cancer. World J. Gastroenterol. 14 6453 - 6457    DOI : 10.3748/wjg.14.6453
Gill C , Rowland IR 2003 Cancer, p. 19-53. In Mattila-Sandholm T, Saarela M (eds.). Functional Dairy Products. CRC Press Boca Raton.
Goldin BR , Gorbach SL , Saxelin M , Barakat S , Gualtieri L , Salminen S 1992 Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. Dig. Dis. Sci. 37 121 - 128    DOI : 10.1007/BF01308354
Goldin BR , Gualtieri LJ , Moore RP 1996 The effect of Lactobacillus GG on the initiation and promotion of DMHinduced intestinal tumors in the rat. Nutr. Cancer 25 197 - 204    DOI : 10.1080/01635589609514442
Hatakka K , Holma R , El-Nezami H , Suomalainen T , Kuisma M , Saxelin M 2008 The influence of Lactobacillus rhamnosus LC705 together with Propionibacterium freudenreichii ssp. shermanii JS on potentially carcinogenic bacterial activity in human colon. Int. J. Food Microbiol. 128 406 - 410    DOI : 10.1016/j.ijfoodmicro.2008.09.010
Howarth GS , Wang H 2013 Role of endogenous microbiota, probiotics and their biological products in human health. Nutrients 5 58 - 81    DOI : 10.3390/nu5010058
Humblot C , Murkovic M , Rigottier-Gois L , Bensaada M , Bouclet A , Andrieux C 2007 Beta-glucuronidase in human intestinal microbiota is necessary for the colonic genotoxicity of the food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in rats. Carcinogenesis 28 2419 - 2425    DOI : 10.1093/carcin/bgm170
IARC 1993 Some Naturally Occurring Substances - Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. International Agency for Research on Cancer Lyon, France.
Kim DH , Jin YH 2001 Intestinal bacterial beta-glucuronidase activity of patients with colon cancer. Arch. Pharm. Res. 24 564 - 567    DOI : 10.1007/BF02975166
Knasmuller S , Steinkellner H , Hirschl AM , Rabot S , Nobis EC , Kassie F 2001 Impact of bacteria in dairy products and of the intestinal microflora on the genotoxic and carcinogenic effects of heterocyclic aromatic amines. Mutat. Res. 480-481 129 - 138    DOI : 10.1016/S0027-5107(01)00176-2
Kumar M , Verma V , Nagpal R , Kumar A , Behare PV , Singh B , Aggarwa PK 2012 Anticarcinogenic effect of probiotic fermented milk and chlorophyllin on aflatoxin-B(1)-induced liver carcinogenesis in rats. Br. J. Nutr. 107 1006 - 1016    DOI : 10.1017/S0007114511003953
Lidbeck A , Nord CE , Gustafsson JA , Rafter J 1992 Lactobacilli, anticarcinogenic activities and human intestinal microflora. Eur. J. Cancer Prev. 1 341 - 353    DOI : 10.1097/00008469-199208000-00002
Liu C , Zhang ZY , Dong K , Guo XK 2010 Adhesion and immunomodulatory effects of Bifidobacterium lactis HN019 on intestinal epithelial cells INT-407. World J. Gastroenterol. 16 2283 - 2290    DOI : 10.3748/wjg.v16.i18.2283
Lubbs DC , Vester BM , Fastinger ND , Swanson KS 2009 Dietary protein concentration affects intestinal microbiota of adult cats: a study using DGGE and qPCR to evaluate differences in microbial populations in the feline gastrointestinal tract. J. Anim. Physiol. Anim. Nutr. (Berl.) 93 113 - 121    DOI : 10.1111/j.1439-0396.2007.00788.x
Mariucci G , Villarini M , Moretti M , Taha E , Conte C , Minelli A 2010 Brain DNA damage and 70-kDa heat shock protein expression in CD1 mice exposed to extremely low frequency magnetic fields. Int. J. Radiat. Biol. 86 701 - 710    DOI : 10.3109/09553001003789588
Moretti M , Marcarelli M , Villarini M , Fatigoni C , Scassellati-Sforzolini G , Pasquini R 2002 In vitro testing for genotoxicity of the herbicide terbutryn: cytogenetic and primary DNA damage. Toxicol. In Vitro 16 81 - 88    DOI : 10.1016/S0887-2333(01)00092-3
Nakagama H , Ochiai M , Ubagai T , Tajima R , Fujiwara K , Sugimura T , Nagao M 2002 A rat colon cancer model induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP. Mutat. Res. 506-507 137 - 144    DOI : 10.1016/S0027-5107(02)00160-4
Ng SC , Hart AL , Kamm MA , Stagg AJ , Knight SC 2009 Mechanisms of action of probiotics: recent advances. Inflamm. Bowel Dis. 15 300 - 310    DOI : 10.1002/ibd.20602
Nowak A , Śliżewska K , Klewicka E 2011 Effect of probiotic lactobacilli on faecal enzyme and genotoxic activity in human faecal water in the presence of the carcinogen PhIP in vitro. Int. J. Dairy Technol. 65 300 - 307    DOI : 10.1111/j.1471-0307.2011.00760.x
Oba S , Shimizu N , Nagata C , Shimizu H , Kametani M , Takeyama N 2006 The relationship between the consumption of meat, fat, and coffee and the risk of colon cancer: a prospective study in Japan. Cancer Lett. 244 260 - 267    DOI : 10.1016/j.canlet.2005.12.037
Ohgaki H 2000 Carcinogenicity in animals and specific organs, Rodents, p. 198-228. In Nagoo M, Sugimura T (eds.). Food Borne Carcinogenesis: Heterocyclic Amines. Wiley, New York NY.
Orrhage K , Sillerstrom E , Gustafsson JA , Nord CE , Rafter J 1994 Binding of mutagenic heterocyclic amines by intestinal and lactic acid bacteria. Mutat. Res. 311 239 - 248    DOI : 10.1016/0027-5107(94)90182-1
Png CW , Linden SK , Gilshenan KS , Zoetendal EG , McSweeney CS , Sly LI 2010 Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am. J. Gastroenterol. 105 2420 - 2428    DOI : 10.1038/ajg.2010.281
Pool-Zobel BL , Neudecker C , Domizlaff I , Ji S , Schillinger U , Rumney C 1996 Lactobacillus- and Bifidobacteriummediated antigenotoxicity in the colon of rats. Nutr. Cancer 26 365 - 380    DOI : 10.1080/01635589609514492
Rafter J 2004 The effects of probiotics on colon cancer development. Nutr. Res. Rev. 17 277 - 284    DOI : 10.1079/NRR200484
Rolfe RD 2000 The role of probiotic cultures in the control of gastrointestinal health. J. Nutr. 130 396S - 402S
Saikali J , Picard C , Freitas M , Holt P 2004 Fermented milks, probiotic cultures, and colon cancer. Nutr. Cancer 49 14 - 24    DOI : 10.1207/s15327914nc4901_3
Sekihashi K , Yamamoto A , Matsumura Y , Ueno S , Watanabe-Akanuma M , Kassie F 2002 Comparative investigation of multiple organs of mice and rats in the comet assay. Mutat. Res. 517 53 - 75    DOI : 10.1016/S1383-5718(02)00034-7
Senesse P , Boutron-Ruault MC , Faivre J , Chatelain N , Belghiti C , Meance S 2002 Foods as risk factors for colorectal adenomas: a case-control study in Burgundy (France). Nutr. Cancer 44 7 - 15    DOI : 10.1207/S15327914NC441_2
Shah S 2007 Dietary factors in the modulation of inflammatory bowel disease activity. MedGenMed 9 60 -
Singh NP , McCoy MT , Tice RR , Schneider EL 1988 A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175 184 - 191    DOI : 10.1016/0014-4827(88)90265-0
Sugimura T , Wakabayashi K , Nakagama H , Nagao M 2004 Heterocyclic amines: mutagens/carcinogens produced during cooking of meat and fish. Cancer Sci. 95 290 - 299    DOI : 10.1111/j.1349-7006.2004.tb03205.x
Tavan E , Cayuela C , Antoine JM , Cassand P 2002 Antimutagenic activities of various lactic acid bacteria against food mutagens: heterocyclic amines. J. Dairy Res. 69 335 - 341    DOI : 10.1017/S002202990200540X
Tavan E , Cayuela C , Antoine JM , Trugnan G , Chaugier C , Cassand P 2002 Effects of dairy products on heterocyclic aromatic amine-induced rat colon carcinogenesis. Carcinogenesis 23 477 - 483    DOI : 10.1093/carcin/23.3.477
Tice RR , Agurell E , Anderson D , Burlinson B , Hartmann A , Kobayashi H 2000 Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35 206 - 221    DOI : 10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J
Trotta F , Caldini G , Dominici L , Federici E , Tofalo R , Schirone M 2012 Food borne yeasts as DNA-bioprotective agents against model genotoxins. Int. J. Food Microbiol. 153 275 - 280    DOI : 10.1016/j.ijfoodmicro.2011.11.009
Verdenelli MC , Ghelfi F , Silvi S , Orpianesi C , Cecchini C , Cresci A 2009 Probiotic properties of Lactobacillus rhamnosus and Lactobacillus paracasei isolated from human faeces. Eur. J. Nutr. 48 355 - 363    DOI : 10.1007/s00394-009-0021-2
Verdenelli MC , Ricciutelli M , Gigli F , Cenci G , Trotta F , Caldini G 2010 Investigations of the antigenotoxic properties of the probiotic Lactobacillus rhamnosus IMC 501 by gas chromatography-mass spectrometry. Ital. J. Food Sci. 4 473 - 478
Villarini M , Caldini G , Moretti M , Trotta F , Pasquini R , Cenci G 2008 Modulatory activity of a Lactobacillus casei strain on 1,2-dimethylhydrazine-induced genotoxicity in rats. Environ. Mol. Mutagen. 49 192 - 199    DOI : 10.1002/em.20367
Villarini M , Fatigoni C , Cerbone B , Dominici L , Moretti M , Pagiotti R 2011 In vitro testing of a laxative herbal food supplement for genotoxic and antigenotoxic properties. J. Med. Plants Res. 5 2533 - 2539
Zhang XB , Ohta Y 1993 Antimutagenicity of cell fractions of microorganisms on potent mutagenic pyrolysates. Mutat. Res. 298 247 - 253    DOI : 10.1016/0165-1218(93)90003-V