Young H. Ju

Estrogenicity of the Isoflavone Metabolite Equol on Reproductive and Non-Reproductive Organs in Mice

Abstract Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight]−1 day−1) or in the diet (0, 500, or 1000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 μM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)−1 day−1. Dietary equol at 500 or 1000 ppm produced total serum equol concentrations of 5.9 and 8.1 μM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8–20 mg (kg body weight)−1 day−1 and 1000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor α when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.

Plant-activation of the bicyclic aromatic amines benzidine and 4-aminobiphenyl

Benzidine and 4‐aminobiphenyl (4‐ABP) are promutagenic bicyclic aromatic amines that are activated into frameshift and base pair substitution mutagens by plant systems. Using the plant cell/microbe coincubation assay, plant‐activated benzidine from 0 to 50 μM induced a concentration‐response in Salmonella typhimurium. At concentrations above 5 μM, plant‐activated benzidine induced frameshift and base pair substitution mutations in the N‐ or O‐acetyltransferase over‐expressing strains, DJ460, YG1024, and YG1029. With plant‐activated 4‐ABP, concentrations above 250 μM induced a significant mutagenic response in strains YG1024 and YG1029. A tobacco cell‐free mixture, TX1MX, activated benzidine and 4‐ABP into mutagenic metabolites in S. typhimurium strains YG1024, YG1029, and DJ460. The mutagenic sensitivities of plant‐activated benzidine and 4‐ABP were the same with two different types of plant activation systems, TX1 suspension cells and TX1MX cell‐free medium. The plant activation of these aromatic amines is mediated by tobacco cell peroxidase. Plant‐activated benzidine and 4‐ABP are converted into intermediates that serve as substrates for bacterial or humanacetylCoA: N‐hydroxyarylamine N‐acetyl‐transferase to generate the ultimate mutagenic products. Environ. Mol. Mutagen. 29:81–90, 1997

Involvement of nitroreductase and O-acetyltransferase on the mutagenicity of plant-activated benzidine and 4-aminobiphenyl

Benzidine and 4‐aminobiphenyl (4‐ABP) are activated by intact plant cells and cell free TX1MX into mutagenic metabolites that induce frameshift and base pair substitution mutations in Salmonella typhimurium. The plant activation of these agents is plant peroxidase‐mediated and bacterial O‐acetyltransferase (OAT) dependent. TX1MX‐activated benzidine and 4‐ABP were analyzed with S. typhimurium frameshift tester strains, YG1021, YG1024, TA98, TA98NR, TA98/1,8‐DNP6, MP219, and base pair substitution tester strains, YG1026, YG1029, TA100, TA100NR, TA100TN:OAT, and MP208. Concentration ranges for benzidine and 4‐ABP were 1–50 μM and 0.1–1 mM, respectively. This study was conducted to determine if the plant‐activation of benzidine and 4‐ABP follows the prostaglandin H synthase‐mediated activation pathway in mammals [Smith et al. (1992): Chem Res Toxicol 5;431–439]. In this model, benzidine is N‐acetylated by S. typhimurium OAT. This acetylated product is a substrate for PHS and is converted into a 4‐nitro product which is catalyzed by nitroreductase into a N‐hydroxy intermediate. The pathway assigns a specific role for nitroreductase in the activation of benzidine. By employing S. typhimurium strains that express different levels of OAT and/or nitroreductase, we determined that the plant‐activation of benzidine and 4‐ABP has an absolute requirement of bacterial OAT activity for the induction of frameshift mutations at hisD3052 and is required for the optimal mutagenic response at hisG46. Nitroreductase also plays a role in the plant activation of these agents. The data suggest that the plant‐activation of benzidine and 4‐ABP generates at least two classes of proximal mutagenic intermediates. One class requires S. typhimurium OAT alone to be transformed into the ultimate mutagen and a second class requires both OAT and nitroreductase. Environ. Mol. Mutagen. 30:330–338, 1997