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.

Synthesis of various kinds of isoflavones, isoflavanes, and biphenyl-ketones and their 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activities.

Forty-eight kinds of isoflavones (8), thirty-one isoflavanes (9), and forty-seven biphenyl-ketones (10, 10') were synthesized from eleven kinds of substituted phenols (11) and six phenylacetic acids (12). Among them, seventy-five compounds are new. The radical scavenging activities of these compounds were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) at pH 6.0. We found that thirty-nine out of forty-three compounds having a catechol moiety on either the A- or the B-ring exhibited a high activity (ED(50)=12-54 microM) similar to that of catechin. In these cases, the remaining part of their structure seemed to have little effect on their activity. Many 6- or 8-hydroxyisoflavanes (9E-I) and their biphenyl-ketone derivatives (10E-H) also showed a high activity (ED(50)=or<50 microM), while all of their corresponding isoflavones (8E-I) were not active at all. The 7-hydroxyisoflavanes having either an additional hydroxyl group at the C5-position (9D) or a methoxy group at the C6-position (9J) presented a high activity (ED(50)=26-32 microM). This study suggests that natural isoflavones have the possibilities of exhibiting antioxidant activities through the hydroxylation at the C6-, C8-, or C3'-position or the formation of the isoflavanes (9) and/or the biphenyl-ketone derivatives (10') by metabolism or biotransformation.

Effects of ethinylestradiol on medaka (Oryzias latipes) as measured by sperm motility and fertilization success.

We investigated the effects of 30-480 ng/L 17alpha-ethinylestradiol (EE(2)) on the sperm motility and fertility of Japanese medaka (Oryzias latipes). Sperm motility was examined by computer-assisted image analysis. In male medaka, the velocity of sperm was found to have increased after 3 weeks of exposure at 60-480 ng/L. This result suggests that higher sperm velocities depleted sperm energy reserves more rapidly and shortened the time for which sperm were viable to fertilize eggs. In a separate experiment that studied whether EE(2) exposure of males affects the fertilization rate or hatchability, sexually mature male medaka were exposed for 3 weeks and subsequently evaluated for their reproductive ability after pairing with unexposed females for 7 days. Exposure of males to EE(2) exerted a potent inhibitory effect on a reproduction parameter (fertilization rate x hatchability), and the highest inhibition was observed at 60 ng/L. The results offer toxicological data for the assessment of EE(2 )exposure in medaka and suggest that short-term exposure to EE(2) might reduce sperm function and fertility in adult male medaka.

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains.

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. alpha-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.

Induction of apoptosis by UV-irradiated chlorinated bisphenol A in Jurkat cells.

Chlorinated derivatives of bisphenol A (ClBPAs) have been detected in wastewater from waste paper recycling plants. We previously reported that bisphenol A (BPA) and ClBPAs [3-chlorobisphenol A, 3,3'-dichlorobisphenol A, and 3,3',5-trichlorobisphenol A] irradiated with ultraviolet (UV) B or UVC (not with UVA) induced inhibition of cell growth, and that 3-hydroxybisphenol A (3-OHBPA) was detected in the photoproducts [Mutou, Y., Ibuki, Y., Terao, Y., Kojima, S., Goto, R., 2006b. Chemical change of chlorinated bisphenol A by ultraviolet irradiation and cytotoxicity of their products on Jurkat cells. Environmental Toxicology and Pharmacology, 21, 283-289]. The formation of hydroxylated BPAs by UV irradiation might contribute to the inhibition of cell growth, but the mechanism of the growth inhibition is not clarified. In this study, we investigated whether BPA and ClBPAs exposed to UVA, UVB, or UVC, and 3-OHBPA could induce the death of Jurkat cells and whether the pattern of cell death was apoptosis. ClBPAs exposed to UVB and UVC induced significant cell death, but those exposed to UVA and BPA did not. The cell death was apoptosis because chromatin condensation and DNA fragmentation were detected. Activation of caspase-3, -8, and -9 and cytochrome c release indicated that ClBPAs exposed to UVB or UVC induced apoptosis via typical apoptotic pathways. In addition, 3-OHBPA induced apoptosis similar to UVB- or UVC-irradiated ClBPA. These results suggested that the photoproducts of ClBPAs generated by UV irradiation, containing 3-OHBPA, contributed to the induction of apoptosis.

Reaction of polycyclic aromatic hydrocarbons adsorbed on silica in aqueous chlorine.

The reaction of polycyclic aromatic hydrocarbons (PAHs) previously adsorbed on silica gel or diatomaceous earth with sodium hypochlorite was carried out to elucidate their reactivity to aqueous chlorine. It was demonstrated that the PAHs adsorbed on silica reacted more rapidly than the PAHs themselves in water, leading to the formation of many chlorinated and oxidized derivatives. A similar reaction in the presence of potassium bromide was found to preferentially produce corresponding brominated derivatives. These reactions seem to proceed through PAHs adsorbed on the silica surface and halogenating agents, the electrophilicity of which may be raised by the catalytic effect of the silanol group of the silica surface. These findings from the environmental viewpoint suggest that the reaction of hydrophobic compounds adsorbed on sediment cannot be neglected.

Formation of chlorinated estrones via hypochlorous disinfection of wastewater effluent containing estrone.

Chlorinated derivatives of estrone (E1) in the effluent of a municipal sewage treatment plant located in Shizuoka prefecture, Japan were detected by gas chromatography/mass spectrometry using electron impact in selected ion monitoring (GC/MS-EI-SIM) analysis. The concentrations of E1, 2-chloroestrone, 4-chloroestrone and 2,4-dichloroestrone in the effluent sample collected in December 2005 were 60.0 ng l(-1), 4.0 ng l(-1), 14.5 ng l(-1), and 9.8 ng l(-1), respectively. In the effluent sample taken in June 2005, 2,4-dichloroestrone was detected at 5.6 ng l(-1) along with 17.6 ng l(-1) of E1. However, only E1 was detected at 5.9 ng l(-1) in the sample in May 2005. To elucidate the behavior of E1 during the disinfection process with sodium hypochlorite in the sewage treatment plant, we carried out a reaction of E1 with sodium hypochlorite in buffer solutions at pH 7 and 9. As E1 was consumed rapidly, chlorinated estrones were produced and relatively fast formation of 2-chloroestrone, 4-chloroestrone, and 2,4-dichloroestrone was observed. Furthermore, 1,4-estradiene-3,17-dione derivatives were formed from the reaction between 2,4-dichloroestrone and sodium hypochlorite.

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.

Change of estrogenic activity and release of chloride ion in chlorinated bisphenol a after exposure to ultraviolet B.

Bisphenol A (BPA) and chlorinated bisphenol A (ClBPAs) were detected in wastewater from waste paper recycling plants. In previous study, we showed the acute cytotoxicity of oxidized products of BPA and ClBPAs generated by ultraviolet (UV) irradiation. However, estrogenic activities of these photoproducts have not been studied. Therefore, we investigated change of estrogenic activities of BPA and ClBPAs [3-chlorobisphenol A (3-ClBPA), 3,3'-dichlorobisphenol A (3,3'-diClBPA) and 3,3',5-trichlorobisphenol A (3,3',5-triClBPA)] after UVB irradiation using yeast two-hybrid assay. The agonist activities of ClBPAs were higher than that of BPA in the absence of S9. ClBPAs irradiated with UVB lost agonist activities. The addition of S9 also completely erased the activity. The antagonist activities of BPA and ClBPAs with or without UVB irradiation were not detected both in the absence or presence of S9. UVB irradiation (0-100 J/cm2) decreased the agonist activity of 3,3'-diClBPA in proportion to increase of released chloride ion. The agonist activity was completely lost at 50 J/cm2 of UVB, of which dose could dissociated almost all chlorine. These findings suggested that UVB irradiation could decrease the estrogenic activity of chlorinated compounds, which was due to the selective release of chloride ion.

Isolation and identification of non-chlorinated phenylbenzotriazole (non-ClPBTA)-type mutagens in the Ho River in Shizuoka Prefecture, Japan.

We previously identified 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA) congeners as major mutagens in water concentrates from several rivers that flow in three different areas, i.e. Kyoto, Aichi, and Fukui Prefectures, in Japan. In synthesis studies, these PBTAs were shown to be formed from corresponding dinitrophenylazo dyes via non-chlorinated derivatives (non-ClPBTAs). However, only non-ClPBTA-1, i.e. 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole, had been detected as a minor contaminant in the Nishitakase River in Kyoto. In this study, analysis of mutagens in water concentrate from the Ho River, which flows through an area with a textile dyeing industry in Shizuoka Prefecture, Japan, allowed the isolation of four compounds (I, II, III, and IV). These four mutagens were identified as 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-2), 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-3), 2-(2-acetylamino-4-amino-5-methoxyphenyl)-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-4), and 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-7) by spectral data and co-chromatography using synthesized standards. Non-ClPBTA-3 and -7 were highly mutagenic in Salmonella typhimurium YG1024, inducing 159,000 and 178,000 revertants/microg, respectively, in the presence of S9 mix. Like PBTAs, non-ClPBTAs might have been produced from azo dyes during industrial processes in dyeing factories and released into rivers.

In vitro and in vivo analysis of the thyroid system-disrupting activities of brominated phenolic and phenol compounds in Xenopus laevis.

We investigated the effects of the brominated phenolic and phenol compounds, some of which are brominated flame retardants, on the binding of (125)I-3,3',5-L-triiodothyronine ((125)I-T(3)) to purified Xenopus laevis transthyretin (xTTR) and to the ligand-binding domain of X. laevis thyroid hormone receptor beta (xTR LBD), on the induction of a T(3)-responsive reporter gene in a recombinant X. laevis cell line (XL58-TRE-Luc) and on T(3)-induced or spontaneous metamorphosis in X. laevis tadpoles. Of the brominated phenolic and phenol compounds tested, 3,3',5-tribromobisphenol A and 3,3'-dibromobisphenol A were the most potent competitors of (125)I-T(3) binding to xTTR and the xTR LBD, respectively. Structures with a bromine in either ortho positions with respect to the hydroxy group competed more efficiently with T(3) binding to xTTR and the xTR LBD. 3,3',5-Tribromobisphenol A and 3,3',5,5'-tetrabromobisphenol A, at 0.1-1.0 microM, exerted both T(3) agonist and antagonist activities in the T(3)-responsive reporter gene assay. Sera obtained from fetal bovine and bullfrog tadpoles weakened the T(3) agonist and antagonist activities of 3,3',5-tribromobisphenol A, but not the T(3) antagonist activity of o-t-butylphenol, for which xTTR has no significant affinity. The T(3) agonist and antagonist activities of 0.5 microM 3,3',5-tribromobisphenol A were confirmed in the in vivo, short-term gene expression assay in premetamorphic X. laevis tadpoles using endogenous, T(3)-responsive genes as molecular markers. Our results suggest that 3,3',5-tribromobisphenol A affects T(3) binding to xTTR and xTR and that it interferes with the intracellular T(3) signaling pathway.

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.

Transformation of pyrene in aqueous chlorination in the presence and absence of bromide ion: kinetics, products, and their aryl hydrocarbon receptor-mediated activities.

To assess the endocrine-disrupting activity stemming from the presence of pyrene in drinking water, the kinetics of chlorination of pyrene was investigated at room temperature, the products of its aqueous chlorination with and without bromide ion were identified, and their aryl hydrocarbon receptor (AhR)-mediated activities were determined. It was found that the presence of bromide ion greatly promoted the reaction rate of chlorination of pyrene accompanied with the formation of brominated products. While the main product was 1-Cl-pyrene without the addition of bromide ion, di-Br-pyrene and 1-Br-pyrene became the main products in the presence of bromide ion. GC-MS and NMR analysis identified three structures of dibromopyrene in chlorination with the addition of bromide ion as 1,3-di-Br-pyrene, 1,6-di-Br-pyrene, and 1,8-di-Br-pyrene, and their molar ratio was determined to be approximately 0.3:1:1. Finally, 1-Br-pyrene, 1,3-di-Br-pyrene, a mixture of 1,6-di-Br-pyrene and 1,8-di-Br-pyrene (di-Br-pyrene), 1-Cl-pyrene, and a mixture of 1,6-di-Cl-pyrene and 1,8-di-Cl-pyrene (di-Cl-pyrene) were fractionated by HPLC, and their AhR-mediated activities were assessed by a yeast assay. It was found that the effective molar concentrations (or mass concentration) showing half-maximal transcriptional response, EC50, for pyrene, 1-Br-pyrene, 1-Cl-pyrene, di-Cl-pyrene, and di-Br-pyrene were 5632 (1.14), 3089 (0.86), 1942 (0.46), 597.2 (0.21), and 147.3 (0.04) nM (mg/L), respectively.

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.

The contribution of azo dyes to the mutagenic activity of the Cristais River.

To verify whether dyes emitted within the discharge of a dye processing plant were contributing to the mutagenicity repeatedly found in the Cristais River, Sao Paulo, Brazil, we chemically characterized the following mutagenic samples: the treated industrial effluent, raw and treated water, and the sludge produced by a Drinking Water Treatment Plant (DWTP) located approximately 6 km from the industrial discharge. Considering that 20% of the dyes used for coloring activities might be lost to wastewaters and knowing that several dyes have mutagenic activity, we decided to analyze the samples for the presence of dyes. Thin layer chromatographic analysis indicated the presence of three prevalent dyes in all samples, except for the drinking water. This combination of dyes corresponded to a commercial product used by the industry, and it tested positive in the Salmonella assay. The structures of the dye components were determined using proton magnetic resonance and mass spectrometric (MS) methods, and the dyes were tested for mutagenicity. The blue component was identified as the C.I. Disperse Blue 373, the violet as C.I. Disperse Violet 93, and the orange as C.I. Disperse Orange 37. The dyes showed mutagenic responses of 6300, 4600, and 280 revertants/microg for YG1041 with S9 respectively. A bioassay-directed fractionation/chemical analysis showed that the C.I. Disperse Blue 373 contributed 55% of the mutagenic activity of the DWTP sludge. We showed that these dyes contributed to the mutagenic activity found in the Cristais River environmental samples analyzed and are indirectly affecting the quality of the related drinking water. Therefore, we believe that this type of discharge should be more thoroughly characterized chemically and toxicologically. Additionally, human and ecological risks associated with the release of dye processing plant effluents should be more fully investigated, especially where the resultant water is taken for human consumption.

Estrogenic activity of alkylphenols, bisphenol S, and their chlorinated derivatives using a GFP expression system.

Alkylphenol ethoxylates, widely used non-ionic surfactants, are biodegraded into alkylphenols such as nonylphenol (NP) and t-octylphenol (OP), short-chain ethoxylates such as NP-monoethoxylate (NP1EO) and NP-diethoxylate (NP2EO), and alkylphenoxy carboxylic acids such as 4-t-octylphenoxyacetic acid (OP1EC). Bisphenol S (BPS) is more heat-stable and photo-resistant than bisphenol A (BPA), and therefore replaces BPA. These chemicals could be chlorinated during wastewater treatment. We synthesized these compounds and their chlorinated derivatives to estimate their estrogenic activities using a GFP expression system. The EC(50) ranking of NP-related compounds was NP > ClNP > diClNP > NP1EO > ClNP1EO > NP2EO. The estrogenic activity of OP1EC was 10 times less potent than parent OP. Furthermore, BPS showed comparable estrogenic activity with BPA. The EC(50) ranking of BPS-related compounds was BPA ≥ BPS > triClBPS > diClBPS > ClBPS. Other tested BPS derivatives had no estrogenic activity. Chlorination of the tested chemicals did not enhance their estrogenic activity, in contrast to certain chlorinated BPAs. Thus, our results demonstrated that chlorinated derivatives of NP, OP, and BPS, even if artificially produced during wastewater processing, were less estrogenic than their parent chemicals, known as endocrine disruptors.

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.

Simultaneous determination of 2-phenylbenzotriazole-type mutagens, PBTA-1 through -8, in river water by liquid chromatography-tandem mass spectrometry.

We describe a method for the simultaneous determination of eight kinds of phenylbenzotriazole-type mutagens (PBTA-1, -2, -3, -4, -5, -6, -7 and -8) in river water based on liquid chromatography-tandem mass spectrometry (LC/MS/MS). The application of dopant-assisted atmospheric pressure photoionization (APPI) for the detection of the PBTAs was studied. The APPI technique provided higher PBTA signal intensities than those obtained with an electrospray ionization (ESI) source, and the APPI method was used for the determination of the PBTAs. A solid-phase extraction procedure was used for the extractions of PBTA-1 through -8 from river water. The procedure was rapid and the relative standard deviations were below 15%. The detection limits of PBTA-1 through -8 in river water using the proposed method were found to range from 0.04 to 0.5 ng L(-1) and PBTAs were successfully detected in river water at sub-ng L(-1) levels.

Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay.

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) are two compounds, which show strong mutagenicity toward bacteria, that have been identified as major mutagens in river water in Japan. In the present study, we examined the genotoxicity of PBTA-6 and ADDB in goldfish (Carassius auratus) by the micronucleus test and single-cell gel electrophoresis (comet assay). The frequencies of micronuclei in gill cells gradually increased until 96h after i.p. injection of PBTA-6 and ADDB at doses of 50mg/kg body weight, and then decreased 144h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1-100mg/kg body weight, giving significantly high frequencies at doses of 50 and 100mg/kg body weight. On the other hand, no significant increase was observed in the peripheral erythrocytes of goldfish exposed to PBTA-6 or ADDB. In the comet assay, values of DNA tail moment and tail length in peripheral erythrocytes increased significantly until 6h after the i.p. injection of PBTA-6 (50mg/kg body weight), only to decrease by 9h after injection. Both the DNA tail moment and tail length were dose-dependently increased by injections of PBTA-6 at doses ranging from 1 to 50mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3h after an i.p. injection of ADDB and persisted for up to 6h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.