Lactobacillus- and bifidobacterium-mediated antigenotoxicity in the colon of rats

Lactic acid bacteria (LAB) are proposed to have several beneficial effects, including the inactivation of carcinogens. We have studied the potential of Lactobacillus acidophilus (from a commercially available yogurt), Lactobacillus gasseri (P79), Lactobacillus confusus (DSM20196), Streptococcus thermophilus (NCIM 50083), Bifidobacterium breve and Bifidobacterium longum (from human infant stool) to prevent the induction of DNA damage by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 7.5 mg/kg body wt) in colon cells of the rat. Using the new technique of single cell microgel electrophoresis, all investigated strains were antigenotoxic toward MNNG after a single dose of 10(10) viable cells/kg body wt p.o. eight hours before the carcinogen. One-half and one-tenth of this initial dose resulted in a loss of protective activity. High doses of heat-treated L. acidophilus strains were also not antigenotoxic. One mechanism of the preventive effect could be that bacterial metabolites or components are responsible. Accordingly, selected examples were investigated in vitro in colon cells of the rat. Metabolically active L. acidophilus cells, as well as an acetone extract of the culture, prevented MNNG-induced DNA damage. Different cell fractions from L. acidophilus (cytoplasm, cell wall skeleton, cell wall) were devoid of antigenotoxic activity, whereas the peptidoglycan fraction and whole freeze-dried cells were antigenotoxic. As a second carcinogen, 1,2-dimethylhydrazine (DMH) was used. A dose- and time-response study was first performed to assess the effects of DMH in several segments of the gastrointestinal (GI) tract. Exposure for 16 hours to 15 or 25 mg DMH/kg body wt p.o. induced DNA damage in cells of the distal colon of rats, whereas no cytotoxicity was seen. Pretreatment orally with LAB on four consecutive mornings before DMH gavage (8 hours after the last LAB application) revealed that L. acidophilus, L. confusus, L. gasseri, B. longum, and B. breve inhibited the genotoxic effect of DMH. One of four S. thermophilus and one of three Lactobacillus delbrueckeii ssp. bulgaricus strains were also protective. Heat-treated L. acidophilus did not inhibit DMH-induced genotoxicity. A few aliquots of the colon cells were processed immunohistochemically for the presence of the "proliferation cell nuclear antigen" (PCNA). DMH treatment did not increase PCNA, nor was there any modulation by LAB. The effect of L. acidophilus on foreign compound-metabolizing enzymes (Phase I and Phase II) in liver and colon cells of rats revealed only one parameter to be modulated, namely, a two- to three-fold increase in the levels of NADPH-cytochrome P-450 reductase. The meaning of this finding, in terms of possible chemoprevention by LAB, remains unclear. In conclusion, our studies show that most, but not all, LAB tested could strongly inhibit genotoxicity in the GI tract of the rat and that viable LAB organisms are required for the protective effect in vivo. The comet assay technique is a powerful tool to elucidate such in vivo antigenotoxic activities in tumor target tissues.