Potential of 2, 4‐dichlorophenoxyacetic acid conjugates as promutagens in the salmonella/microsome mutagenicity test

Abstract

Hepatic S9 preparations from Aroclor 1254 induced rats and 3‐methylcholanthrene induced woodchucks were used to investigate, in vitro, the mutagenic potential of five amino acid conjugates of 2, 4‐Dichlorophenoxyacetic acid (alanine, aspartic acid, leucine, methionine and tryptophan). Five strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535, TA1538) were utilized for this purpose. Dose‐response effects producing a two‐fold increase of revertants over spontaneous levels were not observed with either S9 preparation indicating that the amino acid conjugates are not promutagens in these assays.     

The aqueous sulfite-nitrite system: Investigations on the mutagenicity of some known products

Some known reaction products of the two commonly used food additives, sulfite and nitrite, were examined for mutagenicity using the Salmonella/mammalian-microsome test. Potassium nitrosodisulfonate, potassium aminetrisulfonate and potassium hydroxylaminemonosulfonate were not mutagenic over a dose range of 0.01 – 10 mg/plate in the strains his G 46, TA 100 and TA 98. Potassium hydroxylaminedisulfonate showed a weak mutagenic activity in his G 46 and TA 100 with microsomal activation. Hydroxylamine-O-sulfonic acid was only weakly mutagenic in the excision-repair proficient strain his G 46 in the presence of S9.     

Mutagenicity of chloroaniline/lignin metabolites in the Salmonella/microsome assay

Chloroanilines are constituents of many agrochemicals and have been found to be metabolized to succinic acid conjugates, e.g., succinamides and succinimides. The mutagenic potential of five chloroanilines and their succinamides and succinimide derivatives have been tested with two strains of Salmonella typhimurium (TA98 and TA100) with and without rat hepatic microsomal fraction. None of the compounds produced a dose response effect with a two-fold increase in revertants indicating that these compounds are not mutagens or promutagens in these assays.     

Formation of mutagens by photolysis of amino acids in neutral aqueous solution containing nitrite or nitrate ion

Nine amino acids, i.e., alanine, threonine, cysteine, glutamic acid, arginine, proline, tryptophan, phenylalanine and tyrosine, were irradiated with UV light in water containing nitrite or nitrate ion under neutral conditions. The mutagenicities of the ether extracts and the residual water layers of the reaction mixtures were assayed with and without S-9 mix using $\text{Salmonella typhimurium}$ TA98 and TA100. Three aromatic amino acids, tryptophan, phenylalanine and tyrosine, were found to give direct-acting and frameshift mutagens by irradiation in aqueous nitrite solution. Among them, the ether extract of tryptophan exhibited the strongest mutagenicity toward TA98. In the case of irradiation in aqueous nitrate solution, only the ether extract of tryptophan exhibited weak mutagenicity toward TA98 without S-9 mix. The effects of nitrite concentration, irradiation time and pH on mutagen formation from tryptophan and some characteristics of the produced mutagens were examined.     

Mutagenicity of chloroaniline/lignin metabolites in the salmonella/microsome assay

Abstract

Chloroanilines are constituents of many agrochemicals and have been found to be metabolized to succinic acid conjugates, e.g., succinamides and succinimides. The mutagenic potential of five chloroanilines and their succinamides and succinimide derivatives have been tested with two strains of Salmonella typhimurium (TA98 and TA100) with and without rat hepatic micro‐somal fraction. None of the compounds produced a dose response effect with a two‐fold increase in revertants indicating that these compounds are not mutagens or promutagens in these assays.     

Mutagenicity and identification of products formed by aqueous ozonation of humic acids of different origins

Ozonated soil-humic acid induced direct mutagenicity on TA98, TA100 and TA104, while not the ozonated reagent- and wastewater-humic acids. Oxygen radicals generated from mutagens such as glyoxal and hydrogen peroxide identified as the ozonation products might, in part, contribute to the mutagenicity of ozonated soil-humic acid.     

Mutagenicity of ambient air pollutants collected near aluminum industries

Mutagenicity has been tested in air samples collected in the summer and in the winter near four Norwegian aluminum plants. The samples were separated into a particulate and a volatile fraction and tested for mutagenicity by a quantitative reversion assay which showed that the suspended particles were clearly mutagenic. The volatile part of the air pollutants were cytotoxic to the bacteria and showed only marginal mutagenicity. The particulate fractions were tested more extensively in the Ames Salmonella mutagenicity test, in two laboratories, using the strains TA 98 and TA 100 with and without enzymatic activation (S9). The mutagenicity was relatively high compared to particulate fractions from other areas with industry and dense traffic. The highest mutagenicity was found in TA 100 with enzymatic activation and the lowest in TA 100 without S9. The mutagenicity was influenced by wind speed and direction during sampling. The mutagenic activity was also determined in the nitroreductase deficient strains TA 98NR and TA 98/1.8DNP. A larger reduction in the activity was found compared to samples from other areas, thus indicating a difference in the sample composition.     

Carbaryl-A selective genotoxicant

Mutagenic effects of carbaryl, a contact insecticide with slight systemic properties, have been investigated employing histidine reversion assay in Salmonella typhimurium strains and in vivo chromosomal aberrations in root meristems of Allium cepa. A detailed investigation revealed that carbaryl did not enhance significantly the frequency of histidine revertants in any of the strains of Salmonella i.e. frameshift mutagen tester (TA98), base pair substitution tester strain (TA1535) and ochre mutant strain (TA102). The supplementation with S9 mix did not have any appreciable effect. S14 prepared from wheat seedlings also did not enhance the reversion frequency significantly. However, carbaryl induced both clastogenic and physiological types of chromosomal aberration. The spectrum of chromosomal aberrations included c-mitosis, stickiness, vagrant chromosomes, polyploidy multi-polarity, delayed anaphases, end to end joining of chromosomes, chromosome breaks, ring chromosomes and anaphase bridges. The frequency of chromosomal aberrations was reduced by transferring the carbaryl treated bulbs to distilled water for 24 and 48 h. Similarly, recovery in the mitotic index was noticed by such transfer. The differences between the two assays may be attributed to differences in the metabolism of the test organisms.     

Evaluation of beta-naphthoxyacetic acid for mutagenic activity in the Salmonella/mammalian microsome assay

Beta-naphthoxyacetic acid (BNOA) is used as a plant growth regulator on tomatoes and strawberries. It is the active ingredient in Blossom-Set and Berry-Set, two plant hormone sprays for fruit-set. The mutagenic activity of BNOA was evaluated in four strains of Salmonella typhimurium (TA97, TA98, TA100 and TA1535) in the presence and absence of liver microsomal and cytosolic enzymes derived from Aroclor induced rats. BNOA did not produce any significant increase (p less than 0.05) in the reversion of any of the four tester strains in the standard plate incorporation assay. Results of the agar overlay toxicity tests indicates that the chemical shows toxic effects at concentrations above 500 micrograms/plate. It was concluded that under the conditions of these tests, BNOA did not exhibit any mutagenic activity.     

Chemical and biological profiles of sediments as indicators of sources of contamination in Hamilton Harbour. Part II: bioassay-directed fractionation using the Ames Salmonella/microsome assay

Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using a bioassay-directed fractionation approach. Sample extracts were fractionated using an alumina/Sephadex gel clean-up procedure to afford non-polar aromatic fractions which were characterized using chemical analyses and the Ames/microsome bacterial assay in Salmonella typhimurium strains YG1025 with the addition of oxidative metabolism (S9), and YG1024 without S9. Non-polar aromatic fractions of selected samples were separated by normal phase HPLC into 1-min fractions which were subjected to bioassay analyses. The bioassays using strain YG1025+S9, a TA100-type strain, were performed to assess genotoxicity arising from the presence of polycyclic aromatic hydrocarbons (PAH). Fractions which exhibited mutagenic activity contained PAH with molecular masses of 252, 276 and 278 amu; these fractions contained over 80% of the genotoxicity attributable to PAH. Individual compounds identified using Gas Chromatography-Mass Spectrometry analyses in these active fractions included benzo[a]pyrene, indeno[cd]pyrene and dibenz[a,h]anthracene. The YG1025+S9 mutagenic activity profiles were similar for all samples. Mutagenic activity profiles generated using strain YG1024-S9, a TA98-type strain sensitive to compounds characteristic of mobile source emissions, were very different. The mutagenic activities in strain YG1024-S9 were greatest for harbour-suspended sediment samples collected from sites impacted by a major tributary. Suspended sediments collected near areas known to contain high levels of coal tar-contamination in the bottom sediments contained higher levels of genotoxic PAH than suspended sediments collected from other areas of the harbour.     

Mutagenicity tests with gallic and tannic acid in the Salmonella/mammalian microsome assay

Gallic acid, tannic acid mixture and a purified fraction of tannic acid were evaluated for possible mutagenic activity in three strains of Salmonella typhimurium, TA98, TA100, and TA1535. These chemicals were not mutagenic either before or after activation with rat and woodchuck microsomal and cytosolic enzymes. However, tannic acid mixture and tannic acid fraction both gave a significantly (p = 0.05) dose-related reduction in the number of the revertant colonies, compared to the normal spontaneous revertants with no apparent toxic effects in the background lawn. With an agar diffusion assay, the chemicals exhibited toxic effects at 5000 micrograms/disc.     

A level change in mutagenicity of Japanese tap water over the past 12 yr

A relative comparison study of mutagenicity in Japanese tap water was conducted for 1993 and 2005 surveys. It intended to assess the effects of advanced water treatment installations to water works, improvement of raw water quality and improvement of residual HOCl concentration controlling. Sampling points (taps) were the same in both surveys. The results of 245 samples obtained by the Ames Salmonella mutagenicity test (Ames test) were analyzed. The Ames tests were conducted by using Salmonella typhimurium TA98 and TA100 strains with and without exogenous activation (S9). With the exception of TA100-S9, the other conditions needed no discussion as a factor in the mutagenicity level change. The average mutagenicity in 1993 and 2005 under the conditions of TA100-S9 were 2600 and 1100 net revertant L⁻¹, respectively. This indicated that the mutagenicity level of Japanese tap water decreased during the 12-yr period. Particularly a remarkable decrease in mutagenicity was observed in the water works where the advanced water treatments were installed during the 12-yr period. The advanced water treatments were effective in decreasing the mutagenicity of tap water. Mutagenicity also decreased in the water works with conventional water treatments; the improvement of residual HOCl concentration controlling was also considered to be effective in decreasing the mutagenicity of tap water.     

Evaluation of chlordimeform and degradation products for mutagenic and DNA-damaging activity in Salmonella typhimurium and Escherichia coli

The mutagenic activity of chlordimeform and two of its breakdown products, 4-chloro-o-toludine and 4-chloro-N-formyl-o-toluidine were determined with five histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, TA100) and five tryptophan dependent strains of E. coli WP2 (WP2, WP2uvrA, WP67, CM611, CM571) with and without rat liver microsomal enzymes. 4-chloro-o-toluidine increased the number of the reversions of the S. typhimurium strain TA1535 more than two fold over spontaneous at the concentration of 400 micrograms/plate. The results of the DNA repair tests in the Salmonella TA1538/TA1978 and E. coli multirepair deficient systems showed that both breakdown products were active in inducing damage not repaired in at least one repair deficient strain while chlordimeform itself was inactive.     

Photochemical mutagen formation from aerobically treated night-soil effluent and the contributive substances

Effluents from a night-soil treatment plant, where night-soil was aerobically treated by an activated sludge process, were irradiated with a UV lamp excluding short wavelengths less than 300 nm as a model of exposure to sunlight and the mutagenicities of the ethylacetate extracts from the irradiated effluents were assayed using Salmonella typhimurium TA98. The extracts exhibited mutagenicity toward S. typhimurium TA98 in the absence of rat liver S9 fraction only when the effluents were fortified with nitrite ion (more than 6 ppm) by over aeration or by artificial addition of nitrite, indicating that a limiting factor for mutagen formation is nitrite ion concentration. Nine organic-N-containing compounds as models of the organic components in the effluent were also irradiated and direct-acting potent mutagens were found to be produced from such compounds having indole moiety as indole, oxindole, tryptophan and tryptamine.     

Vectorization of agrochemicals: amino acid carriers are more efficient than sugar carriers to translocate phenylpyrrole conjugates in the <Emphasis Type="Italic">Ricinus</Emphasis> system

Producing quality food in sufficient quantity while using less agrochemical inputs will be one of the great challenges of the twenty-first century. One way of achieving this goal is to greatly reduce the doses of plant protection compounds by improving the targeting of pests to eradicate. Therefore, we developed a vectorization strategy to confer phloem mobility to fenpiclonil, a contact fungicide from the phenylpyrrole family used as a model molecule. It consists in coupling the antifungal compound to an amino acid or a sugar, so that the resulting conjugates are handled by active nutrient transport systems. The method of click chemistry was used to synthesize three conjugates combining fenpiclonil to glucose or glutamic acid with a spacer containing a triazole ring. Systemicity tests with the Ricinus model have shown that the amino acid promoiety was clearly more favorable to phloem mobility than that of glucose. In addition, the transport of the amino acid conjugate is carrier mediated since the derivative of the L series was about five times more concentrated in the phloem sap than its counterpart of the D series. The systemicity of the L-derivative is pH dependent and almost completely inhibited by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). These data suggest that the phloem transport of the L-derivative is governed by a stereospecific amino acid carrier system energized by the proton motive force.     

Effect of fuel properties on mutagenic activity in extracts of heavy-duty diesel exhaust particulate.

The effect of varying fuel properties on the emission of mutagenic materials was studied in diesel exhaust particles from a heavy duty engine run under transient speed and load conditions while using nine fuels varying in aromatics, sulfur and boiling point. Mutagenic activity of the soluble organic fraction (SOF) of the particulate was determined using the Ames Salmonella test system with strain TA98 with and without S9 activation. Increasing mutagenic activity relative to fuel consumed (mutants/lb fuel) or to engine work output (mutants/hp-h) was correlated with increasing fuel aromatics (p less than 0.05), but not with fuel sulfur. Increased fuel sulfur levels were correlated with increased amounts of SOF but with decreasing mutagenic activity of the SOF (mutants/microgram SOF) (p less than 0.05). As a result, mutants/hp-h were essentially the same for high- and low-sulfur fuels with high aromatics. No association was found between the fuels' boiling points and the mutagenic activity of the SOF. Mutagenic activity with S9 was generally lower than without, but the correlations were not changed.     

Effect of Fuel Properties on Mutagenic Activity in Extracts of Heavy-duty Diesel Exhaust Particulate

The effect of varying fuel properties on the emission of mutagenic materials was studied in diesel exhaust particles from a heavy duty engine run under transient speed and load conditions while using nine fuels varying in aromatics, sulfur and boiling point. Mutagenic activity of the soluble organic fraction (SOF) of the particulate was determined using the Ames Salmonella test system with strain TA98 with and without S9 activation. Increasing mutagenic activity relative to fuel consumed (mutants/lb fuel) or to engine work output (mutants/hp-h) was correlated with increasing fuel aromatics (p < 0.05), but not with fuel sulfur. Increased fuel sulfur levels were correlated with increased amounts of SOF but with decreasing mutagenic activity of the SOF (mutants/microgram SOF) (p < 0.05). As a result, mutants/hp-h were essentially the same for high- and low-sulfur fuels with high aromatics. No association was found between the fuels’ boiling points and the mutagenic activity of the SOF. Mutagenic activity with S9 was generally lower than without, but the correlations were not changed.     

Evaluation of chlordimeform and degradation products for mutagenic and DNA‐damaging activity in salmonella typhimurium and escherichia coli

Abstract

The mutagenic activity of chlordimeform and two of its breakdown products, 4‐chloro‐o‐toludine and 4‐chloro‐N‐formyl‐o‐toluidine were determined with five histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, TA100) and five tryptophan dependent strains of E. coli WP₂. (WP₂, WP₂uvrA, WP67, CM611, CM571) with and without rat liver microsomal enzymes. 4‐chloro‐o‐toluidine increased the number of the reversions of the S. typhimurium strain TA1535 more than two fold over spontaneous at the concentration of 400 μg/plate.

The results of the DNA repair tests in the Salmonella TA1538/TA1978 and E. coli multirepair deficient systems showed that both breakdown products were active in inducing damage not repaired in at least one repair deficient strain while chlordimeform itself was inactive.     

Urban airborne particulate: genotoxicity evaluation of different size fractions by mutagenesis tests on microorganisms and comet assay

The genotoxic effects of different size fractions of airborne particulate (Total, PM₁₀ and PM_2.5), extracted with acetone or toluene, were evaluated by: the Ames plate test (TA98 and TA100 strains, w/o S9), gene conversion and reversion (w/o endogenous metabolic activation) in the Saccharomyces cerevisiae D7 strain, and the comet assay on human leukocytes. The data on human leukocytes confirm the sensitivity of the comet assay and its applicability to assess genotoxicity in environmental samples. The PM_2.5 fraction of airborne particulate generally shows the highest concentration of DNA-damaging compounds. Genotoxic response, in all the test systems applied, is highly dependent on extraction solvent used. Acetone seems to extract compounds with more similar genotoxic responses in the three test systems used than toluene extracts. Toluene appears to extract air pollutants genotoxic on yeast and leukocytes but is mainly cytotoxic on Salmonella.     

How Plants Cope with Foreign Compounds. Translocation of xenobiotic glutathione conjugates in roots of barley (Hordeum vulgare) (9 pp)

Background, Aim and Scope Numerous herbicides and xenobiotic organic pollutants are detoxified in plants to glutathione conjugates. Following this enzyme catalyzed reaction, xenobiotic GS-conjugates are thought to be compartmentalized in the vacuole of plant cells. In the present study, evidence is presented for long range transport of these conjugates in plants, rather than storage in the vacuole. To our knowledge this is the first report about the unidirectional long range transport of xenobiotic conjugates in plants and the exudation of a glutathione conjugate from the root tips. This could mean that plants possess an excretion system for unwanted compounds which give them similar advantages as animals. Materials and Methods: Barley plants (Hordeum vulgare L. cv. Cherie) were grown in Petri dishes soaked with tap water in the greenhouse. - Fluorescence Microscopy. Monobromo- and Monochlorobimane, two model xenobiotics that are conjugated rapidly in plant cells with glutathione, hereby forming fluorescent metabolites, were used as markers for our experiments. Their transport in the root could be followed sensitively with very good temporal and spatial resolution. Roots of barley seedlings were cut under water and the end at which xenobiotics were applied was fixed in an aperture with a thin latex foil and transferred into a drop of water on a cover slide. The cover slide was fixed in a measuring chamber on the stage of an inverse fluorescence microscope (Zeiss Axiovert 100). - Spectrometric enzyme assay. Glutathione S-transferase (GST) activity was determined in the protein extracts following established methods. Aliquots of the enzyme extract were incubated with 1-chloro-2,4-dinitrobenzene (CDNB), or monochlorobimane. Controls lacking enzyme or GSH were measured. - Pitman chamber experiments. Ten days old barley plants or detached roots were inserted into special incubation chambers, either complete with tips or decapitated, as well as 10 days old barley plants without root tips. Compartment A was filled with a transport medium and GSH conjugate or L-cysteine conjugate. Compartments B and C contained sugar free media. Samples were taken from the root tip containing compartment C and the amount of conjugate transported was determined spectro-photometrically. Results: The transport in roots is unidirectional towards the root tips and leads to exsudation of the conjugates at rates between 20 and 200 nmol min-1. The microscopic studies have been complemented by transport studies in small root chambers and spectroscopic quantification of dinitrobenzene-conjugates. The latter experiments confirm the microscopic studies. Furthermore it was shown that glutathione conjugates are transported at higher rates than cysteine conjugates, despite of their higher molecular weights. This observation points to the existence of glutathione specific carriers and a specific role of glutathione in the root. Discussion: It can be assumed that long distance transport of glutathione conjugates within the plant proceeds like GSH or amino acid transport in both, phloem and xylem. The high velocity of this translocation of the GS-X is indicative of an active transport. For free glutathione, a rapid transport-system is essential because an accumulation of GSH in the root tip inhibits further uptake of sulfur. Taking into account that all described MRP transporters and also the GSH plasmalemma ATPases have side activities for glutathione derivatives and conjugates, co-transport of these xenobiotic metabolites seems credible. - On the other hand, when GS-B was applied to the root tips from the outside, no significant uptake was observed. Thus it can be concluded that only those conjugates can be transported in the xylem which are formed inside the root apex. Having left the root once, there seems to be no return into the root vessels, probably because of a lack of inward directed transporters. Conclusions: Plants seem to possess the capability to store glutathione conjugates in the vacuole, but under certain conditions, these metabolites might also undergo long range transport, predominantly into the plant root. The transport seems dependent on specific carriers and is unidirectional, this means that xenobiotic conjugates from the rhizosphere are not taken up again. The exudation of xenobiotic metabolites offers an opportunity to avoid the accumulation of such compounds in the plant. Recommendations and Perspectives: The role of glutathione and glutathione related metabolites in the rhizosphere has not been studied in any detail, and only scattered data are available on interactions between the plant root and rhizosphere bacteria that encounter such conjugates. The final fate of these compounds in the root zone has also not been addressed so far. It will be interesting to study effects of the exuded metabolites on the biology of rhizosphere bacteria and fungi.