Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene. Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.     

Influence of inhibition of the metabolic activation on the mutagenicity of some nitrosamines, triazenes, hydrazines and seniciphylline in Drosophila melanogaster

It is determined to what extent certain inhibitors of the xenobiotic metabolizing enzyme systems have an influence on the mutagenicity of various pro-mutagens in Drosophila. 1-Phenylimidazole (PhI) is used as an inhibitor of the cytochrome P-450 (P-450) mediated monooxygenase activities. Iproniazid (Ipr) is a typical monoamine oxidase (MAO) inhibitor which as well seems capable of inhibiting to a certain extent P-450 mediated metabolism. N, N-Dimethyl benzylamine (N, N-DMB) is used as a competitive substrate for the N-oxidizing flavin-containing dimethylaniline monooxygenase (FDMAM). The enzyme-inhibiting activities of PhI and Ipr were determined in vitro using microsomes obtained from Drosophila larvae and adults. Both compounds were capable of inhibiting benzo[a]pyrene (BP) hydroxylation and p-nitroanisole (p-NA) demethylation, although for Ipr 100-fold higher concentrations were required compared to PhI. As model-mutagens were used: the nitrosamines dimethylnitrosamine (DMN) and diethylnitrosamine (DEN), the triazenes 1-(2,4,6-trichlorophenyl)-3,3-dimethyltriazene (Cl3PDMT), 1-(3-pyridyl)-3,3-dimethyltriazene (PyDMT) and dacarbazine (DTIC), the hydrazines procarbazine (PCZ), 1,1-dimethylhydrazine (1,1-DMH) and 1,2-dimethylhydrazine (1,2-DMH) as well as the pyrrolizidine alkaloid seniciphylline (SPh). Simultaneous or pretreatment with Ipr results in a clear decrease of the mutagenicity of Cl3PDMT, while PhI pretreatment leads to an increased mutagenicity. This indicates that these two inhibitors do not inhibit the same enzyme or isozyme. For SPh too, Ipr pretreatment results in some decrease of the mutagenicity. This is in contrast to DEN, where the activation is clearly inhibited by PhI while Ipr has only a minor effect. For DMN, DTIC and PCZ both Ipr and PhI pretreatment caused considerable decreases of the mutagenicity. Inhibition of the FDMAM catalyzed activity by N,N-DMB resulted in an increase of mutagenicity with Cl3PDMT, in a moderate decrease of mutagenicity with DTIC, and a marked decrease with DMN, which was strongly inhibited. In contrast to the clear-cut mutagenicity of PCZ, 1,1-DMH and 1,2-DMH are not mutagenic in Drosophila. No change was observed upon inhibition of the various metabolizing activities. Apart from using strain differences in metabolizing activities and enzyme induction, enzyme inhibition can also be used to determine the influence of metabolism on the in vivo mutagenicity of promutagens in Drosophila.     

Microbial catabolic activities are naturally selected by metabolic energy harvest rate

The fundamental trade-off between yield and rate of energy harvest per unit of substrate has been largely discussed as a main characteristic for microbial established cooperation or competition. In this study, this point is addressed by developing a generalized model that simulates competition between existing and not experimentally reported microbial catabolic activities defined only based on well-known biochemical pathways. No specific microbial physiological adaptations are considered, growth yield is calculated coupled to catabolism energetics and a common maximum biomass-specific catabolism rate (expressed as electron transfer rate) is assumed for all microbial groups. Under this approach, successful microbial metabolisms are predicted in line with experimental observations under the hypothesis of maximum energy harvest rate. Two microbial ecosystems, typically found in wastewater treatment plants, are simulated, namely: (i) the anaerobic fermentation of glucose and (ii) the oxidation and reduction of nitrogen under aerobic autotrophic (nitrification) and anoxic heterotrophic and autotrophic (denitrification) conditions. The experimentally observed cross feeding in glucose fermentation, through multiple intermediate fermentation pathways, towards ultimately methane and carbon dioxide is predicted. Analogously, two-stage nitrification (by ammonium and nitrite oxidizers) is predicted as prevailing over nitrification in one stage. Conversely, denitrification is predicted in one stage (by denitrifiers) as well as anammox (anaerobic ammonium oxidation). The model results suggest that these observations are a direct consequence of the different energy yields per electron transferred at the different steps of the pathways. Overall, our results theoretically support the hypothesis that successful microbial catabolic activities are selected by an overall maximum energy harvest rate.     

Inactivation of cytochrome cd1 by hydrazines

The dissimilatory nitrite reductase, cytochrome cd1, from Pseudomonas aeruginosa (ATCC 19429) was irreversibly inactivated by methyl- or phenylhydrazine but was only reduced by hydrazine itself. The reaction required oxygen and several turnovers, approximately four, of the cytochrome acting to transfer reducing equivalents from phenylhydrazine to oxygen. The reaction with methyl- or phenylhydrazine altered the visible spectrum of the cytochrome. Bands characteristic of reduced heme c appeared plus new features that were not characteristic of either oxidized or reduced heme d1. Extraction of the heme from phenylhydrazine-treated cytochrome yielded a covalently modified form of the original heme d1. Visible, 1H NMR, and mass spectra were obtained on the purified modified heme and on the metal-free esterified derivative. The spectroscopic data indicate that the modification was the regiospecific substitution of the 5 meso-proton by a phenyl group.     

Screening of safrole, eugenol, their ninhydrin positive metabolites and selected secondary amines for potential mutagenicity.

The mutagenicity of safrole, eugenol, the secondary amines, with which they combine during metabolism, and the ninhydrin positive urinary metabolites of safrole and eugenol was tested. The panel of tests included the direct bacterial assay, a microsomal mutagenesis assay and a host-mediated assay. With the direct bacterial assay employing four mutant strains of Salmonella typhimurium (TA1530, TA1531, TA1532, TA1964), all the compounds gave negative results. In the microsomal mutagenesis assay, employing the same four mutant strains, safrole and safrole metabolite II were mutagenic with strains TA1530 and TA1532. Dimethylamine was also found to be a weak mutagen in the microsomal mutagenesis assay with strain TA1530. Safrole and safrole metabolite II were also mutagenic in the host-mediated assay with strains TA1950 and TA1952. Negative results were observed for safrole metabolites I and III, eugenol, eugenol metabolites I and II, piperidine, pipecolic acid, proline, and pyrrolidine in all three assay systems.     

Microbial transformation of phytosterols mixture from rice bran oil unsaponifiable matter by selected bacteria

Rice bran sample (12 Kg) was extracted and rice bran oil (RBO ≅ 76.8 g) was saponified. The resulted unsaponifiable matter of RBO (RBO unsap) was qualitatively and quantitatively estimated using different chromatographic analyses. RBO, produced 9.65% unsaponifiable matter with the following contents, cholesterol, 6.75%; stigmasterol, 3.4%; β. sitosterol, 10.23% and campesterol, 4.2%, in addition to unknown phytosterols, hydrocarbons and waxes. Microbial transformation process started by screening of 35 bacterial strains, locally isolated from rice bran, air and soil, using RBO unsap as a carbon and an energy source to produce some pharmaceutically useful C₁₈ and C₁₉ steroids. Moraxella ovis was the most potent isolate for its highest capability to utilize RBO unsap and selectively degrade the phytosterols side-chain producing androst-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), testosterone (T) and estrone (E). The RBO unsap was the best carbon and energy source. Maximum production of the desired products was observed in 36 h, pH 7 and at 30°C by M. ovis.     

Sensitivity of different E. coli and Salmonella strains in mutagenicity testing calculated on the basis of selected literature

Two microbial screening test systems for gene (point) mutations, the Salmonella typhimurium assay (TA1535, TA1537, TA1538, TA98 and TA100) and the Escherichia coli WP2 reverse-mutation system (WP2, WP2uvrA, WP2pKM101 and WP2uvrApKM101), were compared with regard to sensitivity toward a broad spectrum of compounds that cause base-pair or frameshift mutations and that have known carcinogenic qualities. Based on available published literature we found that all 44 carcinogens and 9 non-carcinogens examined in both test systems also met with criteria for data acceptance drawn up by us. The results obtained are: firstly, that the Salmonella assay is decidedly better validated than the E. coli WP2 test; and secondly, that the E. coli test system sensitivity (91%) is fully on a par with the sensitivity of the Salmonella assay (72%). This last is in divergence from earlier reports, e.g. Brusick et al. (1980), and this difference must be ascribed to the new plasmid-containing strains. The many compounds not tested in the E. coli department result in fewer false negatives in the E. coli test system and their omission constitutes a bias in favour of the E. coli assay. By eliminating compounds that are negative in Salmonella and dropped from the WP2 analysis owing to insufficient data, the sensitivity of the Salmonella system is raised to 84% as compared with 91% for the WP2 assay. The results further indicate that some of the tester strains are superfluous, and show an exceedingly sensitive test can be performed by combining the best tester strains from the two test systems.     

Inhibition of monoamine oxidase by substituted hydrazines

1. The initial rate of inhibition of monoamine oxidase by phenethylhydrazine was shown to be similar, in pH-dependence and kinetic properties, to the oxidation of that compound by monoamine oxidase. 2. The time-course of irreversible inhibition of monoamine oxidase by phenethylhydrazine lags behind that of reversible inhibition. 3. Hydralzine was shown to be a reversible competitive inhibitor of monoamine oxidase, but phenylhydrazine is an irreversible inhibitor. Inhibition by the latter compound is not affected by the absence of oxygen, and the presence of substrate exerts no protective action. 4. Hydrazine does not inhibit monoamine oxidase unless a substrate and oxygen are present. 5. Phenethylidenehydrazine was found to be a time-dependent inhibitor of monoamine oxidase and the rate of inhibition was hindered by increasing oxygen concentration. 6. A mechanism for the inhibition of the enzyme by phenethylhydrazine is proposed in which the product of oxidation of this compound is a potent reversible inhibitor and an irreversible inhibitor of the enzyme. A computer simulation of such a mechanism predicts time-courses of inhibition that are in reasonable agreement with those observed experimentally.     

Carbon radicals in the metabolism of alkyl hydrazines

The metabolism of phenelzine (2-phenylethylhydrazine) by rat liver microsomes yields phenylacetaldehyde, 2-phenylethanol, and ethylbenzene. A carbon radical is formed during the oxidative metabolism of phenelzine that reacts with the prosthetic heme of cytochrome P-450 and irreversibly inactivates the enzyme. The radical has been spin-trapped, isolated, and shown by mass spectrometry to be the 2-phenylethyl radical. The metal-free pophyrin derived from the prosthetic heme group has been isolated and identified as N-(2-phenylethyl)protoporphyrin IX. The metabolism of phenelzine, an alkyl hydrazine, thus yields a carbon radical that inactivates cytochrome P-450, is converted to a hydrocarbon by hydrogen atom abstraction, and reacts with spin traps or (presumably) alternative cellular targets.     

Fluroxene mutagenicity.

The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-1-napthylamine, was tested for mutagenicity using four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-1-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, but not its stabilizer, was mutagenic to strains TA1535, TA100 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was not mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.     

The mechanism of inactivation of dopamine beta-hydroxylase by hydrazines

Dopamine beta-hydroxylase is inactivated by phenyl-, phenethyl-, benzyl-, and methylhydrazine, but not by hydrazine itself. With phenyl-, methyl-, and phenethylhydrazine, the rate of inactivation decreases in the presence of ascorbate and increases in the presence of tyramine. Reduction of the enzyme-bound copper occurs with all of the hydrazines tested. In the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone the carbon-centered radicals generated from each compound are trapped. This is consistent with reduction of the enzyme-bound copper by the hydrazine-containing compounds, resulting in formation of the hydrazine cation radical. Homolytic cleavage of the carbon-nitrogen bond then generates a carbon-centered radical which reacts with the enzyme, resulting in inactivation. Inactivation with [14C]phenylhydrazine results in the incorporation of 0.94 molecule of label per enzyme subunit. Benzylhydrazine behaves as a mechanism-based inhibitor of the enzyme. Both benzyl- and phenethylhydrazine are substrates for dopamine beta-hydroxylase. The second-order rate constant for inactivation of dopamine beta-hydroxylase by benzylhydrazine in the presence of ascorbate is increased about 4-fold when the benzylic hydrogens are replaced with deuterium. The apparent Vmax shows an observed deuterium kinetic isotope effect of 13 +/- 2. The partition ratio for product formation versus inactivation is 11-fold less for alpha,alpha-d2-benzylhydrazine. These results are interpreted in terms of a model where inactivation is due to abstraction of an electron from nitrogen instead of abstraction of a hydrogen atom from the benzylic carbon.     

Aspects of chloroquine mutagenicity

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP2, WP2hcr, WP6 and WP67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP2, WP2hcr; WP6 and WP67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 micrograms/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.     

Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains.

The use of juvenile Artemia as feed in aquaculture and in the pet shop industry has been getting more attention during the last decade. In this study, the use of selected bacterial strains to improve the nutritional value of dry food for Artemia juveniles and to obtain control of the associated microbial community was examined. Nine bacterial strains were selected based on their positive effects on survival and/or growth of Artemia juveniles under monoxenic culture conditions, while other strains caused no significant effect, significantly lower rates of survival and/or growth, or even total mortality of the Artemia. The nine selected strains were used to preemptively colonize the culture water of Artemia juveniles. Xenic culture of Artemia under suboptimal conditions yielded better survival and/or growth rates when they were grown in the preemptively colonized culture medium than when grown in autoclaved seawater. The preemptive colonization of the culture water had a drastic influence on the microbial communities that developed in the culture water or that were associated with the Artemia, as determined with Biolog GN community-level physiological profiles. Chemotaxonomical characterization based on fatty acid methyl ester analysis of bacterial isolates recovered from the culture tanks was performed, and a comparison with the initially introduced strains was made. Finally, several modes of action for the beneficial effect of the bacterial strains are proposed.