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Probiotic lactic acid bacteria detoxify N-nitrosodimethylamineAdriana Nowaka, Sławomir Kuberskib & Zdzisława Libudzisza

a Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and FoodSciences, Lodz University of Technology, Lodz, Polandb Faculty of Process and Environmental Engineering, Division of Molecular Engineering, LodzUniversity of Technology, Lodz, PolandAccepted author version posted online: 10 Jul 2014.Published online: 30 Aug 2014.

To cite this article: Adriana Nowak, Sławomir Kuberski & Zdzisława Libudzisz (2014) Probiotic lactic acid bacteria detoxifyN-nitrosodimethylamine, Food Additives & Contaminants: Part A, 31:10, 1678-1687, DOI: 10.1080/19440049.2014.943304

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Probiotic lactic acid bacteria detoxify N-nitrosodimethylamine

Adriana Nowaka*, Sławomir Kuberskib and Zdzisława Libudzisza

aInstitute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology,Lodz, Poland; bFaculty of Process and Environmental Engineering, Division of Molecular Engineering, Lodz University of Technology,Lodz, Poland

(Received 24 April 2014; accepted 7 July 2014)

Humans can be exposed to N-nitroso compounds (NOCs) due to many environmental sources, as well as endogenousformation. The main nitrosamine found in food products and also synthesised in vivo by intestinal microbiota isN-nitrosodimethylamine (NDMA). It can cause cancer of the stomach, kidney and colon. The effect of four probioticLactobacillus strains on NDMA was studied under different culture conditions (24 h in MRS, 168 h in modified MRS N,and 168 h in phosphate buffer). HPLC and GC-TEA methods were used for NDMA determination in supernatants. Theinfluence of lactic acid bacteria on NDMA genotoxicity was investigated by means of the comet assay. Additionally, theeffect of NDMA (2–100 µg ml–1) on the growth and survival of the probiotic strains was studied. The results indicate thatthe bacteria decreased NDMA concentration by up to 50%, depending on the culture conditions, time of incubation, NDMAconcentration, pH and bacterial strain. Lb. brevis 0945 lowered the concentration and genotoxicity of NDMA mosteffectively by up to 50%. This could be due to either adsorption or metabolism. The growth and survival of the bacteriawas not affected by any of the tested NDMA concentrations.

Keywords: N-nitrosodimethylamine; Lactobacillus; probiotics; detoxification

Introduction

Humans are exposed to nitrosamines through the diet,tobacco smoke and many environmental sources, aswell as from endogenous formation (which contributes45–75% of total exposure) (Jakszyn & Bingham et al.2006). The treatment of food with nitrite is suspected tobe the causative factor of the formation of nitrosamines(Sindelar & Milkowski 2012). The precursor aminesnecessary for the nitrosation reaction are found widelyin the human diet. Red and processed meats induce theendogenous formation of nitroso compounds in thegastrointestinal tract. This process is facilitated byheme. Of particular interest are endogenously formedN-nitroso compounds (NOCs), many of which areknown to be carcinogens. Heme becomes easily nitro-sylated under the anaerobic and reducing conditions ofthe gastrointestinal tract to form nitrosyl heme, whichis an NO donor and can act as a nitrosating agent(Kuhnle et al. 2007; Gunter et al. 2010; Bryan et al.2012).

Endogenous nitrosamines can primarily be formedby bacteria in the oral cavity after ingestion of nitrate,which is reduced to nitrite (Dubrow et al. 2010). Alsoacid-catalysed nitrosation is thought to be a possiblemechanism of in vivo formation of N-nitroso com-pounds in the acidic environment of the stomach,where nitrite reacts with the degradation products ofamino acids (Habermeyer & Eisenbrand 2009).

Up to 1000 species of bacteria may be present in thehuman large intestine. The microbiota can be classifiedinto either health-promoting (mainly the generaBifidobacterium and Lactobacillus) or potentially harmful,some of which can contribute to colorectal cancer (CRC)due to the activation of genotoxic metabolites and carci-nogenic substances in the colon (Zhu et al. 2013). Nitrate,ingested via the diet and drinking water, is readilyconverted to nitrite in the human colon due to the activityof nitrate reductases [EC 1.7.1.] (Enzyme Nomenclature,www.chem.qmul.ac.uk/iubmb/enzyme/) of the colonmicrobiota at neutral pH. The anaerobic reduction ofnitrate to nitrite is the most important route of nitratedissimilation by human colon bacteria. Bacterial strainsbelonging to the genera Escherichia, Pseudomonas,Proteus, Klebsiella and Neisseria have been shown toN-nitrosate nitrogenous precursors in vitro, and N-nitrosa-tion activity is dependent upon the presence of nitrate andnitrite reductase genes (Hughes & Rowland 2000; Zhuet al. 2013). In the colon, nitrites can react with primary,secondary and tertiary amines, amides, glycocholic acid,indoles and phenols to form a variety of nitrosamines(Shephard et al. 1987; Goldin 1990; Krul et al. 2004). Ithas been discovered that E. coli A10 catalyses in vivo theformation of N-nitrosodimethylamine (NDMA) in thecolon (Suzuki & Mitsuoka 1984).

Animal studies suggest that in vivo formation ofnitrosamines is not considerable (but it still needs

*Corresponding author. Email: adriana.nowak@p.lodz.pl

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confirmation), and from a cancer risk perspective pre-formed N-nitroso compounds consumed in items such asbeer, cured meat and fish are considered to be the mainsources of exposure and are significant (WCRF/AICR1997; Habermeyer & Eisenbrand 2009; Ferguson 2010).The IARC stated that there is limited evidence in experi-mental animals for the carcinogenicity of nitrite per se, butthere is sufficient evidence in experimental animals for thecarcinogenicity of nitrite in combination with amines oramides (IARC 2010; SKLM 2014). Mutations induced bycompounds generated by endogenous nitrosation are simi-lar to those found in colorectal tumours, which supportsthe thesis that endogenous nitrosation may be one causa-tive mechanism. According to the IARC, nitrate or nitriteingested by humans under conditions that result in endo-genous nitrosation is probably carcinogenic to humans(Group 2A) (IARC 2010; SKLM 2014). The formationof N-nitrosamines in meat and meat products can occur asa result of various processing techniques such as smoking,salting and curing (Habermeyer & Eisenbrand 2009).Curing is achieved by treating meat with curing salt mix-tures consisting of sodium chloride, sodium/potassiumnitrite and sodium/potassium nitrate. Many foods containhigh amounts of nitrosamines. In humans, certain nitrosa-mines cause cancer of the liver, stomach, oesophagus,kidney, bladder, colon, rectum, pancreas and respiratorytract (Gangolli et al. 1994; Loh et al. 2011; Keszei et al.2013). The major effect of nitrosamines is alkylation ofDNA and the induction of GC-AT transitions in genes(Kuhnle et al. 2007; Dubrow et al. 2010).

Probiotics positively affect the gut microbiota andhuman health by several mechanisms, e.g. modulation ofimmunological parameters, reduction of inflammation,binding of mutagens, inhibition of colon bacterial enzymeactivity and inhibition of bacterial adherence to themucosa (Zhu et al. 2013). Little is known about the inter-action of probiotic bacteria with NDMA.

The objective of the present study was to determinewhether probiotic Lactobacillus strains are able to bind ordegrade NDMA and whether this is correlated with itsdetoxification in a genotoxicity assay. Little is knownabout the ability of the strains to grow and survive in thepresence of NDMA, and so this was also studied.

Materials and methods

Bacterial strains

The following probiotic Lactobacillus strains were used:Lb. rhamnosus ŁOCK 0900, Lb. rhamnosus ŁOCK 0908,Lb. casei ŁOCK 0919, and Lb. brevis 0945(Aleksandrzak-Piekarczyk et al. 2013a, 2013b, 2014).The first three strains are of human origin and licensed(numbers P-382760, P-382761 and P-382762); they pos-sess full probiotic documentation and are deposited at the

Institute of Immunology and Experimental Therapy,Polish Academy of Sciences, Wroclaw (numbersB/00019, B/00020 and B/00021). Lb. brevis ŁOCK 0945is of plant origin and possesses full probiotic documenta-tion (it is in the course of licensing); it is deposited atthe Department of Molecular Microbiology, NationalMedicines Institute, Warsaw. Additionally the commercialprobiotic strain Lb. casei DN 114001 (the Actimel strain)from DANONE (France) was studied.

To maintain the activity of the strains, 24-h cultures inMRS were frozen at –20°C with the addition of 20%glycerol. Before application, the bacteria were activatedtwice in liquid MRS (Merck KGaA, Dermstadt, Germany)and incubated at 37°C for 24 h. Stock cultures were storedat 4–5°C. Inocula (3%) consisted of 24-h cultures ofbacteria in MRS, with a cell density of 109 CFU ml–1

(colony forming units ml–1).

Carcinogen

N-nitrosodimethylamine (Sigma-Aldrich LaborchemikalienGmbH, Seelze, Germany) was diluted in water to obtain astock solution of 0.1%, and it was stored at 4–5°C. Theapplied NDMA concentrations were from 2 to 100 µg ml–1,depending on the experiment. To avoid evaporation of thecompound, each culture of bacteria was sealed with a butylrubber top.

Culture conditions

MRS broth

To determine the influence of NDMA on the growth oflactobacilli during 24 h, the cells were incubated in liquidMRS at 37°C under anaerobic conditions with NDMA,which was added at concentrations of 2, 20 and100 µg ml–1. The controls were bacterial cultures withoutNDMA. To evaluate the influence of the carcinogen on thegrowth of bacteria, the number of living cells was countedusing Koch’s plate method. A total of 1 ml of each culturewas diluted in sterile saline (0.85% NaCl) and serial dilu-tions were poured on plates along with MRS (with 1.5%agar). The number of cells was determined at time ‘0’ andafter 24-h incubation in CFU ml–1. Every concentrationwas plated in quadruplicate and the standard deviation(SD) was calculated each time. Differences between themeans were compared using one-way analysis of variance(ANOVA) (p < 0.01).

Modified MRS broth

In order to ‘force’ the bacteria to use NDMA as a nitrogensource, the medium (MRS broth) was modified and namedMRS N. In MRS N, the amount of yeast extract wasreduced from 4 g l–1 (0.4%) to 1 g l–1 (0.1%), while

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meat extract, peptone and ammonium citrate wereremoved. Moreover, the impact of the microbial growthphase on NDMA decrease was determined. MRS N wasinoculated with bacteria, and NDMA was added at a con-centration of 10 µg ml–1. The cultures were incubated at37°C for 168 h under anaerobic conditions. The controlswere bacterial cultures without NDMA. The concentrationof NDMA in the supernatants was monitored every 4 h(from 0 to 24 h) and then every 48 h (from 24 to 168 h)using HPLC. At the same time, the number of living cellswas counted using Koch’s plate method (for each point,SD and the variability coefficient were calculated).Bacterial cultures were diluted in sterile saline (serialdilutions in 0.85% NaCl), plated using MRS broth (with1.5% agar), and incubated at 37°C for 48 h under anaero-bic conditions. Each dilution of the culture was plated inquadruplicate. After incubation, the colonies werecounted, the results were reported as log CFU ml–1 andthe growth curves of bacteria in the presence of NDMAwere obtained.

Incubation of bacteria with NDMA in phosphate buffer

In order to evaluate the survival of non-growing lactobacilliin the presence of NDMA, 24-h cultures in MRS werecentrifuged (10 700g for 10 min), washed with 20 ml ofsterile phosphate buffer (pH 6.2–6.3) and again centrifuged.Next, the cells were resuspended in the buffer at a concen-tration of 109 CFU ml–1 with 2, 20 or 100 μg ofNDMA ml–1, and incubated at 37°C under anaerobic con-ditions for 168 h (7 days). The control samples were cellsuspensions of each strain without the addition of NDMA.

The pour plate method was used to estimate the num-ber of living cells: every 24–48 h the bacteria were platedusing MRS (with the addition of 1.5% agar) and incubatedat 37°C under anaerobic conditions for 24 h. Colonieswere counted after each 48 h of incubation, and in thisway survival curves were obtained for each strain andmutagen concentration. Differences between the meanswere compared using one-way ANOVA (p < 0.01).

In vitro binding test

In order to determine if the cell walls of lactobacilli canbind NDMA (10 µg ml–1) the cultivation of bacteria inMRS was prolonged to 168 h. Two fractions were pre-pared: culture supernatants and cells. The cells werecentrifuged (12 000g, 15 min), washed twice with steriledistilled water, suspended in water and disintegrated byultrasonic vibrations for 5 min (impulse length 6 s,amplitude 50) at 0°C (in an ice bath). The cell debriswere separated by centrifugation and the concentration ofNDMA in intracellular extracts (supernatants) and mem-brane extracts (pellets) was measured (the boundfraction).

HPLC

Prior to HPLC analysis, NDMA was extracted from allsamples for 1 min with carbon tetrachloride (1:1). Theinitial concentrations of NDMA in the samples were: 2,20 and 100 µg ml–1 in MRS and in phosphate buffer, and10 µg ml–1 in MRS N. NDMA levels were quantifiedusing an HPLC apparatus (Thermo Separation Products;Thermo Fisher Scientific Inc., Waltham, MA, USA),equipped with a UV 6000 LP Photodiode Array Detectorand an ACE-5 C18 column (4.6 mm × 15 cm) with a pre-column. The mobile phase contained methanol and water(20:80, by volume) with the addition of n-butyl methylether (1 ml l–1), and the flow rate was 0.5 ml min–1.Absorbance was measured at 230 nm at RT.

GC-TEA

NDMAwas extracted by low-temperature vacuum distilla-tion according to the method recommended by the FoodSafety and Inspection Service (FSIS) 1991. The distilledextracts were quantitatively analysed on a GC (Varian,model 1440; Varian Medical Systems, Inc., Palo Alto,CA, USA) interfaced with a thermal energy analyser(TEA, model 502A; Thermo Electron Corporation,Waltham, MA, USA). Quantification of nitrosamines wascarried out by analysis of known amounts of the internalstandard N-nitrosodiisopropylamine (NDiPA) (a certifiedstandard from Chem-Services), which was added to thesamples prior to extraction. GC-TEA conditions were asfollows: column: 2.7 m × 3 mm i.d. packed with 15%Carbowax 20 M-TPA on a Chrom W-HP 80/100 mesh;column temperature: 155–170°C; injection port tempera-ture: 200°C; carrier gas: He at 25–30 ml min–1; TEAfurnace temperature: 475°C; vacuum: 0.3–0.4 Torr, andvelocity of oxygen: 7–15–20 ml min–1. The methodenables the identification and simultaneous quantificationof seven nitrosamines at a level above 1 pg ml–1, depend-ing on the analysed material and compound.

The analysis was conducted at the Laboratory ofRadiological Protection and Isotopic Research, NationalVeterinary Research Institute in Puławy, Poland.

HL60 cell culture and treatment

The human promyelocytic leukaemia cell line (HL60) wasused as the target. These cells demonstrate high stabilityand are used in many experiments as model cells. Thecells were cultured in RPMI 1640 medium (Sigma) withthe addition of 10% FBS (Invitrogen; Life TechnologiesPolska Sp. z o. o., Warsaw, Poland), 1% L-glutamine,100 IU ml–1 penicillin and 100 µg ml–1 streptomycin (allSigma). The cells were incubated in a CO2 incubator at37°C in 5% CO2 for 7 days to become fully differentiated.After reaching confluence, the cells were sub-cultivated

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every week. The medium was changed every 3–4 days.The cell suspension was transferred to a 15 ml Falcontube, centrifuged (182g, 5 min), decanted and resuspendedin RPMI 1640. After determination of cell count andviability by trypan blue exclusion (min. 85%), the cellswere ready to use.

Single-cell gel electrophoresis assay (SCGE)

The final concentration of HL60 cells in each sample wasadjusted to 1 × 106 cells ml–1. The cells were incubated at37°C for 1 h with sterile filtered (0.45 µm pore size)supernatants, after cultivation of lactobacilli in MRS andincubation in phosphate buffer with 10 µg NDMA ml–1.The positive controls were samples with NDMA withoutbacteria. The negative controls were supernatants of lacto-bacillus cultures in MRS or suspensions of the bacteria inphosphate buffer without NDMA.

The comet assay was performed under alkaline condi-tions (pH > 13) according to the procedure of Singh et al.(1988) with some modifications (Klaude et al. 1996; Blasiak& Kowalik 2000). After incubation, the cells were centri-fuged (182g, 15 min, 4°C), decanted, suspended in 0.75%LMP agarose (Sigma), layered onto slides pre-coated with0.5% agarose, and lysed at 4°C for 1 h in a buffer consistingof 2.5 M NaCl, 1% Triton X-100, 100 mM EDTA and10 mM Tris, at pH 10. After lysis, the slides were placedin an electrophoresis unit and DNA was allowed to unwindfor 20 min in an electrophoretic solution containing300 mM NaOH and 1 mM EDTA. Electrophoresis wasconducted at 4°C for 20 min at an electric field strength of0.73 V cm–1 (300 mA). The slides were then neutralisedwith 0.4 mol l–1 Tris, stained with 1 µg ml–1 DAPI (4′,6-diamidino-2-phenylindole) and covered with cover slips.The objects were examined at 200× magnification under afluorescence microscope (Nikon, Japan) connected to avideo camera and a personal computer-based image analysissystem Lucia-Comet v.7.0 (Laboratory Imaging, Prague,Czech Republic). Fifty images were randomly selectedfrom each sample and the percentage of DNA in thecomet tail was measured. Two parallel tests with aliquotsof the same sample were performed for a total of 100 cellsand the mean percentage of DNA in the tail was calculatedand taken as a measure of DNA damage. Comet data wereanalysed using two-way ANOVA, while a particular modeof interaction × time was used to compare the effectsinduced by chemicals for this mode of interaction. Theresults are presented as means ± SEM.

The effectiveness of probiotics in decreasing NDMAgenotoxicity after incubation in MRS (24 h) and phos-phate buffer (168 h) was calculated according to thefollowing equation:

PE ¼ M � P

M� 100%

where PE is probiotic effectiveness; M is the genotoxicityof NDMA; and P is the genotoxicity of NDMA in thepresence of a probiotic strain.

Results

Influence of NDMA on the growth and survival ofprobiotic bacteria

The cell harvest in cultures with NDMA and in the con-trols after 24-h growth in MRS was from 109 to1010 CFU ml–1. The highest harvest was obtained forthe commercial strain Lb. casei DN 114001 (1 × 1010

CFU ml–1) and the lowest for Lb. rhamnosus 0908(1 × 109 CFU ml–1) (Figure 1). It is important to notethat NDMA did not influence the morphology of bacteriaeven at a concentration of 100 µg ml–1. Thus, none of thetested NDMA concentrations was toxic for lactobacilli.

NDMA (2–100 µg ml–1) was not found to affect thesurvival of the probiotic strains during 168-h incubation inphosphate buffer (Table 1).

Lactobacillus-induced decrease in NDMA concentration

In MRS

In order to determine whether active growing cells areable to reduce the concentration of NDMA, the bacteriawere cultured in MRS for 24 h. A drop in NDMA con-centration was confirmed with the GC-TEA method. Atthe initial NDMA concentration of 2 µg ml–1 MRS, adecrease of 0.40–0.92 µg ml–1 was found in the presenceof all strains after 24-h cultivation (Table 2), but at theinitial NDMA concentration of 20 µg ml–1 the decreaseamounted to about 6 µg ml–1 in the presence of two strainsonly: Lb. rhamnosus 0908 and Lb. casei DN 114001(Table 2 and Figure 2). At the initial NDMA concentrationof 100 µg ml–1 no change was found.

Figure 1. Number of living cells of Lactobacillus strains after24-h cultivation in MRS with NDMA (error bars denote ±SD;results from three replications). Results are not significantlydifferent from the control, ANOVA (p < 0.05).

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NDMA adsorbing ability was checked after pro-longed cultivation in MRS broth (168 h) with an initialNDMA concentration of 10 µg ml–1. The amount ofNDMA in culture supernatants decreased most consid-erably in the case of Lb. brevis 0945 (Table 3), and itamounted to 6.09 µg ml–1. After sonication, the amountof NDMA in intracellular extracts ranged from0.60 µg ml–1 (Lb. casei DN 114001) to 1.05 µg ml–1

(Lb. rhamnosus 0908) (Table 3), while in membraneextracts it ranged from 0.15 to 0.26 µg ml–1, dependingon the strain. The total amount of NDMA was close toinitial 10 µg ml–1 in the case of three strains (0900,0908 and 0919). For Lb. brevis 0945, the total amountwas 6.91 µg ml–1, so the recovery of NDMAwas 69.1%.

In MRS N

The ability of the bacteria to lower NDMA concentrationwas weak and depended on the growth phase and strain ofbacteria (Figure 3). During the logarithmic phase ofgrowth (8–12 h), lactobacilli were able to decrease

NDMA by about 0.3–0.8 µg ml–1 MRS N (Table 4).However, in the stationary phase, NDMA concentrationincreased to the initial level (10 µg ml–1). The amount ofNDMA decreased again in the death phase in the culturesof Lb. rhamnosus 0900, Lb. casei DN 114001 and Lb.brevis 0945 by about 0.6–0.9 µg ml–1. For Lb. rhamnosus0908 and Lb. casei 0919, NDMA content remained at thesame level throughout 168 h. The effect of the bacteria onNDMA concentration in MRS N is shown in Figure 3 forLb. brevis 0945 and Lb. casei DN 114001.

Table 1. Number of living cells of Lactobacillus strains (logCFU ml–1) after 168-h incubation with NDMA in phosphatebuffer.

StrainTime(h)

Number of bacteria(log CFU ml–1) ± SD

0 2 100

Lb. rhamnosus0900

0 9.53 ± 0.47 9.36 ± 0.06 9.41 ± 0.11168 7.36 ± 0.04 7.53 ± 0.06 7.72 ± 0.14

Lb. rhamnosus0908

0 9.54 ± 0.07 9.55 ± 0.01 9.49 ± 0.18168 6.78 ± 0.14 6.69 ± 0.13 6.60 ± 0.21

Lb. casei DN114001

0 9.58 ± 0.06 9.65 ± 0.31 9.63 ± 0.16168 8.04 ± 0.08 7.88 ± 0.10 7.84 ± 0.28

Lb. casei 0919 0 9.51 ± 0.04 9.51 ± 0.04 9.43 ± 0.08168 7.54 ± 0.09 8.00 ± 0.12 7.89 ± 0.08

Lb. brevis0945

0 9.70 ± 0.05 9.84 ± 0.06 9.92 ± 0.02168 7.09 ± 0.10 7.04 ± 0.06 7.11 ± 0.12

Note: Results are not significantly different from the control (‘0’ for eachstrain); ANOVA (p < 0.05).

Table 2. NDMA concentration (± SD) after 24-h cultivation of Lactobacillus strains in MRS and after 168-h incubation in phosphatebuffer (GC-TEA and HPLC).

Initial NDMA(μg ml–1)

Strain

Lb. rhamnosus 0900 Lb. rhamnosus 0908 Lb. casei DN 114001 Lb. casei 0919 Lb. brevis 0945

2.00 MRS 1.13 ± 0.10 1.23 ± 0.07 1.60 ± 0.00 1.08 ± 0.13 1.38 ± 0.01Buffer 1.45 ± 0.02 1.48 ± 0.05 1.78 ± 0.03 1.58 ± 0.13 1.58 ± 0.01

20.00 MRS 20.00 14.10 ± 0.01 13.90 ± 0.01 19.90 ± 0.01 20.00Buffer 20.00 20.00 17.40 ± 0.02 18.80 ± 0.02 11.70 ± 0.02

Figure 2. Chromatogram (A) of NDMA standard (2 µg ml–1)and (B) after cultivation of Lb. brevis 0945 in MRS with2 µg ml–1 of NDMA (GC-TEA).

Table 3. Amount of NDMA in membrane extracts, intracellularextracts and supernatants after 168-h culture of probioticLactobacillus strains in MRS (± SD) (the initial concentrationin each culture was 10 µg ml–1).

Strain

NDMA concentration (μg ml–1) SD ± 0.09

Membraneextracts

Intracellularextracts Supernatants Total

Lb. rhamnosus0900

0.22 0.74 8.94 9.90

Lb. rhamnosus0908

0.26 1.05 8.70 10.01

Lb. casei DN114001

0.15 0.60 7.57 8.32

Lb. casei 0919 0.25 0.84 8.84 9.93Lb. brevis 0945 0.15 0.67 6.09 6.91

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In phosphate buffer

Subsequently, to determine whether non-growing lacto-bacilli can bind NDMA, they were incubated in phos-phate buffer for 168 h. At the initial NDMAconcentration of 2 µg ml–1, NDMA content decreasedto 1.45 µg ml–1 (Lb. rhamnosus 0900), and it was 0.22–

0.55 µg ml–1 lower, depending on the strain. At ahigher concentration (20 µg ml–1), a decrease wasobserved in the presence of three strains, and forLb. brevis 0945 the decline amounted to 8.3 µg ml–1

(Table 2). At the initial NDMA concentration of100 µg ml–1 there was no change in NDMA content.

Figure 3. Impact of the growth stage of Lb. brevis 0945 (A) and Lb. casei DN 114001 (B) on the concentration of NDMA aftercultivation in MRS (the initial concentration of NDMA was 10 µg ml–1); ■, control (CFU ml–1); ●, 10 µg ml–1 (CFU ml–1); and ▲,NDMA concentration (µg ml–1). Error bars denote ±SD (results from three replications).

Table 4. Changes in NDMA concentration (± SD) during 168-h cultivation of bacteria in MRS N (the initialconcentration of NDMA was 10 µg ml–1) (HPLC).

Strain

Time (h)

0 4 8 12 24 48 96 168

NDMA concentration (μg ml–1) ± 0.2

Lb. rhamnosus 0900 10.0 9.5 9.6 9.8 10.0 10.0 9.2 9.2Lb. rhamnosus 0908 10.0 9.5 9.5 9.7 10.0 10.0 10.0 10.0Lb. casei DN 114001 10.0 9.0 9.2 10.0 10.0 10.0 10.0 9.4Lb. casei 0919 10.0 9.7 9.6 9.8 10.0 10.0 10.0 10.0Lb. brevis 0945 10.0 9.6 9.6 9.6 10.0 10.0 9.2 9.1

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The drop in NDMA concentration was confirmed withGC-TEA method (Table 2).

Genotoxicity of NDMA in the presence of probiotics

Lactobacilli reduced the genotoxicity of NDMA. Thedegree of detoxification depended on the incubation timeand medium used. The values for negative controls were0.97 ± 0.13 (for MRS) and 0.40 ± 0.07 (for phosphatebuffer). Detoxification was more efficient for non-growingcells (after 168-h incubation in phosphate buffer) than foractive cells (after 24 h growth in MRS). Lb. rhamnosus0908 and Lb. brevis 0945 decreased the genotoxicity ofNDMA in MRS most effectively (Table 5). All strains(except Lb. casei DN 114 001) significantly decreasedthe amount of DNA damage in phosphate buffer, withLb. casei 0919 being the most effective (71.3%).

Discussion

The most common nitrosamine found in food and drinkingwater worldwide is NDMA, which is classified as ‘prob-ably carcinogenic to humans’ (Lijinsky 1999; Omaye2004; Nawrocki & Andrzejewski 2011). Cured meat pro-ducts and smoked and salted fish are important foodsources of NDMA.

In most industrialised countries, the estimated averagedaily intake of all volatile N-nitrosamines is approxi-mately 0.2–0.3 μg/person, or 3.3–5.0 ng kg–1 body weight(Habermeyer & Eisenbrand 2009). In the case of NDMA,the mean daily intake from food depends on the countryand dietetic habits, and amounts to 1.8 µg from dried fishin Japan; 0.53 µg from meat products in the UK andGermany (Tricker & Preussmann 1991); 0.19 µg inFrance (for 1987–92) (Biaudet et al. 1994); 0.07 µg inFinland and Northern European countries (Dich et al.1996); and 0.114 µg in the Spanish cohort (Jakszyn &Agudo et al. 2006) – while the range of TDI was estimatedto be from 4.0 to 9.3 ng kg–1 day–1 (which is 0.240–0.558 µg day–1 for a person weighing 60 kg) (Fitzgerald& Robinson 2007). It is important to note that NDMA andother nitrosamines have been detected in beer, drinking

water, vegetables, cigarette smoke, air, cosmetics andmany other items, so actual human exposure to thesecarcinogens is impossible to estimate.

Grill et al. (1995) investigated the influence ofNDMA, NPIP (N-nitrosopiperidine), and NPYR (N-nitrosopyrrolidine) on the growth of bifidobacteria (24 hin TPY (tryptone, peptone, yeast extract) medium). In theconcentration range of 2–200 µg ml–1, the nitrosamineshad no effect on the growth of Bifidobacterium longumBB536. In our study, NDMA concentrations in the rangeof 2–100 µg ml–1 did not influence the growth or survivalof Lactobacillus strains (by either inhibition or stimula-tion), so it may be concluded that the bacteria are notaffected by the compound in the colon for up to 168 h;they remain alive and can be active in the presence of evenunrealistically high concentrations of NDMA. What isimportant is that the amount of total N-nitroso compoundsin the colon increases with the consumption of meatproducts and is positively correlated with intestinal transittime, which varies greatly in individuals and depends onmany factors (e.g. chronic constipations). Slow colonictransit time (more than 72 h) is associated with a highprevalence of CRC (de Kok & van Maanen 2000). Thelonger the transit time, the longer the contact of nitrosa-mines with colon epithelium and intestinal microbiota,which can interact with the compounds – positively ornegatively for human health. Taking into considerationcolonic transit time, the incubation of bacteria in phos-phate buffer and their cultivation in MRS N was pro-longed to 168 h.

In the present study, two NDMA measurement meth-ods were used: HPLC and, additionally, GC-TEA as arecommended system to confirm the results. The decreasein NDMA concentration depended on incubation time,medium and strain. At a low initial concentration ofNDMA in MRS (2 µg ml–1) all strains decreased theircontent after 24-h cultivation, but at a higher concentration(20 µg ml–1) a decrease was observed only in the presenceof Lb. rhamnosus 0908 and DN 114001. In phosphatebuffer, this effect was weaker than in MRS, with theexception of Lb. brevis 0945, for which an almost 41.5%decline was noted at the initial NDMA concentration of20 µg ml–1. At the initial NDMA concentration of100 µg ml–1 there was no reduction in the amount ofNDMA, so in further experiments an NDMA concentra-tion of 10 µg ml–1 was used.

The concentration of amino acids, proteins and nitro-gen in human colon is not high. The amount of proteinentering the caecum from the ileum is 12–18 g day–1, andthis is determined by the total amount of protein in the dietand its physical form. Total nitrogen that enters the largeintestine each day is 0.5–4 g and comprises 10–15% urea,ammonia, nitrate and free amino acids, 48–51% proteinand 34–42% peptides. Faecal nitrogen is 2–4 g day–1

(MacFarlane et al. 1992; Cummings & MacFarlane

Table 5. Effectiveness of lactobacilli in decreasing NDMAgenotoxicity after incubation in MRS and phosphate buffer.

Strain

Effectiveness of probiotic strain (%)

MRS Phosphate buffer

Lb. rhamnosus 0900 2.4 49.3*Lb. rhamnosus 0908 32.0* 37.1*Lb. casei DN 114001 13.1 22.1Lb. casei 0919 12.7 71.3*Lb. brevis 0945 49.8* 34.2*

Note: *Significantly different from positive control, ANOVA (p < 0.05).

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1997; Hughes & Rowland 2000). The protein in ilealeffluent is largely composed of pancreatic enzymes,mucous, exfoliated epithelial cells and dietary residues,whilst in faeces it is at least 50% bacterial nitrogen. Therest are bacterial secretory products, proteins and peptidesreleased during bacterial cell lysis in the distal bowel.Bacterial protein synthesis is a major component of nitro-gen metabolism in the colon (MacFarlane et al. 1992;Hughes & Rowland 2000).

The tested probiotic strains are able to decreaseNDMA concentration in two ways: by absorption andmetabolism. In MRS N (with limited nitrogen sources),the lower capability of decreasing NDMA levels could becaused by lower numbers of bacteria (108 CFU ml–1 inMRS N). The highest decrease was observed in the loga-rithmic, early stationary and death phases of growth. Atthe end of incubation time (168 h), NDMAwas totally (forLb. rhamnosus 0908 and Lb. casei 0919) or partially (forLb. rhamnosus 0900, DN 114001 and Lb. brevis 0945)desorbed to the medium. Adsorptive ability can be con-nected with pH. In MRS, the pH is more acidic (about3.8–4.0) than in MRS N (pH 5.0–5.5), because of the highfermentative activity of bacteria, or than in phosphatebuffer (with a constant pH of 6.2–6.3). A lower pH canenhance the adsorption of carcinogens (Bolognani et al.1997). The ability to bind harmful compounds from thediet is a very important feature of probiotics. In the studyby Peltonen et al. (2000), the aflatoxin-binding capacity ofLactobacillus and Bifidobacterium strains was found torange from 5.8% to 31.3%. After the adsorption of carci-nogens by bacteria, they are excreted via faeces and colonepithelial cells are no longer exposed to them. In thepresent study, NDMA elimination was observed either inculture supernatants or in membrane extracts, and it wasthe most significant for two strains: Lb. brevis 0945 andLb. casei DN 114001. It seems that this process wasgenus- and strain-specific. A 69.1% recovery of totalNDMA was observed for Lb. brevis (at 10 µg ml–1 initialconcentration). This could indicate NDMA metabolism orirreversible binding. Also intracellular extracts had theability to reduce the amount of NDMA, especially in thecase of Lb. rhamnosus 0908; therefore, an enzymaticreaction taking place in intracellular extracts could beinvolved in nitrosamine elimination. The presented resultsare consistent with Grill et al. (1995) who reported thatBifidobacterium longum BB536 metabolised NDMA,NPIP and NPYR in vitro as a result of the enzymaticactivity of intracellular extracts in phosphate buffer. Inthe case of NDMA at a concentration of 2 µg ml–1, theauthors observed 16% degradation, weak elimination at aconcentration of 20 µg ml–1 (0.5–1%), and no change inthe case of 200 µg ml–1. Thus, the ability to decreaseNDMA concentration is negatively correlated with itselimination. The reason for that could be slow transportof NDMA to the bacterial cell. Rowland & Grasso (1975)

showed that intestinal bacteria (Lactobacillus,Bifidobacterium, E. coli, Streptococcus) can effectivelydegrade NDMA, but it was a slow process, and after20 h only 1.75% of the compound was transformed intoits precursor – dimethylamine (DMA), nitrites and othervolatile compounds. They also concluded that the higherthe nitrosamine concentration, the slower the metabolism(degradation was most effective at a concentration of0.5 µmol and amounted to 20%). Dunn et al. (1998)demonstrated that after supplementation of two strains ofLb. acidophilus (NCFM and BG2F04), they loweredDMA and NDMA levels in the blood and body fluids byabout 50% in patients with small bowel bacterialovergrowth.

In the present study, the genotoxicity of NDMA after24-h incubation in MRS was significantly decreased(p < 0.05) by Lb. rhamnosus 0908 and Lb. brevis0945. This was correlated with a drop in NDMA con-centration. All strains (except Lb. casei DN 114001)decreased NDMA levels after 168-h incubation in phos-phate buffer (p < 0.05). The detoxifying and anti-geno-toxic properties of probiotics can be pH dependent, andat neutral and basic pH they have a tendency to be morereversible (Commane et al. 2005). In the present study,these properties were more pronounced at pH close toneutral (phosphate buffer). The inhibitory effects of lacticacid bacteria (LAB) (40 strains isolated from fermentedmilk) on the mutagenicity of nitrosamines (NDMA,NPYR, NPIP and NDEA – N-nitrosodiethanolamine)were investigated in vitro using a Salmonella typhimur-ium TA 98 test by Hosono et al. (1990). LAB inhibitedto the greatest extent the mutagenicity of NDEA, to alesser degree that of NDMA, but did not have any effecton NPIP or NPYR. LAB also inhibited the genotoxicityof nitrosamines in vitro, which was a species-dependentproperty: while Lb. casei and Lactococcus lactisdecreased the mutagenicity of nitrosated beef (by morethan 85%), Lb. confuses and Lb. sake did not (Gill &Rowland 2003). It was demonstrated that the anti-geno-toxic activity of probiotics toward faecal water was posi-tively correlated with cell density, and live cells loweredgenotoxicity more effectively than dead cells (Burns &Rowland 2004), and the former is in agreement with thepresented study. Hebels, Brauers, et al. (2011) reportedsignificant association between urinary NOC excretionand micronuclei frequency in human lymphocytes aswell as gene expression changes in NOC-exposedCaco-2 cells associated with malignant cell transforma-tion. Whole blood transcriptomes of 30 individuals werescreened for transcription patterns correlating with NOCexposure markers and micronuclei frequency in lympho-cytes. Various genes were analysed as potential transcrip-tomic biomarkers. The association between NOCexcretion and micronuclei frequency suggested anincreased cancer risk for humans exposed to NOCs.

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Incubation of human blood cells with NOCs induced theformation of micronuclei, a biomarker of DNA damage(Hebels, Jennen, et al. 2011).

In conclusion, the tested LAB were resistant (couldgrow and survive) to even high concentrations(100 µg ml–1) of NDMA for a long time (up to 168 h).LAB also decreased NDMA content (by adsorption and/ordegradation). This process was influenced by the mediumused, incubation time, phase of growth (depending on thestrain), cell density, pH and NDMA concentration. NDMAdegradation was pronounced in the intracellular fractionand weak in membrane extracts. A decrease in NDMAwas in most cases correlated with a decrease in genotoxi-city, and this effect was stronger under less acidic condi-tions (close to neutral pH). Thus, the studied probioticsdetoxify NDMA in a species- and strain-specific manner.

The detoxifying activity of LAB toward diet carcino-gens could be an additional property of probiosis in thehuman gastrointestinal tract and could probably contributeto diminishing the risk of CRC. Because in vitro systemsare often uncertain, particular attention should be given toestablish accessible biomarkers that reflect the endogenousformation of carcinogenic nitrosamines and study theimpact of probiotics on urinary excretion of genotoxicNOCs or their intermediates.

AcknowledgementsThe comet assay was conducted with the help of ProfessorJanusz Błasiak, Faculty of Molecular Genetics, University ofLodz. The analysis of NDMA with GC-TEA was conductedin the Laboratory of Radiological Protection and IsotopicResearch, National Veterinary Research Institute, Puławy; thanksto Professor Bogdan Kowalski.

FundingThe scientific work was financed by the Ministry of Science andHigher Education as a research project [grant number 2 P06T043 27].

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