Identification of junk buyers' contribution to recycling of household waste in Hanoi, Vietnam, through a physical composition analysis.

Even in developing countries, the amount of containers and packaging waste are increasing in line with population concentration and lifestyle changes in urban areas. This can cause serious problems for the disposal of municipal solid waste. Through a physical composition analysis of household waste in Hanoi, the capital of Vietnam, this study aimed to identify the contribution made by junk buyers to recycling. Interviews on the handling of recyclable waste by households were conducted. About 232 kg of recyclable waste was sampled from a total of 115 households, and about 230 kg of municipal solid waste was sampled from a total of 101 households and sorted into 69 categories for measurement by volume and weight. The interview survey revealed that a high proportion of households tended to routinely store recyclable waste for sale or donation to junk buyers. Junk buyers accounted for 8.8% of recycling by weight or 26.0% by volume according to the results of the physical composition analysis. In addition, the results suggested that containers and packaging waste accounted for the largest proportion of household waste by volume. Junk buyers recycled 25.5% by weight of containers and packaging waste. In the formulation of new plans for municipal solid waste management to improve the current situation and handle future challenges, the role of the informal sector should be monitored carefully and reliable data on recyclable waste should be collected continuously.

Genotoxicity of colloidal fullerene C60.

Previous genotoxicity tests of aqueous fullerene C60) suspension (aqu-C60) yielded both positive and negative results. In the present study, aqu-C60 elicited positive responses in two bacterial genotoxicity tests, the Bacillus subtilis Rec-assay and the umu test at concentrations as low as 0.048 mg/L and 0.43 mg/L, respectively. In mammalian cell experiments, aqu-C60 showed a significant growth inhibitory effect on human hepatocarcinoma HepG2 cells at 0.46 mg/L. The level of the oxidative DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine, measured by liquid chromatography tandem mass spectrometry, was slightly but not significantly increased in HepG2 cells treated with 0.46 mg/L for 24 h, whereas the level of the lipid peroxidation-related DNA lesion α-methyl-γ-hydroxy-1,N²)-propano-2'-deoxyguanosine was not changed. Under the same conditions, we did not detect any bulky DNA adducts, as measured by ³²P-postlabeling/polyacrylamide gel electrophoresis analysis. Our data suggest that aqu-C60 has DNA-damaging potential and that the DNA damage is not due to covalent DNA adduct formation by C60 itself.

Aryl hydrocarbon receptor and estrogen receptor ligand activity of organic extracts from road dust and diesel exhaust particulates.

A wide variety of contaminants derived from diesel and gasoline engines, tire, asphalt, and natural organic compounds is found in road dust. Polycyclic aromatic compounds (PACs) are the important toxic targets among various contents in road dust and diesel exhaust particulates (DEPs), and endocrine-disrupting activity of PACs was suggested. In the present study, aryl hydrocarbon receptor (AhR) ligand activity was confirmed in the extract of both road dust and DEPs. In the separation of the extracts for both road dust and DEPs with reversed-phase HPLC, it was found that polar fractions contributed to significant AhR ligand activity in both a mouse hepatoma (H1L1) cell system and a yeast system. Furthermore, the contribution of these polar fractions was higher in DEPs than in road dust, probably because of the greater concentration of oxy-PAHs in DEPs than in road dust. The contribution of contaminants associated with the polar region to AhR ligand activity was also evident following the separation of road dust with normal-phase HPLC. Additionally, remarkable estrogen receptor (ER) ligand activity was detected in the highly polar region separated with normal-phase HPLC. It is suggested that many unknown AhR or ER ligand active compounds are contained in the polar region.

Application of the adductome approach to assess intertissue DNA damage variations in human lung and esophagus.

Methods for determining the differential susceptibility of human organs to DNA damage have not yet been explored to any large extent due to technical constraints. The development of comprehensive analytical approaches by which to detect intertissue variations in DNA damage susceptibility may advance our understanding of the roles of DNA adducts in cancer etiology and as exposure biomarkers at least. A strategy designed for the detection and comparison of multiple DNA adducts from different tissue samples was applied to assess esophageal and peripherally- and centrally-located lung tissue DNA obtained from the same person. This adductome approach utilized LC/ESI-MS/MS analysis methods designed to detect the neutral loss of 2'-deoxyribose from positively ionized 2'-deoxynucleoside adducts transmitting the [M+H](+)>[M+H-116](+) transition over 374 transitions. In the final analyses, adductome maps were produced which facilitated the visualization of putative DNA adducts and their relative levels of occurrence and allowed for comprehensive comparisons between samples, including a calf thymus DNA negative control. The largest putative adducts were distributed similarly across the samples, however, differences in the relative amounts of putative adducts in lung and esophagus tissue were also revealed. The largest-occurring lung tissue DNA putative adducts were 90% similar (n=50), while putative adducts in esophagus tissue DNA were shown to be 80 and 84% similar to central and peripheral lung tissue DNA respectively. Seven DNA adducts, N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG), 1,N(6)-etheno-2'-deoxyadenosine (varepsilondA), alpha-S- and alpha-R-methyl-gamma-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-PdG(1), 1,N(2)-PdG(2)), 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxy-pyrimido[1,2-a]purine-(3H)-one (8-OH-PdG) and the two stereoisomers of 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (6-OH-PdG) were unambiguously detected in all tissue DNA samples by comparison to authentic adduct standards and stable isotope dilution and their identities were matched to putative adducts detected in the adductome maps.

Aryl hydrocarbon receptor ligand activity of polycyclic aromatic ketones and polycyclic aromatic quinones.

Polycyclic aromatic ketones (PAKs) and polycyclic aromatic quinones (PAQs) are oxygenated polycyclic aromatic hydrocarbons (PAHs), and reports about the aryl hydrocarbon receptor (AhR) ligand activities of these compounds are few. In this study, activation of AhR by 41 polycyclic aromatic compounds (PACs), focusing especially on PAKs and PAQs, was determined by measuring beta-galactosidase activity from a reporter plasmid in yeast engineered to express human AhR and the AhR nuclear translocator proteins and by measuring luciferase activity from mouse hepatoma (H1L1) cells (chemical-activated luciferase expression [CALUX] assay). The PACs used in these experiments included 11 PAKs, seven PAQs, and 21 PAHs. In this study, the PAKs 11H-benzo[a]fluoren-11-one (B[a]FO), 11H-benzo[b]fluoren-11-one (B[b]FO) and 7H-benzo[c]fluoren-7-one and the PAQs 5,12-naphthacenequinone, 1,4-chrysenequinone, and 7,12-benz[a]anthracenequinone showed high AhR activities in H1L1 cells, although these values were not as high as that for benzo[a]pyrene (B[a]P). These PAKs and PAQs showed significantly stronger activities in yeast cells relative to B[a]P. It was predicted that PAKs such as B[a]FO and B[b]FO occupied 0.06% to 1.3% of the total induction equivalents, and each contribution matched the contribution of PAHs such as B[a]P, chrysene, and benz[a]anthracene in gasoline exhaust particulates and airborne particulates using data of CALUX assay.

Development of the adductome approach to detect DNA damage in humans.

The development of new strategies designed to detect DNA damage caused by oxidative stress and other means may advance our understanding of the roles of such types of damage in the etiology of cancers, in aging processes, and as biomarkers of exposure. A DNA adduct detection method that uses liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) to detect multiple DNA adducts in human lung tissue is reported herein. This adductome analysis strategy is designed to detect the neutral loss of 2 -deoxyribose from positively ionized 2 -deoxynucleoside adducts in multiple reaction ion monitoring mode (MRM) transmitting the [M + H](+) > [M + H - 116](+) transition over a total of 374 transitions in the mass range from m/z 228.8 to m/z 602.8. Data analysis is optimized and coupled with a comprehensive manual screening process designed to minimize the number of artifactual adducts appearing in the final analysis. In the final analysis, putative adducts were organized into an adductome map and unambiguous confirmation of selected oxidative adducts were made by stable isotope dilution and comparison to authentic standards. The future applications of this method are discussed.

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases.

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.

4-oxo-2-hexenal, a mutagen formed by omega-3 fat peroxidation, causes DNA adduct formation in mouse organs.

To identify mutagens formed in a model reaction of lipid peroxidation, linolenic acid methyl ester and hemin were reacted with dG. As a result, a 4-oxo-2-hexenal-dG adduct (dG*) was identified in the model reaction mixture. The 4-oxo-2-hexenal (4-OHE) showed mutagenic activity in the Salmonella typhimurium strains TA100 and TA104. After 4-OHE was orally administered to mice, dG, 4-OHE-dC- and 4-OHE-5-methyl-dC adducts were detected in esophageal, stomach and intestinal DNA. In the vapor phase released from the methyl linolenate-hemin model system, and in the smoke released during the broiling of fish, 4-OHE was detected by GCMS. The 4-OHE seems to be produced by the auto-oxidation of omega-3 polyunsaturated fatty acids. These results provide a warning to workers dealing with omega-3 fats, who may be exposed to this volatile mutagen.

Comparison of gene expression patterns between 2,3,7,8-tetrachlorodibenzo-p-dioxin and a natural arylhydrocarbon receptor ligand, indirubin.

Indirubin is a natural arylhydrocarbon receptor (AhR) ligand isolated from human urine. We previously reported that it was more potent than the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a yeast assay system. Here we compared gene expression changes in HepG2 cells exposed to 10 nM of indirubin or TCDD using nylon-membrane-based cDNA arrays with 1176 genes to elucidate the toxic differences at the transcriptional level. The gene expression profiles for TCDD and indirubin were very similar. The number of up-regulated genes (fold change > or =2.0) was 11 and 4 and the number of down-regulated genes (fold change < or =0.5) was 17 and 21 in TCDD-treated and indirubin-treated cells, respectively. Cytochrome P450 (CYP) 1A1, 1A2, 19A1, insulin-like growth factor binding protein 1 (IGFBP1), and IGFBP10 were confirmed to be up-regulated using real-time reverse transcription polymerase chain reaction. CYP1A1 and CYP1A2 mRNAs were induced by as little as 1 pM of indirubin, whereas they were not induced by 10 pM of TCDD. In the time-course experiment, CYP1A1 mRNA was induced by indirubin transiently. Indirubin was also metabolized by CYP1A1 and lost its ligand activity. Indirubin would appear to be a good substrate of CYP1A1 given its low dissociation constant. Our results suggest that indirubin rapidly activates its own metabolism via AhR-mediated induction of CYP1A1 and this characteristic is consistent with the notion that indirubin is a physiological ligand of AhR.

An electrospray ionization-tandem mass spectrometry method for identifying chlorinated drinking water disinfection byproducts.

Identification of chlorinated drinking water disinfection byproducts (DBPs) was investigated by using electrospray ionization-mass spectrometry/mass spectrometry (ESI-MS/MS). Chlorine-containing compounds were found to form chloride ion fragments by MS/MS, which can be used as a 'fingerprint' for chlorinated DBPs. Instrumental parameters that affect the formation of chloride ions by ESI-MS/MS were examined, and appropriate conditions for use in finding specific structural information were evaluated. The results show that maximizing the formation of chloride ions by MS/MS required a relatively high collision energy and collision gas pressure; also, limiting the scan range to m/z 30-40 allowed improved sensitivity for detection; but obtaining structural information required the use of lower collision energies. The conditions obtained were demonstrated to be effective in identifying chlorinated DBPs in a standard sample with relatively low concentrations of each component and in a chlorinated humic substance sample. Sample pretreatment techniques including ultrafiltration and size exclusion chromatography appeared to be helpful for identifying highly polar or high molecular weight chlorine-containing DBPs by ESI-MS/MS.

Analysis of estrogen-like compounds in the environment by high performance liquid chromatography bioassay.

To identify chemicals with endocrine-disrupting activity easily, we developed a new bioassay system, consisting of bioassay using genetically modified yeast expressing human estrogen receptor and high performance liquid chromatography (HPLC), in which advantages of instrumental analysis and bioassay are combined. The peaks in the mixture of these estrogen-like compounds analyzed using an HPLC bioassay were similar to those obtained by analysis using an HPLC-UV detector. Underground water and sea sediment were analyzed by an HPLC bioassay, and detected a few estrogen-like compounds, respectively. Estrogen-like compounds and yeast-growth inhibitors can be separated by HPLC-bioassay.

Integrative assessment of potential effects of dioxins and related compounds in wild Baikal seals (Pusa sibirica): application of microarray and biochemical analyses.

We have previously indicated that accumulation of chlorinated dioxins and related compounds (DRCs) induced cytochrome P450 (CYP) 1A1, 1A2 and 1B1 isozymes in the liver of wild Baikal seals (Pusa sibirica). Here we attempt to assess the potential effects of DRCs triggered by the induction of these CYP1 isozymes in this species, using an integrative approach, combining gene expression monitoring and biochemical assays. To screen genes that may potentially respond to the exposure of DRCs, we constructed a custom cDNA oligo array that can target mRNAs in Baikal seals, and monitored hepatic mRNA expression levels in the wild population. Correlation analyses between the hepatic total 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) and mRNA levels supported our previous findings that high accumulation of DRCs induces the transcription of CYP1A1, CYP1A2 and CYP1B1 genes. In addition, our integrative assessment indicated that the chronic exposure to DRCs may alter the hepatic transcript levels of genes related to oxidative stress, Fe ion homeostasis, and inflammatory responses. The expression levels of CYP1A2 showed significant positive correlations with levels of malondialdehyde, a biomarker of lipid peroxidation, and of etheno-dA, a DNA adduct, suggesting that the lipid peroxidation may be enhanced through the production of reactive oxygen species (ROS) triggered by CYP1A2 induction. Moreover, there was a positive correlation between heme oxygenase activities and malondialdehyde levels, suggesting the prompted heme degradation by ROS. Fetuin-A levels, which are suppressed by inflammation, showed a significant negative correlation with TEQ levels, and hepcidin levels, which are conversely increased by inflammation, had significant positive correlations with malondialdehyde and etheno-dA levels, implying the progression of inflammation by DRC-induced oxidative stress. Taken together, we propose here that wild Baikal seals may suffer from effects of chronic exposure to DRCs on the induction of CYP1 isozymes, followed by increased oxidative stress, heme degradation and inflammation.

Biodegradation of high molecular weight lignin under sulfate reducing conditions: lignin degradability and degradation by-products.

This study is designed to investigate the biodegradation of high molecular weight (HMW) lignin under sulfate reducing conditions. With a continuously mesophilic operated reactor in the presence of co-substrates of cellulose, the changes in HMW lignin concentration and chemical structure were analyzed. The acid precipitable polymeric lignin (APPL) and lignin monomers, which are known as degradation by-products, were isolated and detected. The results showed that HMW lignin decreased and showed a maximum degradation capacity of 3.49 mg/l/day. APPL was confirmed as a polymeric degradation by-product and was accumulated in accordance with HMW lignin reduction. We also observed non-linear accumulation of aromatic lignin monomers such as hydrocinnamic acid. Through our experimental results, it was determined that HMW lignin, when provided with a co-substrate of cellulose, is biodegraded through production of APPL and aromatic monomers under anaerobic sulfate reducing conditions with a co-substrate of cellulose.

Metabolic enzyme induction by HepG2 cells exposed to oxygenated and nonoxygenated polycyclic aromatic hydrocarbons.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) such as polycyclic aromatic quinones and polycyclic aromatic ketones as well as polycyclic aromatic hydrocarbons (PAHs) are abundant in the atmospheric environment. In this study, mRNA induction of six metabolic enzymes including P4501A1, 1A2, and 1B1, aldo-keto reductase 1C1 (AKR1C1), NAD(P)H-dependent quinone oxidoreductase 1 (NQO1), and glutathione S-transferase M1 (GSTM1) were examined in detail in human hepatoma (HepG2) cells exposed to environmentally relevant 13 PAHs and seven oxy-PAHs. Most PAHs such as benzo[a]pyrene (B[a]P) showed significant induction of P4501A1 and 1A2 mRNA, while induction by oxy-PAHs such as 5,12-naphthacenequinone (NCQ) and 11H-benzo[b]fluoren-11-one (B[b]FO) occurred less strongly. AKR1C1 mRNA was significantly induced by oxy-PAHs, 11H-benzo[a]fluoren-11-one (B[a]FO), NCQ, cyclopenta[cd]pyren-3(4H)-one (CPPO), and B[b]FO and also by P450s-inducing PAHs such as B[a]P, benzo[k]fluoranthene (B[k]FA), and dibenz[a,h]anthracene (DB[a,h]A). Both chemical-dependent and time-dependent induction patterns of NQO1 mRNA were of the mixed types of P4501A1 and AKR1C1. The tendency for the decrease of GSTM1 mRNA was observed when exposed to PAHs B[a]P and B[k]FA.

Isolation and identification of xenobiotic aryl hydrocarbon receptor ligands in dyeing wastewater.

Dyeing wastewater collected in Kyoto city, Japan, was investigated for the occurrence of aryl hydrocarbon receptor (AhR) ligands by using an AhR-responsive reporter gene assay. Concentrated extracts of wastewater samples elicited a dose-dependent increase in AhR ligand activity, and several hydrophobic HPLC fractions of the extracts were highly effective in inducing AhR ligand activity. Three potential AhR ligands were isolated from these fractions and identified to be Disperse Red 92, Disperse Yellow 64, and 3'-hydroxybenzo[b]quinophthalone by using HPLC and LC-MS/MS. Disperse Red 92, which has also been detected in the treated effluent from a sewage plant receiving the wastewater, is an anthraquinone disperse dye showing weak AhR binding affinity in the assay. Disperse Yellow 64 and 3'-hydroxybenzo[b]quinophthalone are quinoline disperse dyes capable of activating the AhR at nanomolar concentrations. In particular, Disperse Yellow 64 is a highly potent AhR ligand that was 3 times more effective in inducing AhR ligand activity than beta-naphthoflavone in the assay. Quinoline disperse dyes are suggested to be a new class of xenobiotic AhR ligands which pose a danger to aquatic biota and human health.

Characterization of polycyclic aromatic hydrocarbons (PAHs) in different size fractions in deposited road particles (DRPs) from Lake Biwa area, Japan.

Deposited road particles (DRPs) collected from 13 heavily traveled roadways in an urban area of Southern Lake Biwa, Japan, were analyzed for polycyclic aromatic hydrocarbons (PAHs) in seven different particle size fractions (< or = 20 to 1000-2000 microm) and evaluated for the aryl hydrocarbon receptor (AhR) ligand activity by using a yeast bioassay. The mean compositions of individual PAHs to total PAH concentrations in different particle size fractions were 19-21% for pyrene, which was the most dominant component, 14-16% for fluoranthene, and 7-13% for benzo[g,h,i]perylene, which were the next dominant components. The total PAH distribution pattern in different particle size fractions of DRPs was different from the organic matter distribution pattern which increased with decreasing particle size of DRPs, and could be explained by the differences in their sources. Moreover, AhR ligand activities were observed in the DRP extracts of all size fractions. The activity of the DRP extracts from the smallest size fraction was approximately 5 times more potent than that of the largest size fraction in a yeast AhR ligand activity assay. The mean contribution (%) of benzo[k]-fluoranthene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene to the AhR activity of DRP extracts in all size fractions was 1.43-4.11%, 1.63-3.53%, 0.63-1.69%, and 0.31-1.42%, respectively. Although the contribution of PAHs to AhR ligand activity presented in the DRP extracts was relatively low (below 10%), it may be best to remove these DRPs before discharge to receiving water environments.

Use of chlorophyll fluorescence of Closterium ehrenbergii and Lemna gibba for toxic effect evaluation of sewage treatment plant effluent and its hydrophobic components.

In this study fluorometric methods using an alga, Closterium ehrenbergii, and a higher plant, Lemna gibba, were employed to evaluate the toxicity of sewage treatment plant (STP) effluent and its hydrophobic components. Fluorescence parameters such as the operational photosystem II quantum yield at steady state of electron transport, the nonphotochemical quenching, and the complementary area were modified in the presence of hydrophobic components, particularly with C. ehrenbergii. It was found that C. ehrenbergii was a suitable species to be used in a hydrophobic components bioassay, since this alga was 400 times more sensitive than L. gibba to hydrophobic components. Results indicate that hydrophobic STP effluent components are less toxic as a constituent of the STP effluent than when they are extracted from the effluent. We also demonstrated here that in addition to the growth inhibition test, fluorometric methods can be usefully employed in bioassays for the toxic effect evaluation of pollutants present in municipal wastewater treatment plant effluents.

Activation of the aryl hydrocarbon receptor by methyl yellow and related congeners: structure-activity relationships in halogenated derivatives.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological action of many environmental compounds. Methyl yellow (4-dimethylaminoazobenzene; MY) is a principal azo-dye, and structurally related compounds were subjected to analysis of structure-activity relationships as AhR ligands by using a yeast AhR signaling assay. The effects of halogen-substitution among 23 halogenated MYs on the AhR ligand activity can be summarized as follows: enhancement by halogen-substitution at the ortho-position (2'- and 6'-position), and reduction by substitution at the para-position (4'-position). The greatest enhancement of the ligand activity was observed in 2',6'-dichlorinated MY (13.5-fold of MY), and its AhR ligand activity was very close to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the present assay system. In the study of compounds structurally related to MY, benzanilide (BA) showed almost the same AhR ligand activity as azobenzene and trans-stilbene. Furthermore, 4'-chlorobenzanilide, in which the length of the molecule is similar to that of MY, enhanced the AhR ligand activity by ortho(2')-chlorine-substitution, and the AhR ligand activity of 2',4'-dichlorobenzanilide was similar to that of 2'-chloro-MY. These results suggest that the amide bond is equivalent to the -N=N- or -CH=CH- double bond for recognition as the ligand by AhR in 1,2-diphenyl-1,2-ene derivatives.

Activation of the human Ah receptor by aza-polycyclic aromatic hydrocarbons and their halogenated derivatives.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor through which dioxins and carcinogenic polycyclic aromatic hydrocarbons cause altered gene expression and toxicity. Ten aza-polycyclic aromatic hydrocarbons (aza-PAHs), consisting of nitrogen substituted naphthalenes, phenanthrenes, chrysenes, and benzo[a]pyrenes (BaPs), were subjected to analysis of their structure-activity relationships as an AhR ligand by using a yeast AhR signaling assay, in which AhR ligand activity was evaluated as lacZ units. Most of the aza-PAHs showed similar or more potent AhR ligand activities than the corresponding parent PAHs. About a 100-fold increased in ligand activity was observed in 10-azaBaP compared with BaP. Halogen-substitution effects on AhR ligand activity in aza-polycyclic aromatics were also investigated with quinoline, benzo[f]quinoline (BfQ), benzo[h]quinoline (BhQ) and 1,7-phenanthroline (1,7-Phe). Position-specific induction of AhR ligand activity was observed in aza-tricyclic aromatic compounds, BfQ, BhQ, and 1,7-Phe, and the ratio of the ligand activities (lacZ units/microM) of monochlorinated and monobrominated aza-tricyclic aromatic compounds to those of the corresponding parent non-halogenated compounds ranged from 2.2- to 254-fold. Greatest enhancement of ligand activity was observed in 2-brominated BfQ (2-Br-BfQ), and its ligand activity was higher than that of BaP. These results suggest that even monohalogenation markedly enhances AhR ligand activity in aza-PAHs.

Mutagenic events induced by 4-hydroxyequilin in supF shuttle vector plasmid propagated in human cells.

Increased incidence of breast, ovarian and endometrial cancers are observed in women receiving estrogen replacement therapy (ERT). Equilin and equilenin are the major components of the widely prescribed drug used for ERT. These equine estrogens are metabolized primarily to 4-hydroxyequilin (4-OHEQ) and 4-hydroxyequilenin, respectively, which are autoxidized to react with DNA, resulting in the various DNA damages. To explore the mutagenic potential of equine estrogen metabolites, a double-stranded pMY189 shuttle vector carrying a bacteria suppressor tRNA gene, supF, was exposed to 4-OHEQ and transfected into human fibroblast. Plasmids containing mutations in the supF gene were detected with indicator bacteria and mutated colonies obtained were analyzed by automatic DNA sequencing. The proportion of plasmids with the mutated supF gene was increased dose-dependently. The majority of the 4-OHEQ-induced mutations were base substitutions (78%); another 22% were deletions and insertions. Among the base substitutions, 56% were single base substitutions and 19% were multiple base substitutions. The majority (86%) of the 4-OHEQ-induced single base substitutions occurred at the C:G site. C:G --> G:C and C:G --> A:T mutations were detected preferentially with lesser numbers of C:G --> T:A transitions. Sixty-two percent of base substitutions were observed particularly at C:G pairs in (5')-TC/AG-(5') sequences. Using (32)P-post-labeling/gel electrophoresis analysis, 4-OHEN-dC was a major adduct, followed by lesser amounts of 4-OHEN-dA adduct. Mutations observed at C:G pairs may result from 4-OHEN-dC adduct. These results indicated that 4-OHEQ is mutagenic, generating mutations primarily at C:G pairs in (5')-TC/AG-(5') sequences.