Effects Of Bowman-Birk Protease Inhibitor Research Articles

Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens

Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies.

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The chemopreventive effects of Bowman-Birk protease inhibitor (BBI), a soybean polypeptide, and its palmitic acid conjugate, Pal-BBI, on 7,12-dimethylbenz[a]anthracene (DMBA)-induced transformation were investigated by using an in vitro whole organ culture system of mouse mammary glands. The transformation incidence was measured as the number of glands containing nodule-like alveolar lesions (NLAL). Both BBI and Pal-BBI were effective in preventing DMBA-induced transformation, especially when added to the medium during the developing period after the exposure of mammary glands to DMBA, i.e. 35.9 and 53.4% prevention, respectively. However, when only present in the medium before the promotion period, Pal-BBI was effective in decreasing the transformation incidence (32.2%) while BBI was not (10.3%). This difference could possibly be due to the high lipophilicity and tissue retention of Pal-BBI in mammary gland cultures.

Bowman-Birk protease inhibitor and its palmitic acid conjugate prevent 7,12-dimethylbenz[ a]anthracene-induced transformation in cultured mouse mammary glands

The chemopreventive effects of Bowman-Birk protease inhibitor (BBI), a soybean polypeptide, and its palmitic acid conjugate, Pal-BBI, on 7,12-dimethylbenz[ a]anthracene (DMBA)-induced transformation were investigated by using an in vitro whole organ culture system of mouse mammary glands. The transformation incidence was measured as the number of glands containing nodule-like alveolar lesions (NLAL). Both BBI and Pal-BBI were effective in preventing DMBA-induced transformation, especially when added to the medium during the developing period after the exposure of mammary glands to DMBA, i.e. 35.9 and 53.4% prevention, respectively. However, when only present in the medium before the promotion period, Pal-BBI was effective in decreasing the transformation incidence (32.2%) while BBI was not (10.3%). This difference could possibly be due to the high lipophilicity and tissue retention of Pal-BBI in mammary gland cultures.

The Bowman-Birk protease inhibitor enhances clonogenic cell survival of ionizing radiation-treated nucleotide excision repair-competent cells but not of xeroderma pigmentosum cells

Purpose : The radioprotective effect of the Bowman-Birk protease inhibitor (BBI) was previously shown to result from a TP53 dependent mechanism. Whether this effect involves specific DNA repair mechansims is now tested. Material and methods : Normal human fibroblasts were pre-treated with BBI before exposure to X-rays, UVB or to chemical agents (bleomycin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cisplatin). These agents were chosen because of their ability to induce different spectra of DNA damage. The radiometric agent bleomycin primarily induces double-strand breaks (dsb), which are repaired by recombination; MNNG results in alkylated bases which are repaired by base excision repair (BER); cisplatin results in DNA-crosslinks which are repaired mainly by nucleotide excision repair (NER); and finally UVB generates thymine dimers and thymine-cytosine-6-4 products which are also repaired by NER. Cell survival was analysed by colony formation assay and DNA dsb by constant field gel electrophoresis. The combined effect of BBI and X-rays was also tested for XP-fibroblasts, which are defective in NER. Results : For normal human fibroblasts the radioprotective effect of BBI was clearly found by using a delayed plating procedure. The radioprotective effect was found to be unrelated to an altered induction or repair of radiation-induced DNA dsb. Pre-treatment with BBI did not affect cell killing after exposure to bleomycin or MNNG, but resulted in a significant protection of cells exposed to cisplatin or UVB. These results indicate that pre-treatment with BBI did not alter recombination repair or BER, but was able to modify NER. The latter finding was supported by the observation made for XP-cells, where pre-treatment with BBI failed to result in radioprotection after exposure to ionizing radiation. Conclusions : On the basis of these data it is proposed that the radioprotective effect of BBI is the result of an improved nucleotide excision repair mechanism.

Bowman-Birk Inhibitor Suppresses Production of Superoxide Anion Radicals in Differentiated HL-60 Cells

The Bowman-Birk protease inhibitor (BBI) is a soybean-derived protease inhibitor with anticarcinogenic and anti-inflammatory properties. BBI has previously been shown to suppress the release of superoxide anion radicals from purified polymorphonuclear leukocytes. In the present study we evaluated the effect of BBI on the production of superoxide anion radicals in differentiated HL-60 cells. HL-60 cells are human lymphocytic cells that acquire neutrophil-like characteristics when treated with dimethyl sulfoxide or tetradecanoyl phorbol acetate. Superoxide anion radical production by differentiated HL-60 cells was measured in the presence of various concentrations of BBI or BBI concentrate, a soybean extract containing high levels of BBI. BBI was observed to suppress superoxide anion radical production by differentiated HL-60 cells in a dose-dependent manner. Extracts of differentiated HL-60 cells were also observed to produce superoxide anion radicals, but this activity was not affected by the presence of BBI. These results suggest that BBI inhibits superoxide anion radical generation in HL-60 cells but does not act as a simple free radical scavenger.