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The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 77-82
, and
Institute for Nutritional Physiology, Federal Research Centre for Nutrition, D-76131 Karlsruhe, Germany; * Department of Food Science and Technology, Oregon State University, OR 97331-6602, U.S.; Institute for Hygiene and Toxicology, Federal Research Centre for Nutrition, D-76131 Karlsruhe, Germany
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Lactic acid-producing bacteria prevent carcinogen-induced preneoplastic lesions and tumors in rat colon. Because the mechanisms responsible for these protective effects are unknown, two strains of lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus 191R and Streptococcus salivarius ssp. thermophilus CH3, that are used to produce yogurt, were investigated in vitro and in vivo to elucidate their potential to deactivate carcinogens. Using the "Comet assay" to detect genetic damage, we found that L. bulgaricus 191R applied orally to rats could prevent 1,2-dimethylhydrazine-induced DNA breaks in the colon in vivo, whereas St. thermophilus CH3 were not effective. However, in vitro, both strains prevented DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in isolated primary rat colon cells. Extracts prepared from milk fermented with St. thermophilus CH3 were as efficient in deactivating MNNG as was L-cysteine. Isolated metabolites arising from bacteria during fermentation in the colon or in milk [L(+) lactate, D(
) lactate, palmitic acid and isopalmitic acid] were not effective. We postulate that thiol-containing breakdown products of proteins, via catalysis by bacterial proteases, could be one mechanism by which MNNG or other carcinogens are deactivated in the gut lumen resulting in reduced damage to colonic mucosal cells.
Fermented milk products are associated with several human health benefits. In addition to being palatable and nutritious, certain strains of lactic acid bacteria (LAB)4 help to maintain a well-balanced microflora. This positive influence on the ecology of the gut may contribute to alleviation of diarrhea, increased lactose tolerance in susceptible individuals and modulation of the immune response, as has been shown in human intervention trials (Bartram et al. 1994 This protective effect of fermented milk is supported by some model experiments in vitro or in animals that suggest the potential of certain LAB to inactivate carcinogens, thus proffering them a role as cancer preventive agents (Elnezami et al. 1998 Pool-Zobel et al. (1993b) In this study, we have extended the group of LAB to include strains actually used for fermenting yogurt and investigated metabolic fractions arising during fermentation of milk. Acetone extracts of yogurt fermented by Lactobacillus bulgaricus 191R and Streptococcus thermophilus CH3 previously have been shown to be antimutagenic in the S. typhimurium mutagenicity assay, perhaps via production of transient metabolite fractions that could include fatty acids such as palmitic acid (Nadathur et al. 1995 Chemicals and bacterial strains.
N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) from Aldrich (Steinheim, Germany) was dissolved in physiological saline solution for the in vitro studies. 1,2-Dimethylhydrazine (DMH) was obtained from Sigma (Deisenhofen, Germany) and dissolved in physiological saline solution for application to rats in the in vivo studies. Hydrogen peroxide (H2O2) as a 30% solution was obtained from Merck (Darmstadt, Germany) and was diluted to 100 µmol/L. L-Cysteine, L(+) lactate, D( Acetone extracts.
Acetone extracts of LAB cultures, grown individually or together in milk (St. thermophilus CH3 and L. bulgaricus 191R), were prepared to elucidate whether the strains would cause the same type of protective activity during production of yogurt as they did when grown in MRS. The extracts were obtained from 500 mL fermented milk of 24-h cultures, as has been reported previously (Nadathur et al. 1995 Animals.
Male Sprague-Dawley rats were obtained from our own breeding stock at Karlsruhe or from Charles River (Sulzfeld, Germany); rats were used for the experiments at 6-8 wk of age (160-270 g). Permission for the animal studies was obtained from the State Veterinary Office of Karlsruhe, and experiments were done according to the guidelines for the care and use of laboratory animals.
In vivo assays.
For the in vivo studies, animals were housed individually in wire-bottomed cages. On the last 4 d before killing, rats were given LAB in the morning at 0800 h. Twelve hours before LAB application, the animals were deprived of food to allow the bacteria to reach the colon without interactions. For 30 min after the LAB application, rats were given free access to the diet for 2 h, followed by a food-deprivation period of 5.5 h until DMH (15 mg/kg BW) was applied intragastrically at 1600 h; after 30 min, rats were again given free access to the diet until they were killed at 0800 h the next morning (24 h after the last LAB application) for the isolation of colon cells. The exposure time of 16 h for DMH was chosen because previous studies had shown this schedule to be optimal for observing damaging effects of DMH in colon cells (Pool-Zobel 1996 Cell isolation.
The colons were isolated, flushed with prewarmed buffer, filled with digestion solution containing protease (50,000 U/L) (Proteinase K, EC 3.4.21.64 Sigma) and incubated at 37°C for 30 min. Colon cells were shaken free by gentle agitation, centrifuged (at 107 × g for 8 min, 4°C) and resuspended in RPMI 1640 medium (Gibco, Eggenstein-Leopoldshafen, Germany) at a density of 2 × 109 colon cells/L for cytotoxicity and genotoxicity determinations.
In vitro assays.
The isolated colon cells from male Sprague-Dawley rats at a density of 2 × 109 colon cells/L in RPMI 1640 medium (supplemented with 10 g/L glutamine) were incubated with MNNG, the acetone extracts, or the isolated metabolites or both for 30 min at 37°C in a shaking water bath. For the investigation of the selected metabolites [cysteine (0.2-1.6 mmol/L MRS), L(+) and D( Determination of cytotoxicity and genotoxicity.
Cytotoxicity was determined by staining a 100-µL aliquot of the colon cell suspension in serum-free RPMI with an equal volume of trypan blue and counting the colored and uncolored cells in the Neubauer cell chamber.
Evaluation of slides.
Three slides were processed for each animal or in vitro treatment group. One hundred cells of each slide (300 cells per animal or per in vitro concentration) were scored for the parameter "image length" (IL) using interactive image analysis (Perceptives Instruments, Surrey, UK). The images were grouped into classes of different degrees of damage: percentage of cells with DNA < 40 µm, 40-80 µm, 80-120 µm and > 120 µm. In analogy to the "arbitrary units" of Collins et al. (1995), similar units based on the length of images were used for evaluating the results and to weight the degree of DNA damage (image length units; IL-units). Therefore the percentage of cells with DNA 40-80 µm were multiplied by the factor 2, those with 80-120 µm by the factor 3 and > 120 µm by the factor 4, and were added to yield the IL-unit of each treatment group. This value was then used to compare these data with other published data.
Statistical analyses.
All results are expressed as means ± SD (n as indicated). Comparisons were done with the statistical software package (Instat, GraphPad Prism, San Diego, CA). Differences in treatment groups were evaluated by one-way ANOVA followed by a multiple comparison vs. the positive control by the Dunnett post-test. Differences were considered significant at P < 0.05 and very significant at P < 0.01. In the study of palmitic and isopalmitic acids, a two-way ANOVA was performed, with dose of MNNG and palmitic acid or isopalmitic acid as factors tested.
Figures 1 and 2 show in vivo and in vitro effects of LAB in comparison to published data of related strains of different sources by Pool-Zobel et al. (1996)
Increasing public awareness of the probiotic concept of LAB is being promoted by the commercial propagation of LAB added to milk products, not to achieve fermentation in the traditional sense, but for the putative health-related benefits (Roberfroid et al. 1995
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Jiang et al. 1996, Kaila et al. 1992, Marteau et al. 1997). Some epidemiologic studies showed a decreased incidence of colon cancer in people consuming fermented milk products or yogurt (Malhotra 1977, Peters et al. 1992, Young and Wolf 1988), whereas other studies either showed no effect or even pointed to an increased risk (Kampman et al. 1994, Kearney et al. 1996).
, Fernandes et al. 1992, Hosoda et al. 1996, Nakazawa and Hosono 1992, Orrhage et al. 1994, Rafter 1995).
recently demonstrated this latter activity by showing that several LAB strains are antimutagenic in the Salmonella typhimurium mutagenicity assay and prevent DNA damage induced by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1,2-dimethylhydrazine (DMH) in the colon of rats in vivo (Pool-Zobel et al. 1993a, and Pool-Zobel et al. 1996). These latter studies with the Comet assay may be considered of special relevance to cancer prevention because the antigenotoxic effects were observed in vivo after oral application of LAB and carcinogens and were detected in the colon, an important target organ of dietary-related carcinogenesis. A detailed presentation of the relevance of studies on genotoxic and antigenotoxic effects for studying aspects of cancer prevention was presented previously (Pool-Zobel 1996).
and Nadathur et al. 1996). We also investigated LAB metabolites, expected to occur as fermentation products in the gut lumen, for their ability to deactivate the genotoxic/carcinogenic compounds, MNNG, and hydrogen peroxide (H2O2). MNNG, a direct-acting carcinogen with alkylating properties, was chosen as a representative compound for endogenously formed N-nitroso compounds. In 1996, Bingham et al. showed that a diet high in meat could induce a threefold increase of endogenous N-nitroso compound production in humans.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
) lactate, palmitic acid and isopalmitic acid were from Sigma. LAB cultures were obtained from W. E. Sandine, Oregon State University, (St. thermophilus CH3 and L. bulgaricus 191R), or isolated from a commercially available yogurt from Germany (L. acidophilus). The other remaining St. thermophilus strains, A, B, C as well as L. bulgaricus A, B, C, were coded and provided by L. Lievense from Unilever, in the Netherlands. De Man Rogosa and Sharpe broth (MRS) from Merck was used as the growing medium for LAB. All LAB were inoculated into MRS broth and incubated at 25°C for 16 h. The resulting stationary phase cells were centrifugated (at 11,000 × g for 10 min) and the pellets were resuspended in saline solution to yield 1012 cells/L with the use of a photometric calibration curve (Pool-Zobel et al. 1993a and Pool-Zobel et al. 1996). Paracoccus denitrificans was obtained from E. Mayer-Miebach (Institute for Process Engineering, Federal Research Centre for Nutrition, Karlsruhe, Germany) and grown in a modified mineral salt solution (Mayer-Miebach and Schubert 1991). All bacteria were applied intragastrically to rats in physiological saline solution at 1012 cells/L, 10 mL/kg body weight (BW) in the morning on four consecutive days.
). In short, milk was inoculated with St. thermophilus CH3, L. bulgaricus 191R or a mixture of the two strains, and was fermented at 37°C for 24 h. The fermented milks were freeze-dried and portions of the lyophilized powder were extracted with ~2 volumes of acetone. Acetone was removed by vacuum evaporation, and the residue was dissolved in DMSO and stored at 20°C.
). The food restriction model has been published previously in more detail (Pool-Zobel et al. 1996).
) lactate (2.77-22.2 mmol/L MRS)], a preincubation step with MNNG (0.68 mmol/L) was carried out for 60 min before aliquots were added to colon cells. For studying the effect of whole LAB cultures on MNNG, LAB resuspended in fresh MRS were preincubated with MNNG. Bacteria were then removed by centrifugation, and only aliquots of the supernatants were used for colon cell treatment. Aliquots of the preincubation mixtures, containing unidentified metabolites of LAB and MNNG, were mixed with 1 mL of the colon cells to yield a MNNG concentration of 6.8 µmol/L. Treatment of colon cells was then performed for 30 min at 37°C in a shaking water bath. Colon cells were then centrifuged, resuspended in RPMI 1640 medium and processed for cytotoxicity and genotoxicity.
as described in detail (Pool-Zobel et al. 1996). Briefly, the assay involved the plating of colon cells (2 × 105) in 75 µL low melting temperature agarose (7 g/L) on microscope slides after 10 min of covering with another layer of agarose. Three slides from each rat or incubation tube were placed into lysis solution (100 mmol/L Na2 EDTA, 10 mL/L TritonX-100, 2.5 mol/L NaCl) for 1 h, 4°C. The slides were placed in an electrophoresis chamber containing alkaline buffer (1 mmol/L Na2EDTA, 300 mmol/L NaOH) for DNA unwinding. After 20 min, the current was switched on and electrophoresis conducted at 25 V, 300 mA for 20 min. The slides were removed from the alkaline buffer, placed on a tray and washed three times for 5 min with neutralization buffer (0.4 mmol/L Tris base, pH 7.5). Subsequently, each slide was stained with 100 µL ethidium bromide solution (50.7 µmol/L). All steps, beginning with the isolated colonocytes, were conducted under red light.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
, as well as that of an unrelated bacterium, P. denitrificans, introduced as a control for nonspecific bacterial action. For the data generated in vivo, it is evident that L. bulgaricus 191R is antigenotoxic in the DMH-treated rats, whereas St. thermophilus CH3 is not. In contrast to St. thermophilus CH3, St. thermophilus A was antigenotoxic as were L. bulgaricus A and L. bulgaricus 191R (Fig. 1). The average of all strains tested here (first bar) as negative control showed a significant difference from the positive DMH control. Previous studies revealed no differences after LAB application in NaCl compared with NaCl application. Image lengths were 26.93 ± 4 for L. acidophilus (n = 3), 29.9 and 23.1 for L. gasseri, 23.2 and 29.0 for St. thermophilus and 25.1 and 23.7 for L. confusus in comparison to 26.01 ± 3 for NaCl (n = 7).
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Fig 1.
In vivo antigenotoxic activities of various strains of Streptococcus thermophilus and Lactobacillus bulgaricus against 1,2-dimethylhydrazine (DMH) in the Comet assay. Image length (IL) units as parameters of DNA damage were determined in isolated colon cells of rats treated with the corresponding lactic acid bacteria (LAB) strains, 1012 cells/L, 10 mL/kg body weight (BW) in the morning on four consecutive days without DMH (negative control) or with DMH, 15 mg/kg BW (positive control) or with a combination of both. Values are means ± SD; * P < 0.05 or ** P < 0.01 vs. positive control. The bars represent the mean values of rats treated with either of the following: (A) various St. thermophilus strains without DMH (St.th. all) (n = 14) or DMH (n = 22) or the combination of St. thermophilus (St.th. A, B, C, CH3) or Paracoccus denitrificans (P.den.) with DMH (n = 4-6); (B) various L. bulgaricus strains without DMH (L.b. all) (n = 12) or DMH alone (n = 19) or the combination of L. bulgaricus (L.b. A, B, C, 191R) or P. denitrificans (P.den.) with DMH (n = 4-6).
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Fig 2.
In vitro antigenotoxic activities of various strains of Streptococcus thermophilus and Lactobacillus bulgaricus against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the Comet assay. Image length (IL) units as parameters of DNA damage were determined in isolated colon cells of rats treated for 30 min with DeMan, Rogosa and Sharpe broth (MRS) (negative control), MNNG preincubated in MRS, 6.8 µmol/L (positive control), or with the preincubation mixture of MNNG together with several lactic acid bacteria (LAB) strains in MRS. Values are means ± SD; * P < 0.05 or ** P < 0.01 vs. positive control. The bars represent the mean values of isolated colon cells treated with either of the following: (A) MRS (n = 15), or MNNG preincubated in MRS (n = 15) or the preincubation mixture of various St. thermophilus (St.th. A, B, C, CH3) or Paracoccus denitrificans (P.den.) and MNNG (n = 3); (B) MRS (n = 15), or MNNG preincubated in MRS (n = 15) or the preincubation mixture of various L. bulgaricus (L.b. A, B, C, CH3) of P. denitrificans (P.den.) and MNNG (n = 3).
and Pool-Zobel et al. 1996).
). As shown in Figure 2, in vitro St. thermophilus CH3 and L. bulgaricus 191R were effective in reducing the genotoxic effect of MNNG (6.8 µmol/L) in colon cells as well. In comparison, only one of the other three strains of St. thermophilus, St. thermophilus A, and two of the L. bulgaricus strains, L. bulgaricus B and C showed significant protection. The reduced genotoxicity achieved by treating the cells with St. thermophilus CH3 and L. bulgaricus 191R was apparent for the parameter IL-units (shown in the figures) as well as for the percentage of cells with DNA < 40 µm (data not shown). The supernatant of the active cells (incubated in fresh medium) of St. thermophilus CH3 increased the degree of intact colon cells (percentage of cells with DNA < 40 µm) from 38 ± 1 (MNNG + NaCl) to 53 ± 1 (MNNG + supernatant; P < 0.01, n = 3). In the same way, the supernatant of active cells of L. bulgaricus 191R resulted in a higher value of undamaged cells after MNNG-induced genotoxicity (47 ± 6, MNNG + supernatant vs. 33 ± 6 for MNNG + NaCl; P < 0.05, n = 3), whereas the supernatant of active cells of P. denitrificans, an unrelated bacterium, showed no effect (27 ± 4, MNNG + supernatant vs. 30 ± 6 for the MNNG + NaCl-control).
View this table:
Table 1.
In vitro effects of acetone extracts of fermented milk on genotoxicity induced by N-methyl-N'-nitro-N-nitrosoguanidine (MMNG) in rat colon cells1
) lactate were not protective.
View this table:
Table 2.
In vitro effects of palmitic acid (PA) and isopalmitic acid (isoPA) on genotoxicity induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in rat colon cells1,2
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Fig 3.
In vitro effects of L(+) lactate, D( ) lactate and cysteine on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage in rat colon cells in the Comet assay. Image length (IL)-units as parameters of DNA damage were determined in isolated colon cells of rats treated for 30 min in vitro with MNNG 6.8 µmol/L (positive control) and the metabolites L (+) lactate, D () lactate and cysteine. Values are means ± SD, n = 3-4; * P < 0.05, significantly different from positive control as (group 0; MNNG without corresponding metabolite).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The health benefits claimed include well-being and stimulation of immune defense. In Germany, there has been considerable interest in these products, many of which have become available only recently. In light of these presumed health benefits, it is expected that consumption of LAB may increase in the near future. In addition, because probiotic food products are expected to contain considerably higher levels of LAB than traditionally fermented yogurts, and because these are especially selected to survive the gastrointestinal passage, higher levels of LAB in the gut may result. This assumption is supported by human studies showing that the daily intake of fermented milk products with living LAB, especially probiotic strains, resulted in a measurable increase in their number and metabolic activity in the colon (Link-Amster et al. 1994). Furthermore, a more aware intake of certain nondigestible carbohydrates (prebiotics) should also increase LAB in the gut (Delzenne and Roberfroid 1994, Gibson et al. 1995). Therefore, because of an altered nutritional effect of LAB in the general population, research is required to assess actual benefits and possible long-term hazards of these food ingredients.
). On the basis of the knowledge that cancer induction is a complex multistep process initiated by the accumulation of mutations in tumor-suppressor genes and in protooncogenes, studying protective effects by measuring DNA damage in primary cells with the Comet assay is more relevant than performing other standard short-term genotoxicity assays in bacteria or cultivated cells (Pool-Zobel 1996). At the moment, there is no direct evidence that LAB can inhibit carcinogenesis in humans. Most of the data postulating the protective effect of LAB are based on animal studies. However, these experiments emphasize that LAB can influence several mechanisms possibly linked to carcinogenesis such as preventing mutations, binding mutagens, inhibiting bacterial enzymes that form carcinogens from procarcinogens in the colon, decreasing pH in the colon lumen or activating the immune system. In humans, a decrease in fecal mutagenicity was revealed after consumption of L. acidophilus fermented milk together with fried meat (Lidbeck et al. 1992).
). The underlying mechanism of the unknown metabolites could either be due to a binding of reactive intermediates before they reach the DNA or to an activation of enzymes that detoxify MNNG. In comparison to other strains of St. thermophilus and L. bulgaricus selected from other sources (over half of which had no antigenotoxic activity), they scored as LAB with good potential to be protectants. However, in these studies they were isolated and tested directly as viable LAB in culture, which means that no assumptions can be made concerning the protective activities that may be exerted by yogurt products. Such insight would require knowing the numbers of viable cells in the product and the lowest quantities necessary to achieve antigenotoxic effects in vivo (in humans).
previously showed that free palmitic acid and isopalmitic acid, in particular, which may result from lipolysis of milk, were antimutagenic towards MNNG in the S. typhimurium mutagenicity assay. However, neither fatty acid was antigenotoxic against MNNG. Other protective ingredients of the fermented milk could include major products such as cysteine. We have shown here that cysteine may deactivate MNNG. Specific mechanisms of cysteine interaction with MNNG, potentiating its decomposition, are known (Lawley 1990). This deactivation in the colon lumen, resulting in less available carcinogen reaching the colon cells, is a plausible mechanism that may lead to cancer prevention in the colon (at least cancer induced by compounds such as MNNG). Several studies revealed an increase of cysteine as a consequence of the proteolytic system of LAB (Grudzinshaya and Koroleva 1970, Law and Kolstad 1983). The proteolytic activity will probably not be specific for yogurt bacteria because we have also shown other LAB to be antigenotoxic (Pool-Zobel et al. 1996). In addition, it will not be a "typical" feature of all LAB because we have shown that not all are protective. It would appear that protective activities are dependent on the survival of LAB through the intestinal tract, the accumulation of these organisms in the colon and their ensuing metabolic activities. Other compounds potentially associated with inducing genotoxicity in human colon cells as described by Pool-Zobel and Leucht (1997) should now be studied for their interaction with LAB and LAB metabolites in a similar manner. These studies will give us more in-depth knowledge concerning the total deactivating capacities of these important and beneficial bacteria.
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FOOTNOTES |
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Manuscript received 15 June 1998. Initial reviews completed 30 July 1998. Revision accepted 20 October 1998.
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ACKNOWLEDGMENTS |
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The authors thank Martin Knoll, Renate Lambertz and Sudarshan R. Nadathur for excellent technical assistance.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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