Isolation and identification of a novel aromatic amine mutagen produced by the Maillard reaction.

To clarify the formation of mutagens in the Maillard reaction of glucose and amino acids, 20 amino acids were separately incubated with glucose in the presence or absence of hydroxyl radicals produced by the Fenton reaction. After 1 week at 37 degrees C and pH 7.4, the reaction mixtures of glucose and tryptophan with and without the Fenton reagent showed mutagenicity toward Salmonella typhimurium YG1024 in the presence of a mammalian metabolic system (S9 mix). To identify mutagens in the reaction mixture, blue rayon-adsorbed material from a mixture of glucose, tryptophan, and the Fenton reagent was separated by column chromatography using various solid and mobile phases, and one mutagen, which accounted for 18% of the total mutagenicity of the reaction mixture, was isolated. The chemical structure of the mutagen was determined to be 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ) on the basis of ESI mass, high-resolution APCI mass, (1)H NMR, (13)C NMR, and IR spectral analyses and chemical synthesis of the mutagen. The novel aromatic amine showed high mutagenicity toward S. typhimurium TA98 and YG1024 with S9 mix, inducing 857 revertants of TA98 and 6007 revertants of YG1024/microg, respectively. The mutagenicity of ABAQ was comparable to that of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, which is a mutagenic and carcinogenic hetrocyclic amine in cooked meat and fish formed through the Maillard reaction at high temperature.

Estrogenic activity of the chlorinated derivatives of estrogens and flavonoids using a GFP expression system.

Estrogenic chemicals are widely reported to be present in the environment. Their chlorinated derivatives are considered to be produced through the chlorination process in water purification and sewage treatment plants. In this study, several chlorinated derivatives of estrogens and flavonoids, including phytoestrogens, were synthesized by the reaction with hypochlorous acid, and their estrogenic activities were investigated using a devised GFP expression system in human breast carcinoma MCF7 cells. The chlorinated derivatives were less estrogenic than the parent compounds. The EC(50) ranking of estrogen-related compounds was 17ß-estradiol (E2)>4-ClE2>estrone (E1)>4-ClE1>10-Cl-1,4-estradiene-3,17-dione (10-Cl-3,17-dione)>2-ClE2>2-ClE1. 2,4-diClE2, 2,4-diClE1, and 2,4,16,16-tetraClE1 showed lower or no estrogenic activity. Genistein and daidzein are well known as phytoestrogens. 6,8-diCl-genistein, 3',8-diCl-daidzein, (+)-6,8-diCl-naringenin, and 6,8-diCl-apigenin showed lower estrogenic activity than their parent compounds. 3',5',8-triCl-daidzein exhibited no estrogenic activity. No activity was detected in chrysin, (+)-catechin, and their chlorinated derivatives. Similar results were obtained in a cell proliferation assay using MCF7 cells.

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite.

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.

A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of L-cysteine.

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing L-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC-mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and 1H and 13C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and L-cysteine in foods, and might act as a mutagen.

Changes in the mutagenic and estrogenic activities of bisphenol A upon treatment with nitrite.

Bisphenol A (4,4'isopropylidenediphenol: BPA), an endocrine-disrupting chemical, is contained in food-packaging and can-coating agents as well as in dental sealants. Nitrite is present in vegetables, fish and tap water as an ingredient or contaminant, and also in human saliva. Here, we explored the possible generation of genotoxicity from the reactions of BPA and nitrite under acidic conditions, a situation simulating the stomach. We determined the changes in the mutagenic and estrogenic activities of BPA before and after nitrite treatment. Untreated BPA did not exhibit any mutagenicity. However, the mixture of BPA and sodium nitrite after incubation at pH 3.0 showed strong mutagenic activity toward Salmonella typhimurium strains TA 100 and TA 98 either with or without a metabolic activation system (S9 mix). The clastogenic properties of nitrite-treated and untreated BPA were analyzed by a micronucleus test with male ICR mice. A single gastric intubation of nitrite-treated BPA induced a significantly higher frequency of micronucleated reticulocytes (MNRETs) in mice. The results of analysis of electron spin resonance (ESR) suggest that the expression of the mutagenic activity of nitrite-treated BPA is related to the generation of radicals in the reaction mixture. By applying 1H and 13C NMR, AB-MS and APCI/LC/MS, we identified two compounds 3-nitrobisphenol A and 3,3'-dinitro-bisphenol A. These compounds were synthesized by the reaction of BPA with nitric acid. 3,3'-Dinitro-bisphenol induced a significantly greater frequency of MNRETs in male ICR mice. By applying a green fluorescent protein (GFP)-reporter expression system and an estrogen R(alpha) competitor screening kit, we found that nitrite-treated BPA and 3,3'-dinitro-bisphenol A showed weak estrogenic activity compared to that of untreated BPA.

In vitro and in vivo estrogenic activity of chlorinated derivatives of bisphenol A.

The estrogenic activity of bisphenol A (BPA) and its chlorinated derivatives, 2-(3-chloro-4-hydroxyphenyl)-2-(4-hydroxyphenyl)propane (3-ClBPA) and 2,2-bis(3-chloro-4-hydroxyphenyl)propane (3,3'-diClBPA) was assessed by determining their relative binding affinity for the human estrogen receptor-alpha and -beta (ERalpha and ERbeta) and also their uterotrophic activity in ovariectomized female rats. BPA and its chlorinated derivatives were active in competing with [3H]17beta-estradiol for their binding to the human ERalpha and ERbeta proteins. While 3-ClBPA and 3,3'-diClBPA competed more effectively for ERalpha binding than BPA (IC50 values of 2.48x10(-5), 1.28x10(-5), and 1.08x10(-4)M, respectively), they had similar activity as BPA for competing the binding to ERbeta (IC50 values of 1.43x10(-5), 1.87x10(-5), and 2.59x10(-5)M, respectively). To determine the uterotropic activity, three doses (10, 50 and 100 mg/kg/day) of BPA and its derivatives were given to mature ovariectomized Sprague-Dawley rats for 3 consecutive days. Treatment of animals with 50 and 100 mg/kg/day of BPA or its chlorinated derivatives caused a significant increase in the uterine wet weight and the endometrial area. The results of our present study demonstrated that the affinities of 3-ClBPA and 3,3'-diClBPA for ERalpha were higher than the affinity of BPA, although the in vivo estrogenic activity of the two chlorinated BPAs in ovariectomized female Sprague-Dawley rats appeared to be comparable to that of BPA.

Products of aqueous chlorination of 17beta-estradiol and their estrogenic activities.

To assess the estrogenic activity potentially stemming from 17beta-estradiol (E2) in drinking water, ESI-LC-MS was used to identifythe products of its aqueous chlorination under the following conditions: 50 microg/L E2, 1.46 mg/L sodium hypochlorite, pH 7.5, 25 degrees C. Seven products, including 2,4-dichloro-17beta-estradiol, monochloroestrone, 2,4-dichloroestrone, and the four byproducts such as 4-[2-(2,6-dichloro-3-hydroxyphenyl)ethyl]-7alpha-methyloctahydroinden-5-one (product C in the text) were identified in chlorinated E2 solution. The estrogenic activities of the aqueous chlorinated E2 solution at 10, 30, 60, 120, and 180 min contact time were assessed by a yeast two-hybrid system based on the ligand-dependent interaction of two proteins, a human estrogen receptor (ER) and a coactivator. All five solutions elicited transcriptional activation induction. The maximal beta-galactosidase activities induced by the chlorinated solution at 10, 30, and 60 min were similar and slightly lower than those before chlorination, while the activities of the chlorinated solution at 120 and 180 min were about 40% of those before chlorination. Finally, 4-chloro-17beta-estradiol (4-chloro-E2) (we failed to synthesize the 2-chloroestrone (2-chloro-E1)), 2,4-dichloro-17beta-estradiol (2,4-dichloro-E2), and 2,4-dichloroestrone (2,4-dichloro-E1) were synthesized, and product C was fractionated by HPLC. It was found that 4-chloro-E2 elicited strong estrogenic activity, at almost the same level as that of estrone (EC50 = 10(2) nM), while 2,4-dichloro-E2 elicited weaker beta-galactosidase activity compared with that of 4-chloro-E2. The EC50 was ca. 10(3) nM. The maximal beta-galactosidase activity for 2,4-dichloro-E1 was lower than that of 2,4-dichloro-E2, while its EC50 was similar to that of 2,4-dichloro-E2. In addition, product C, 4-[2-(2,6-dichloro-3-hydroxyphenyl)ethyl]-7alpha-methyloctahydroinden-5-one, induced high beta-galactosidase activity at the relatively higher concentration of 3.5 x 10(5) nM. On the basis of the dose-response curve of a single byproduct of chlorinated E2, the estrogenic activity at 120 and 180 min appears to be induced mainly by 2,4-dichloro-E2 and 2,4-dichloro-E1.

Identification of estrogenic activity of chlorinated bisphenol A using a GFP expression system.

A green fluorescent protein (GFP)-reporter vector regulated by an estrogen response element (ERE) was constructed and transfected into human breast carcinoma MCF7 cells. Stable transfectants were selected and their GFP fluorescence intensity was measured using a quantitative fluorescent imaging system. 17ß-estradiol (E(2)) and bisphenol A (BPA) induced a dose-dependent increase in GFP intensity in the cells, reaching maximum response at 5×10(-10) and 10(-5) M, respectively. Using this GFP expression system, we examined the estrogenicity of mono-, di-, tri-, and tetra-chlorinated BPAs, which were detected in wastewater from waste-paper recycling plants using sodium hypochlorite as a bleaching agent. 3-ClBPA and 3,3'-diClBPA showed similar estrogenicities, effective at lower concentrations than parent BPA. On the other hand, the maximum activities of BPA and 3,3',5-triClBPA, whose EC(50) were similar, were higher than other chlorinated BPAs. This is the first demonstration of the estrogenicity of chlorinated BPAs. Since polychlorinated BPAs were not easily biodegraded, chlorinated BPAs might be more severe endocrine disruptors than BPA.