Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells

Seven promutagens belonging to two chemical classes--polycyclic aromatic hydrocarbons (PAHs) (benzo[a]pyrene, dimethyl benz[a]anthracene, 3-methylcholanthrene, fluoranthene) and alkyl nitrosamines (dimethyl, diethyl, and dibutyl nitrosamine)--were studied in Chinese hamster ovary (CHO) cells. Findings of practical importance in the use of Aroclor 1254-induced rat liver homogenate (S9) in the CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay were made. Our novel findings are (1) the inclusion of CaCl2 in the S9 cofactor mixture dramatically decreased the cytotoxicity of S9, and (2) different S9 optimum concentrations were observed for structurally similar promutagens. The inclusion of CaCl2 in the S9 cofactor mixture and the testing of each chemical of unknown S9 requirement at several S9 concentrations are therefore recommended for assaying promutagens in the CHO/HGPRT mutation assay.     

Mutagenicity of the organic fraction of World Trade Center dust

Most studies of the health effects and chemical characterization of the dust resulting from the catastrophic collapse of the World Trade Center (WTC) on September 11, 2001, have focused on the large inorganic fraction of the dust; however, chemical analyses have identified mutagens and carcinogens in the smaller organic fraction. Here, we determined the mutagenicity of the organic fraction of WTC dust in Salmonella. Only 0.74% of the mass of the particulate matter (PM) <53 μm in diameter was extractable organic matter (EOM). Because the EOM was 10 times more mutagenic in TA100 +S9 than in TA98 +S9 and was negative in TA98 −S9, we inferred, respectively, that polycyclic aromatic hydrocarbons (PAHs) played a role in the mutagenicity and not nitroarenes. In TA98 +S9, the mutagenic potency of the EOM (0.1 revertant/μg EOM) was within the range of EOMs from air and combustion emissions. However, the EOM-based mutagenic potency of the particles (0.0007 revertants/μg PM) was 1–2 orders of magnitude lower than values from a review of 50 combustion emissions and various air samples. We calculated that 37 PAHs analyzed previously in WTC EOM were 5.4% of the EOM mass and 0.04% of the PM mass; some air contained 0.3 μg WTC EOM/m³ (0.02 μg PAHs/m³). Populations exposed to WTC dust have elevated levels of prostate and thyroid cancer but not lung cancer. Our data support earlier estimates that PAH-associated cancer risk among this population, for example, PAH-associated lung cancer, was unlikely to be significantly elevated relative to background PAH exposures.     

Synthesis and bacterial mutagenicity of the cyclopenta oxides of the four cyclopenta-fused of isomers of benzanthracene

Many polycyclic aromatic hydrocarbons containing peripherallyfused cyclopenta rings are believed to be activated primarilyby epoxidation of the cyclopenta ring. The cyclopenta epoxidesof a series of four cyclopenta benzanthracene derivatives, benz[e]aceanthrylene-5,6-oxide,benz[j]ace-anthrylene-1,2-oxide, benz(l)anthrylene-1,2-oxideand benz[k]acephenaceanthrylene-4,5-oxide were synthesized fromtheir parent hydrocarbons by formation of the bromohydrin followedby dehydrobromination, and characterized by u.v. – vis,and ¹H n.m.r. spectroscopy and mass spectrometry. The mutagenicityof these compounds was investigated in the Ames plate incorporationassay with Salmonella typhimurium strain TA98. All the oxideswere active without exogenous metabolic activation (170–320His⁺ revertants per nanomole) and also toxic above 0.5 µg/plate.Addition of S9 protein did not increase, and generally decreased,the mutagenicity of the oxides, while toxicity was largely unchanged.These results are consistent with the postulated role of cyclopentaoxides as major contributors to the mutagenicity of the parentcompounds in the Ames assay.     

Physical‐chemical and microbiological characterization,and mutagenic activity of airborne PM sampled in a biomass‐fueled electrical production facility

Biomass combustion is used in heating and electric power generation in many areas of the world. Airborne particulate matter (PM) is released when biomass is brought to a facility, stored, and combusted. Occupational exposure to airborne PM within biomass‐fueled facilities may lead to health problems. In March and August of 2006, airborne PM was collected from a biomass‐fueled facility located in Denmark. In addition, source‐specific PM was generated from straw and wood pellets using a rotating drum. The PM was analyzed for polycyclic aromatic hydrocarbons (PAHs), metals, microbial components, mutagenic activity, and ability to generate highly reactive oxygen species (hROS) in cell‐free aqueous suspensions. PM collected from the boiler room and the biomass storage hall had higher levels of mutagenic activity, PAHs and metals, and a higher hROS generating potential than the source specific PM. The mutagenic activity was generally more potent without S9 activation, and on the metabolically enhanced strain YG1041, relative to TA98. Significant correlations were found between mutagenicity on YG1041 (without S9) and PAH concentration and mutagenicity on YG1041 (with S9) and hROS generating ability. PM collected in March was more toxic than PM collected in August. Overall, airborne PM collected from the facility, especially that from the boiler room, were more toxic than PM generated from straw and wood chips. The results suggest that exposure to combustion PM in a biomass‐fueled facility, which likely includes PM from biomass combustion as well as internal combustion vehicles, may contribute to an elevated risk of adverse health effects. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.     

Disubstituted amino-, nitroso-, and nitrofluorenes: a physicochemical basis for structure-activity relationships in Salmonella typhimurium

Twenty-nine derivatives of fluorene were tested for mutagenic potency in four strains of Salmonella typhimurium with and/or without S9 microsomal activation. The effects of a second functional group on the mutagenic activity of an amino-, nitroso-, and nitrofluorene were correlated with its physical and chemical properties. When the functional group is conjugated by resonance, both inductive and resonance effects are determinants of mutagenic potency. Electron-withdrawing groups such as the halogens (F, C1, Br, and I), nitro, nitroso, and cyano at C-7 increased the mutagenic potency of 2-nitrofluorene. Electron-donating substituents such as hydroxy and amino groups at C-7 decreased the mutagenic potency of 2-amino, 2-nitroso-, and 2-nitrofluorene. Acetylation of a hydroxy or an amino group at C-7 increased the mutagenic potency of 2-nitrofluorene, presumably by decreasing the electron-donating properties of these groups. In contrast, acetylation of a nonresonance-conjugated amino group decreased mutagenic activity. The physical properties of a second functional group are expected to exert their effect(s) at three points in the metabolic activation of 2,7-disubstituted fluorene derivatives: initial reduction of the nitro group (redox effect), stabilization of the hydroxylamine (inductive effect), and stabilization/destabilization of the nitrenium ion (resonance and inductive effects). The relationships between the physical properties of a second functional group and their effects on biological activities of nitro- and aminofluorenes in the Ames Salmonella assay may be of predictive value in a first approximation of both the mutagenic and carcinogenic potency of chemicals with comparable structures such as fluoranthene and biphenyl.     

Use of the spiral Salmonella assay to detect the mutagenicity of complex environmental mixtures

The success demonstrated by the spiral Salmonella assay in a recent study of 20 pure prompted us to examine the effectiveness of this automated bacterial mutagenicity assay for testing complex environmental mixtures. Three sets of combustion emissions were selected for evaluation: automotive diesel exhaust, woodsmoke, and a coal combustion emission. Each sample was tested in the Salmonella mutagenicity assay according to standard protocol (plate incorporation) and spiral assay techniques. In the spiral assay, a specialized plating instrument dispenses the bacteria, test agent, and S9 mix in a spiral pattern onto a minimal agar plate supplemented with histidine and biotin. The components of the assay are administered in such a way that a uniform density of bacteria is exposed to a concentration gradient of the test agent on a single plate. When results are analyzed, a dose-response curve comprised of 13 data points is generated. A comparison of results from the two assays demonstrated the following: 1) Diesel exhaust was generally the most mutagenically potent sample in both assays, followed closely by the coal combustion emission. The woodsmoke sample was only weakly mutagenic in the standard assay but demonstrated higher mutagenic activity in the spiral assay. 2) Samples were more mutagenic on rev/microgram basis in the spiral assay, especially when metabolic activation was added. This disparity presumably was due to differences in the relative amounts of S9 administered across the dose range. 3) The spiral assay required 1/20 the sample mass of the standard assay to test equivalent doses; in addition, for some samples, 50 times more sample mass was required by the standard assay to generate a comparable dose response. 4) Dichloromethane extracts of the complex mixtures could be tested for mutagenicity in the spiral assay, thereby precluding solvent exchange (to dimethylsulfoxide) required by the standard assay for sample/bioassay compatibility.     

Comparative mutagenicity of nitrofluoranthenes in Salmonella typhimurium TA98, TA98NR, and TA98/1,8DNP6

The nitration of fluoranthene, one of the most abundant polycyclic aromatic hydrocarbons (PAH) in diesel fuels, occurs in the laboratory under either electrophilic or free-radical conditions to give nitro-PAH. 3-Nitrofluoranthene (3-NF) is the major product under electrophilic ionic conditions while 2-nitrofluoranthene (2-NF) is the major product under free-radical nitration conditions. The free-radical nitration of fluoranthene also yields 1,2- and 1,3-dinitrofluoranthene (1,2-DNF and 1,3-DNF). Nitration on the 3-position of fluoranthene enhances the mutagenic potency more strongly than on the 2-position. Thus, 3-NF is a more potent mutagen than 2-NF and 1,3-DNF is more potent than 1,2-DNF, an isomer with one near coplanar nitro group and one nitro group substantially out of plane with the fluoranthene skeleton, when tested against Salmonella typhimurium TA98, TA98NR, and TA98/1,8-DNP6. In addition, the activation of these dinitro-PAH to mutagens does not depend on the "classical nitroreductase" and/or O-acetylase, suggesting that they are activated via different pathways. Despite the fact that 3-NF and 1-phenyl-4-nitronaphthalene (1-P-4NN), a non-planar analog of 3-NF, have virtually identical reduction potentials, their mutagenic potencies differ by three orders of magnitude. This finding suggests that when nitro-PAH of varying steric requirements are compared, the reduction potential may not predict mutagenic potency as well as had been previously suggested.     

Identification of mutagens in freshwater sediments by the Ames-fluctuation assay using nitroreductase and acetyltransferase overproducing test strains

Extracts of sediments from an area of concern in the Elbe river basins (Spittelwasser creek) were analyzed with the Ames-fluctuation test and in parallel with gas chromatography/mass spectrometry for compound identification. The standard test strains TA 98 and TA 100 showed mutagenicity mainly in medium-polar fractions of the sediment extracts. PAHs contribute to the overall mutagenic potential of the sample. Especially, cyclopenta[c,d]pyrene that was previously not defined as a priority hazardous substance has to be considered as well. The addition of metabolically competent test strains, which overexpress nitroreductase and acetyltransferase (e.g., YG1041 and YG1042) to the test battery, increased significantly the sensitivity of the Ames test for medium polar to polar genotoxins. The increased mutagenicity that was found in these bacterial strains indicates the presence of nitroarenes and/or aromatic amines. In fact, a number of heterocyclic and nitrogen-substituted aromatic compounds were identified in the sediments of the Spittelwasser creek of which methyl parathion, 1-naphthylamine, and N-phenyl-2-naphthylamine are mutagenic.     

Mutagenicity of an aged gasworks soil during bioslurry treatment

This study investigated changes in the mutagenic activity of organic fractions from soil contaminated with polycyclic aromatic hydrocarbons (PAHs) during pilot‐scale bioslurry remediation. Slurry samples were previously analyzed for changes in PAH and polycyclic aromatic compound content, and this study examined the correspondence between the chemical and toxicological metrics. Nonpolar neutral and semipolar aromatic fractions of samples obtained on days 0, 3, 7, 24, and 29 of treatment were assayed for mutagenicity using the Salmonella mutation assay. Most samples elicited a significant positive response on Salmonella strains TA98, YG1041, and YG1042 with and without S9 metabolic activation; however, TA100 failed to detect mutagenicity in any sample. Changes in the mutagenic activity of the fractions across treatment time and metabolic activation conditions suggests a pattern of formation and transformation of mutagenic compounds that may include a wide range of PAH derivatives such as aromatic amines, oxygenated PAHs, and S‐heterocyclic compounds. The prior chemical analyses documented the formation of oxygenated PAHs during the treatment (e.g., 4‐oxapyrene‐5‐one), and the mutagenicity analyses showed high corresponding activity in the semipolar fraction with and without metabolic activation. However, it could not be verified that these specific compounds were the underlying cause of the observed changes in mutagenic activity. The results highlight the need for concurrent chemical and toxicological profiling of contaminated sites undergoing remediation to ensure elimination of priority contaminants as well as a reduction in toxicological hazard. Moreover, the results imply that remediation efficacy and utility be evaluated using both chemical and toxicological metrics. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Antimutagenic activity of spearmint

The antimutagenic activity of spearmint (Mentha spicata), a popular food flavoring agent, was studied in the Salmonella assay. Spearmint leaves were brewed in hot water for 5 min at concentrations up to 5% (w/v), and the water extracts were tested against the direct-acting mutagens 4-nitro-1,2-phenylenediamine (NPD) and 2-hydroxyamino-3-methyl-3H-imidazo[4,5-f]quinoline (N-OH-IQ) using Salmonella typhimurium strain TA98. Nontoxic concentrations of spearmint extract inhibited the mutagenic activity of N-OH-IQ in a concentration-dependent fashion, but had no effect against NPD. These experiments by design focused on the water extract consumed commonly as an herbal tea, but chloroform and methanol extracts of spearmint also possessed antimutagenic activity against N-OH-IQ. Water extract of spearmint inhibited the mutagenic activity of the parent compound, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), in the presence of rat liver S9; however, the concentration for 50% inhibition (IC50) against IQ was approximately 10-fold higher than in assays with N-OH-IQ minus S9. At concentrations similar to those used in the Salmonella assays, spearmint extract inhibited two of the major enzymes that play a role in the metabolic activation of IQ, namely, cytochromes P4501A1 and 1A2, based on ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase assays in vitro. In vivo, rats were given spearmint water extract (2%; w/v) as the sole source of drinking fluid before, during, and after 2-week treatment with IQ; colonic aberrant crypt foci were inhibited significantly at 8 weeks (P < 0.05, compared with rats given IQ alone). Collectively, these findings suggest that spearmint tea protects against IQ and possibly other heterocyclic amines through inhibition of carcinogen activation and via direct effects on the activated metabolite(s).     

Up-regulation of the glutathione S-transferase system in human liver by polycyclic aromatic hydrocarbons; comparison with rat liver and lung

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the alpha-class selective 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.     

Evaluation of Pluronic Polyol F127 as a vehicle for petroleum hydrocarbons in the Salmonella/microsomal assay

Complex hydrocarbon mixtures have proven difficult to evaluate in in vitro mutagenicity assays owing to their insolubility in aqueous environments. Pluronic Polyol F127 (BASF Wyandotte, Parsippany, NJ), prepared as a 50% (w/w) solution in absolute ethanol, proved effective in emulsifying various petroleum hydrocarbon fractions. Its effectiveness in the Salmonella/microsomal assay was evaluated using model solutions each comprising a polycyclic aromatic hydrocarbon (PAH) dissolved in mineral oil. The PAHs used were benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene. Model solutions were evaluated neat and as emulsions with the Pluronic F127 solution or Tween 80. Similar levels of each PAH were prepared in dimethyl sulfoxide (DMSO) for comparison. Cytotoxicity and mutagenesis were evaluated in the preincubation technique using strain TA97. Little or no cytotoxicity or mutagenesis was evident for model solutions tested neat. However, emulsification of these PAH-laden mixtures with the Pluronic F127 solution yielded cytotoxic and mutagenic responses similar to, or greater than, those observed for PAHs delivered in DMSO. Model mixtures emulsified with Tween 80 were less active. Study results demonstrate that Pluronic F127, prepared as a 50% (w/w) solution in absolute ethanol, is an effective vehicle for evaluating the mutagenic potential of complex hydrocarbon mixtures containing PAHs in the Salmonella/microsomal assay. Since PAHs are a class of insoluble hydrocarbons, the results also suggest the potential usefulness of the Pluronic F127 solution to detect the mutagenicity of other insoluble hydrocarbons and hydrocarbon mixtures.     

Mutagenic activity of the 4,5- and 9,10-dihydrodiols of benzo[J]fluoranthene and their syn- and anti-dihydrodiol epoxides in salmonella typhimurium

The objective of this study was to determine the relative mutagenic activities of the major dihydrodiol metabolites of benzo[j]fluoranthene (B[j]F) and their corresponding syn- and anti-dihydrodiol epoxides. Salmonella typhimurium tester strains TA97a, TA98, and TA100 were used to evaluate the mutagenic potencies of the parent hydrocarbon and these suspect proximate and ultimate mutagenic metabolites. B[j]F and the trans-dihydrodiol metabolites were active only in the presence of an external metabolic activation system (S9) with the exception of the B[j]F-4,5-diol, which was weakly active in TA98 and TA100 in the absence of S9. The B[j]F-4,5-diol was more mutagenic than the B[j]F-9,10-diol in tester strains TA98 and TA100, whereas the opposite effect was observed in TA97a. In the absence of S9, the anti-B[j]F-4,5-diol epoxide was more mutagenic than the syn-B[j]F-4,5-diol epoxide and the syn- and anti-B[j]F-9,10-diol epoxides in tester strains TA97a and TA100. The exceptional mutagenic potency of the anti-B[j]F-4,5-diol epoxide in TA100 resembles that observed by epoxides located within a fjord, or by the anti-diol epoxides of bay region methylated polycyclic aromatic hydrocarbons. In contrast, the mutagenicity of the pseudo bay region dihydrodiol epoxides arising from the B[j]F-9,10-diol more closely resembles that observed with the classical bay region dihydrodiol epoxides of chrysene. In summary, both dihydrodiol metabolites of B[j]F are mutagenic in S. typhimurium, and the relative potency varies among the tester strains. The highest mutagenic response was achieved in tester strain TA100, which detects base-pair substitutions. The most potent direct-acting dihydrodiol epoxide in this tester strain was the anti-B[j]F-4,5-diol epoxide, which agrees with the results of mouse skin painting studies that indicate that the B[j]F-4,5-diol is more tumorigenic that the parent hydrocarbon or the B[j]F-9,10-diol. A cova-lent DNA adduct formed between the anti-B[j]F-4,5-diol epoxide and deoxyguanosine was the major species of DNA adduct formed in S. typhimurium. This adduct corresponds to the major DNA adduct formed in mouse skin following application B[j]F.     

Contribution of ethyl methanesulfonate vapors to the yield of mutations detected in Drosophila melanogaster when the adult feeding technique is used

Complex hydrocarbon mixtures have proven difficult to evaluate in in vitro mutagenicity assays owing to their insolubility in aqueous environments. Pluronic® Polyol F127 (BASF Wyandotte, Parsippany, NJ), prepared as a 50% (w/w) solution in absolute ethanol, proved effective in emulsifying various petroleum hydrocarbon fractions. Its effectiveness in the Salmonella/microsomal assay was evaluated using model solutions each comprising a polycyclic aromatic hydrocarbon (PAH) dissolved in mineral oil. The PAHs used were benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenzanthracene. Model solutions were evaluated neat and as emulsions with the Pluronic® F127 solution or Tween 80. Similar levels of each PAH were prepared in dimethyl sulfoxide (DMSO) for comparison. Cytotoxicity and mutagenesis were evaluated in the preincubation technique using strain TA97. Little or no cytotoxicity or mutagenesis was evident for model solutions tested neat. However, emulsification of these PAH-laden mixtures with the Pluronic® F127 solution yielded cytotoxic and mutagenic responses similar to, or greater than, those observed for PAHs delivered in DMSO. Model mixtures emulsified with Tween 80 were less active. Study results demonstrate that Pluronic® F127, prepared as a 50% (w/w) solution in absolute ethanol, is an effective vehicle for evaluating the mutagenic potential of complex hydrocarbon mixtures containing PAHs in the Salmonella/microsomal assay. Since PAHs are a class of insoluble hydrocarbons, the results also suggest the potential usefulness of the Pluronic® F127 solution to detect the mutagenicity of other insoluble hydrocarbons and hydrocarbon mixtures.     

Study on the mutagenic activity of 13 bioflavonoids with the Salmonella Ara test

The mutagenicity of 13 flavonoids has been investigated with the L-arabinose forward mutation assay of Salmonella typhimurium. Each flavonoid was tested by both plate incorporation and preincubation mutagenesis protocols in the presence or the absence of mammalian metabolic activation (S9 mixture). All flavonoids gave a dose-response relationship and induced a number of AraR mutants considered statistically significant. Their minimum mutagenic doses (MMD) differed markedly in the Ara test, covering a 400-fold range: from 4 nmol for quercetin to 1626 nmol for taxifolin. Flavonols were the strongest mutagens, with mutagenic potencies (MMD-1) representing from 27 to approximately 2% that of quercetin. Comparatively, the mutagenicities of other flavonoids represented only less than or equal to 1%. The data reported in this paper for the Ara forward mutation test suggest structural requirements for mutagenicity of bioflavonoids like those previously reported for the His reverse mutation assay: (i) flavonols with a free hydroxyl at position 3 are the strongest mutagenic flavonoids, (ii) saturation of the 2,3 double bond diminishes the mutagenic potency, and (iii) free hydroxyl groups at positions 3' and 4' influence the non-requirement for metabolic activation. The mutagenic properties of quercetin and rutin in the Ara test support the idea that these flavonols are not the major putative mutagens in complex mixtures such as wine.     

Generation and Characterization of Human Single-Chain Antibodies Against Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. BP is one of the most toxicologically active PAHs and is often used as a prototype for this entire class of contaminants. In order to select anti-BP antibodies, the conjugate of BP with BSA (BP-BSA) was used to screen naïve combinatorial phage library of human scFvs. Seven unique scFvs against BP-BSA were selected after three rounds of selection. Analysis of the genes encoding the scFvs subdivided them to gene families and subfamilies. Homology with the closest germline ranged from 80.21% to 97.57% for heavy chains and 88.89% to 98.57% for the light chains. Four of the seven scFv amino acid residues sequences without stop codons in frame were selected for proteomic analysis with each other. Four scFvs encoded unique non-related proteins with low-sequence identity among them. All CDRs and the boundaries in the CDR3 formation were carried out. Two of the scFvs (T68 and T72) with the highest binding capabilities to PAHs were expressed in E. coli and purified using a nickel resin. The KDs of T68 to BP-BSA, chrysene, pyrene, and benzo[a]anthracene were almost similar, approximately 10^?7?M. The KDs of T72 to benzo[a]anthracene and chrysene were 9.42?×?10^?8?M and 2.63?×?10^?7?M, respectively. The computational models of T68 and T72 active centers were different.     

Optimization of metabolic activation for four mutagens in a bacterial, fungal and two mammalian cell mutagenesis assays

The optimum concentrations of Aroclor-induced rat liver S9 microsomal fraction for the mutagenic activity of the four standard mutagens 2-aminofluorene (2-AF), acriflavine (ACR), benzo[a]pyrene (BP) and cyclophosphamide (CP) were determined in four mutation assays. The four assays were the Ames test using Salmonella typhimurium strain TA100, cycloheximide resistance in the yeast Saccharomyces cerevisiae, the mouse lymphoma TK assay and the human peripheral lymphocyte cytogenetic assay. BP was the only mutagen to be most active at comparable S9 concentrations, of approximately 1%, for all four assays. The optimum S9 concentrations for each of the remaining three mutagens varied substantially between the four assays. ACR was a potent direct-acting mutagen in both mammalian cell assays. The mouse lymphoma TK assay results showed similar optimal values of 1.5% S9 or below for each of the four test agents. The assay with the largest variation of optimal S9 values for the four mutagens was the Ames test in strain TA100, although it also had the widest peaks of activity over the range of S9 concentrations tested. It is likely that the diversity of findings is due to a variety of metabolites affecting the different genetic endpoints that are measured in these assays. Thus from these results it is not possible for bacterial optimization data to be related to other routine in vitro systems. The use of more than one concentration of S9 would contribute useful information.     

Poly(N‐hexyl‐cyclopenta[c]pyrrole) – A Novel 1,3,4‐Alkyl‐substituted Polypyrrole Soluble in Organic Solvents and Redox Conducting

Summary: N‐Hexyl‐cyclopenta[c]pyrrole was synthesized and electrochemically and chemically polymerized to a novel 1,3,4‐alkyl‐substituted polypyrrole. The polymer was characterized in solution and as thin solid film by cyclic voltammetry, UV‐vis, FTIR and NMR spectroscopy, MALDI mass spectroscopy, GPC analysis, electrochemical quartz crystal microbalance, ESR spectroscopy and in situ conductivity. The polymer, with a defect‐free structure and an average degree of polymerization of 13 (electrochemically prepared) and 24 (chemically prepared), is soluble (>1%) in acetone, acetonitrile and chlorinated solvents. Its in situ conductivity as a function of potential and doping charge has the typical features of redox conductivity with a maximum value of ca 1 × 10^?3 S · cm^?1.

Structure of poly(N‐hexyl‐cyclopenta[c]pyrrole).     

Conditions affecting the mutagenicity of trichloroethylene in Salmonella

Trichloroethylene (TCE) is a high production volume chemical frequently stabilized with oxiranes. These oxiranes may be responsible for the mutagenic activity of TCE in Salmonella, which has been occasionally, but not consistently, reported. High purity and oxirane-stabilized TCE samples were tested for their mutagenic potential in Salmonella typhimurium strains TA 1535, TA 98, and TA 100. Stabilized TCE was tested using a preincubation protocol up to a dose level of 10,000 micrograms per plate, but no mutagenic response was observed in either the presence or absence of a supplementary metabolic activation system (S9 mix) derived from Aroclor 1254-induced male rat liver. TCE without oxirane stabilizers also was nonmutagenic when tested in a vapor delivery system at nominal concentrations of up to 20% and using S9 mix derived from either rat or hamster. TCE containing 0.5-0.6% 1,2-epoxybutane did induce mutagenic responses from strains TA 1535 and TA 100 in the presence and absence of S9 mix. The lowest effective dose was about 0.63% in TA 1535 in the absence of S9 mix. Vapor-phase tests with 1,2-epoxybutane showed that an atmospheric concentration of 0.009% could induce 12-fold and 3-fold increases, respectively, in strains TA 1535 and TA 100. These increases would account for the mutagenic activity of the stabilized TCE sample. Epichlorohydrin (another commonly used stabilizer) induced similar increases in mutant numbers at an atmospheric concentration of 0.0009%. The absence of a significant response caused by unstabilized TCE in the presence of S9 mix is probably due to a lack of assay sensitivity, since chloral, a metabolite of TCE, is a mutagen in TA 100 [Haworth et al.: Environ Mutagen [Supplement 1] 5:3-142, 1983].