Anti-mutagenic and immune-stimulatory properties of lactic acid bacteria L. Ebringer,* M. FererWk, Ns Lahitov&, L. KaSmi and D. MichAlkov& Statistkally (Lactolwill~s

Enterococcus TAW. The lOO*C for neutrophils macrophages inactivated

significant antigenotoxic activity was exerted by six of nine strains of lactic acid bacteria tested delin-ueckii subsp. bulgakus, Staphylococcus carnosus, Streptococcus thermqhilus, L rhamnosus, fuecium and EK faecak) against nitrovin and Z-amiuoffuorene in Salmonella ~hjrnu~urn TAX00 and mutagenic activity of both mutagens was substantiaIly deczreased by viable bacteria: cells heated to 15 min were ineffective. lo ui&oP EEL fadurn stimulated the basic metabolic activities of human which were essential for their antimkrobia1 and cytotoxie activity, whereas stimulation of guinea-pig was not so effective. Similar immuno-stimulatory effects were observed with both viable and heatbacteria.

I&J words: Ames test* anti-mutagenicity, immune-stimulatiort, lactic acid bacteria, phagocytic activity,

Several Iactic acid bacteria (FAB) occur as part of the normal microfIora of the gastro-intestinal tract of heakhy humans and other animals but relatively little is known of their effects on their hosts. Lactobacilli implanted in the intestinal tract of experimental animals have a suppressive effect on other members of the gut microflora and can confer protection against harmful bacteria (Sandine 197% Gorbach t9 a/. I%%‘). This suppression is not only due to the inhibitory effect of the lactic acid, I-&U2 and baeteriocins produced by the lactobacil~ (Tagg et @I. I%%: Silva ef al. 1987) but also to competition for adhesion sites on the gut epitheliai surface (Conway & ~1.I&37). Dietary supplements of ~acfobacillus~ci~~~~il~ substantially reduced the activity of human and animal faecal bacterial enzymes, such as j3” glucuronidase, @-glucosidase,and azoreductase, which convert indirect-acting carcinogens to proximal carcinogens, and nitroreductase, which is involved in the synthesis of nitrosamines (Goldin cf al. 1980). Several lactobaciili induce macrophage activation and confer enhanced immune rea sponses (Marteau & Ramboud 1993). There are indications that,some lactobacilli inhibit cholesL. Ebringer, Mu Lahitova and 0. MichAlkov& are with the Institute of Molerxrlsr and Subcellular Biology, Comenius University, Odborarske n&m. 5, SK-8$lg? Bradslava, Slovakia; fax: 427 214!302. M. FerenCfk and L. Ka@ini sre with the Institute of Immunology. Faculty of Medicine, Comenius University, SK-El108 Bratisiava, Slovakia. *Corresponding author.

terol synthesis in animals, i~cI~ding man (F&mn 1977), or lower choIestero1 IeveIs by direct assimilation (GEiland ef ai. 1985; Zacconi ef ~2. 199~). There is a generai belief that most lactobacilli are beneficial (Fuller 1989; Marteau & Ramboud 1993). As one can expect qualitative and quantitative differences, not only between speciesbut also among strains, we decided to evaluate the antigenotoxic properties of several LAB. Professional phagocytes (neutroph&, eosinophiIs, monocytes and ma~ophages) play a pivotal roie in the defence of humans and other animals against invading pathogenic microorganisms; activated macropbages are also essential in defence against several different tumours. The respiratory burst, with activation of oxygen metabolism (FerenZik 1993), and the ability to release lysosomal enzymes and other substancesafter various phagocytic or soIuble stimuli (Feren&k 1986) are the most important antimicrobial and anti-tumour factors of phagocytes. The potential for using LAB for iherapy and immuno~moduIation in man was recently reviewed by Marteau & Ramboud {I%x?~].

Materials S~epfococc~

and Methods

~ker~opkii~s KM 4X7?& ~acfubucilf~ sp. CCM 4179, and ~c~~~aci~l~ sp. CCM 4180 were obtained from the Czechoslovak Culture ColIection of Microorgmisms in Bmo. Sfapkyfucuccus caroms eS-lac-Xl/l, &&xxocc~s fcwcalis eS-lac-ID, L. de&v-we&ii

Anfi-mufagenicify Table i. Effect nitrovin towards

of Enferococcus Sa. typhimurhm

faecium TAlOO

En. beciutn (5 x lO*/piate)

0 I I 0

on the mutagenicity and TA97.

of

No. of hfs’ reve~an~/plate* Sa. fyphiSa. typhimurium TAlOO murhm TA87

+ +

226 1236 592 299

114 959 469 147

Nitrovin-l,5-bis(S-n~tro-2-furyl)-l,4-pentadiene-3-on-aminohy-

drazone hydrochloride. *Values are the means of three separate with three plates (in the presence of S9 mix). Table 2. Effect 2-aminofluorene

of Enterococcus ~aecium towards Sa. typhimurium

2.Aminofluorene t.ud/Wte

En. iaecium (5 X 10S/plate)

0 50 50 0

i+

*Values are with triplicate

experiments,

on the mutagenici~ TAlOO.

each

of

No. of his + revertants/piate’ 202 756 566 290

the means of three separate experiments, plates (in the presence of S9 mix).

each

subsp. bujgaricw es-lac-C and L. rhamnosw eS-lac-IA were obtained from Dr J. Zemek, BioEffect Ltd, Bmo. EHterococcusfaeciwn M-74 was donated by Dr P. Mican, Mediphann GmbH, Hustope?e, near Bmo. The bacteria were propagated in MRS broth (Merck) at 37OC for 24 h. The cells were harvested at 18,500 X g for 15 min, washed several times with sterile phosphate-buffered (0.75 mM) saline, pH 7.4 (PBS), and resuspended in PBS to give 5 X 109 bacteria/ml. Assay for Mutagenicify and .4~ti+ntifagenicify in fhe Ames Tesf Anti-mutagenicity was determined by the procedure described by Ames et al. (1975) for testing mutagenic activity of chemicals. Salmonella fypkim~ri~m TAlOO and TA97 (5 X lO’/plate) were used as test strains. Test bacteria were exposed to the mutagens together with the S9-mix metabolic-activating system, then LAB were added at the same concentrations as the test strain (5 X 10s/plate) and the mixture poured onto the plates. LAB do not grow in this medium in the absence of yeast extract or some other complex medium containing growth factors. were separated from heparinized (2.0 U/ml) peripheral blood of healthy donors by the method of Bqum (1976) using Dextran T-500 (Pharmacia). Contaminating erythrocytes were removed by one or two hypotonic lyses. Isolated granulocytes were washed twice with PBS containing 5 Ll heparin/ml (PBSH) and finally resuspended at a concentration of lO’/ml in PBSH containing 0.1% glucose (PBSHG). Only those suspensions which contained at least 9.5% neutrophilic granulocytes were used. Peritoneal macrophages were obtained from guinea-pigs (mean Human

granuiocytes

of lacfic acid bacteria

weight 4OOg) after intraperitoneal injection of 3Om1 10% (w/v) proteose peptone (Difco) in PBS. After 4 days, the peritoneal exudate was withdrawn from each animal into 40ml PBSH and centrifuged and the cells washed twice with PBSH and resuspended in PBSHG to a concentration of IO’/ml. The release of lysosomal enzymes from human granulocytes or guinea-pig macrophages was examined by incubating 0.5 X 10’ phagocytes in 0.5 ml PBSHG without (controls) or with stimulatory substances: opsonized zymosan (OZ), with fresh homologous serum, at ~ng/phagocyte, or lpg phorbol myristate acetate (PMA; Sigma)/phagocyte. Another set of tubes was set up with En. &Y~UWI at 0.05 to 400 cells/phagocyte. Following 6Omin incubation at 37OC, the tubes were cooled and centrifuged for 1Omin at 2000 X g and 4’C. Cell-free supematants (the first supernatants) were used for the determination of extracellular enzymes. PBSHG, 0.5 ml, containing 0.1% Triton X-100 was added to the sediments and, after cell rupture by six freeze-thaw cycles and centrifugation, these second supernatants were used for the estimation of cell-bound enzyme activities. The ability to stimulate the respiratory burst was evaluated by reduction of 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyltetrazohum chloride (INT; Lachema, Brno), which was assayed by a modi~cation of the method of Lokaj & Obtirkovi (1973, using 96-well microtitration pIates (Feren?ik ef al. 1988). In some wells, INT-reductase activity of resting (non-stimulated) phagocytes was measured. Other wells served to determine the activity in phagocytes stimulated by 02 or PMA and additional wells to determine INT-reductase activity of non-stimulated and stimulated phagocytes in the presence of different concentrations of E. &e&m. Both viable cells and cells inactivated by boiling for 3Omin were used. Granulocytes were isolated from peripheral blood of IO clinically healthy volunteers and macrophages were isolated from eight guinea-pigs. INT-reduction was measured in quadruplicate, parallel samples. The results are generally expressed as fmol formazani neutrophil or macrophage after INT reduction for 30min at 37°C. Some results are given as percentages of the values found in the phagocyte samples incubated without enterococci. Zymosan (ZKL; Tren&, Slovakia) was prepared as a homogeneous suspension at a concentration of lOmg/ml PBS. This suspension was incubated with 0.5 ml of fresh homologous serum. After centri~gation, washing and resuspending in the original volume, opsonized zymosan was obtained. PMA was used at a concentration of lOpg/ml in 0.5% DMSO, Lysozyme (EC 3.2.1.17) was assayed turbidimetrically, using A&cmcucc~ ~ysodeicficus(Difco) as substrate, by the method of Ferenzik ef al. (1980). Myeloperoxidase (MPO; EC 3.11.1.7) was determined with odianisidine (Sigma) as an electron acceptor (Ferentik ef aI. 1980). The statistical significance of the results was evaluated by Student’s f-tests.

Results Anti-mufagenic Properfies of lacfic Acid Bacferia In preliminary experiments it was shown that six out of nine LAB prevented the growth of his’ revertants of Sa. ~phima~~m TAW inducible by nitrovin (lpg/piate, without the S9 mix). Streptocuccus fkermophilus decreased induction of his” revertants by 29%, L. delbrueckii subsp. bulgaricus by 33%‘ Sfa. cawzosus by u%, L. rhamnosus by 56%, En.

L. Ebringer et al. Table 3. INT-reductase activity and wIthout En- faecium.*

of human

Group

neutrophilic

Actlvlty fmol/Ne

Le Le + EF400 Le + EFlOO Le + EF50 Le+EFlO Le + EF5 Le + EFO.5 Le + EFO.05 Le+OZ Le + OZ + EF400 Le+OZ+EFlOO Le + OZ + EF50 Le + OZ + EFlO Le+OZ+EF5 Le + OZ + EFO.5 Le + OZ + EFO.05 Le + PMA Le + PMA + EF400 Le + PMA+ EFlOO Le + PMA + EF50 Le+PMA+EFlO Le + PMA + EF5 Le + PMA + EFO.5 Le + PMA+ EFO.05

6.9 20.8 17.7 12.5 6.8 6.3 8.1 6.5 48.3 84.3 86.5 82.4 72.7 59.8 59.7 49.1 29.3 112.8 105.6 75.5 44.7 35.7 27.0 39.4

l lNT-reductase

activity is expressed The results represent mean values Le-Neutrophils or macrophages; phorbol myristate acetate; NS-not

k k 5 k It k k k in k k k 2 k k k k k k k k k k k

granulocytes

100 301 256 181 99 91 88 94 100 175 179 171 151 124 124 102 100 385 360 257 153 122 92 134

hmuno-sfhulafory Acthify of Enterococcus faecium The effect of different concentrations of E. faecim on INTreductase activity of human neutrophils and guinea-pig peritoneal macrophages is shown in Table 3. Concentrations

World Jownd

of Microbiology

Activity Significance P

Anti-mutagenic and immuno-stimulatory properties of lactic acid bacteria.

Statistically significant antigenotoxic activity was exerted by six of nine strains of lactic acid bacteria tested (Lactobacillus delbrueckii subsp. b...
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