Detection of potential chemopreventive agents in plants

In efforts to prevent cancer growing attention has been paid to the identification of chemical compounds for use as chemopreventive agents. In order to screen and rapidly fractionate plant extracts to isolate potential chemopreventive agents, an assay measuring their effects on the metabolism of benzo(a)pyrene (B(a)P) to water-soluble derivatives in hamster embryo cell cultures was utilized. Activity-directed fractionation of an extract of Trifolium pratense led to the isolation of biochanin A, an isoflavone. Biochanin A was found to inhibit two pathways of metabolism of B(a)P in hamster embryo cells: the cytochrome P-450 system and conjugation of B(a)P-phenols to glucuronides. Biochanin A treatment of hamster embryo cell cultures resulted in a decrease in the binding of B(a)P to DNA, especially through the carcinogenic (+)-anti-B(a)P-7,8-diol-9,10-epoxide. Biochanin A was not mutagenic and was an effective inhibitor of B(a)P-induced mutagenesis in the hamster embryo cell-mediated V79 cell mutation assay. To determine which flavonoids would have the greatest ability to inhibit B(a)P metabolism, a series of isoflavonoids and flavonoids were tested in the hamster embryo cell culture B(a)P metabolism assay. Analysis of structure-activity relationships indicated the importance of the 5- and 7-hydroxyl groups and a 2,3-double bond for activity and that flavonoids were more active than the corresponding isoflavonoids. Studies on the metabolism of B(a)P by hepatic microsomes from rats induced with various classes of cytochrome P-450 inducers demonstrated that those flavonoids active in inhibiting the metabolic activation of B(a)P in cells were potent inhibitors of $\beta$-naphthoflavone-induced cytochrome P-450. No correlation was found with effects on noninduced or phenobarbital-induced cytochrome P-450. Studies with a series of glucosinolates demonstrated that specific glucosinolates can increase the activation of B(a)P in hamster embryo cell cultures, illustrating the importance of caution in considering their effects on various classes of carcinogens in the selection of compounds as potential chemopreventive agents. The above studies demonstrate the ability of the B(a)P metabolism in hamster embryo cell culture assay to allow the isolation and characterization of potential chemopreventive agents from plants. These results indicate that based upon inhibition of B(a)P activation and relatively low toxicity, the flavonoids acacetin and kaempferide are promising candidates for testing as chemopreventive agents

Nitroreduction of 4-nitropyrene is primarily responsible for DNA adduct formation in the mammary gland of female CD rats

We determined whether DNA adducts derived from 4-nitropyrene (4-NP) are formed via nitroreduction or ring oxidation. DNA adduct markers derived from both pathways were prepared and, consequently, were compared with those obtained in vivo in rats treated with 4-NP. Following in vitro reaction of 9,10-epoxy-9,10-dihydro-4-nitropyrene (4-NP-9,10-epoxide), an intermediate metabolite derived from ring oxidation of 4-NP, with calf thymus DNA (average level of binding in two determinations was 8.5 nmol/mg of DNA), DNA was enzymatically hydrolyzed to deoxyribonucleosides and the DNA hydrolysates were analyzed by HPLC. Electrospray mass and 1H NMR spectra of the major products indicated that these adducts are deoxyguanosine (dG) derivatives that resulted from N2-dG substitution at the 9- or 10-position of the pyrene nucleus. However, these adducts were not detected in vivo in the rat mammary gland and liver following the administration of 4-NP. Nitroreduction of 4-NP catalyzed by xanthine oxidase in the presence of DNA resulted in three major putative DNA adducts (level of binding of 12.0 ± 1.1 nmol/mg of DNA, n = 4) designated as peak 1 (46%), peak 2 (25%), and peak 3 (17%). Although peak 1 was further resolved into peaks 1a and lb, both were unstable and gradually decomposed to peak 2, and the latter was unequivocally identified as pyrene- 4,5-dione. On the basis of electrospray mass spectral analysis, peak 3 was tentatively identified as a deoxyinosine-derived 4-aminopyrene adduct. None of the adducts derived from nitroreduction of 4-NP catalyzed by xanthine oxidase coeluted with the synthetic standard N-(deoxyguanosin-8-yl)-4- aminopyrene prepared by reacting dG with N-acetoxy-4-aminopyrene. Nevertheless, HPLC analysis of the hydrolysates of liver and mammary DNA obtained from rats treated with [3H]-4-NP yielded four radioactive peaks, all of which coeluted with the markers derived from the nitroreduction pathway. These results indicate that nitroreduction is primarily responsible for DNA adduct formation in the liver and, especially, in the mammary gland which is the organ susceptible to carcinogenesis by this environmental agent

Efficacy and Safety of <i>Lactobacillus Plantarum</i> C29-Fermented Soybean (DW2009) in Individuals with Mild Cognitive Impairment: A 12-Week, Multi-Center, Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Early intervention using dietary supplements may be effective in alleviating cognitive impairment among individuals with mild cognitive impairment (MCI). This study investigated the efficacy and safety of Lactobacillus plantarum C29-fermented soybean (DW2009) as a nutritional supplement for cognitive enhancement. One hundred individuals with MCI were randomly assigned to take DW2009 (800 mg/day, n = 50) or placebo (800 mg/day, n = 50) for 12 weeks. The primary outcome measure was change in the composite score of cognitive functions related to memory and attention, measured by computerized neurocognitive function tests. Associations between changes in serum brain-derived neurotrophic factor (BDNF) levels and cognitive performance for each treatment group were evaluated. Compared to the placebo group, the DW2009 group showed greater improvements in the combined cognitive functions (z = 2.36, p for interaction = 0.02), especially in the attention domain (z = 2.34, p for interaction = 0.02). Cognitive improvement was associated with increased serum BDNF levels after consumption of DW2009 (t = 2.83, p = 0.007). The results of this clinical trial suggest that DW2009 can be safely administered to enhance cognitive function in individuals with MCI. Increased serum BDNF levels after administering DW2009 may provide preliminary insight into the underlying effects of cognitive improvement, which suggests the importance of the gut-brain axis in ameliorating cognitive deficits in MCI