Generation of mutagenicity by ozonation of humic substances' components.

Components of humic substances, such as vanillin, syringaldehyde, vanillic acid and di-n-butylphtalate, were ozonated and subjected to the mutagenicity assay using Salmonella typhimurium TA 98 and 100 with and without S9 mix. The strong mutagenic activity was found on all components except di-n-butylphtalate by strain TA 100 with and without S9 mix. Substances with strong mutagenic activity in ozonated vanillin were water-soluble and were slightly extracted with benzene, dichloromethane and ethyl acetate. Following gel chromatography on Sephadex G-10, the strong mutagens generated by ozonation were found with molecular weights greater than 300.

Mutagenicity from ozonation of humic substances.

Eight structural components of humic substances were ozonated. Mutagenic activity was found using TA100 with and without S9 mix for all ozonated components. p-Hydroxybenzaldehyde was chosen as an important component and ozonation products were determined by gas chromatography-mass spectrometry (GC-MS). Aldehydes, ketones and carboxylic acids were identified as the ozonation products. Among these products, acetaldehyde, formaldehyde, glyoxal, methylglyoxal and glyoxylic acid were recognized to be mutagenic. Furthermore, p-hydroxybenzaldehyde was first ozonated and then chlorinated. A great variety of chlorinated organic compounds, many of which are known mutagens, have been identified by GC-MS in the ether extract. The same compounds have previously been reported as chlorination products of humic substances. Aldehydic products by ozonation were identified from ozonation followed by chlorination of humic substances and p-hydroxybenzaldehyde.

Aldehydes as mutagens formed by ozonation of humic substances.

Humic substances and p-hydroxybenzaldehyde, one of their components, were ozonated and quantitative analysis of the mutagenic aldehydes (formaldehyde, acetaldehyde, glyoxal, glyoxylic acid and methylglyoxal) was performed. Glyoxal and glyoxylic acid were the main mutagenic compounds. The ozone-treated solutions were flowed through a granular activated carbon (GAC) column and the KMnO4 consumed of the effluent decreased to about 40-50%. Most of the aldehydes formed by ozonation reduced, but glyoxal increased.

Modulating effect of tanshinones on mutagenic activity of Trp-P-1 and benzo[a]pyrene in Salmonella typhimurium.

The modulating effects of the Chinese medicinal plant 'Tan-shen', the radix of Salvia miltiorrhiza Bunge, on the mutagenic activities of Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) and B(a)P (benzo[a]pyrene) were investigated using Salmonella typhimurium TA98. Ether- and hot water-extracted 'Tan-shen' enhanced both mutagens at low concentrations, but suppressed them at high concentrations. Extracts by ether treatment were more effective than those extracted by hot water. Dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA were isolated from the ether extract by high performance liquid chromatography (HPLC) and were recognized to be the mutagenic modulators. 4 tanshinones enhanced the mutagenicity of Trp-P-1 by 8-24-fold at 20 micrograms/plate and the enhancement was reduced at the higher concentration. Dihydrotanshinone I suppressed Trp-P-1 activity completely at 100 micrograms/plate.

Mutagenicity of the components of ozonated humic substance.

Eight components of humic substances were ozonated. Mutagenic activity was found with TA100 with and without S9 mix for all ozonated components. Ozonated products of p-hydroxybenzaldehyde were separated into five fractions by silica gel chromatography and each fraction subjected to mutagenicity assay. Mutagenic activity was found in the chloroform and chloroform-acetone (1:1) fractions. The compounds in these fractions were identified, and aldehydes such as formaldehyde, acetaldehyde, glyoxal, glyoxylic acid and methyl glyoxal were found to be mutagenic. Mutagenic compounds are present in the polar fraction.

Mutagenicity of ozonation and chlorination products from p-hydroxybenzaldehyde.

p-Hydroxybenzaldehyde, a component of soil humic substances, was ozonated and chlorinated. The ether extract and the residue were subjected to the Ames assay; mutagenic activities were identified. The non-ionic resin CSP800 and the anion exchange resin CHPA25 were used for separation of mutagenic compounds. The compounds in the water layer were not adsorbed on CSP800 or CHPA25 and exhibited strong mutagenic activity. Mutagenic activity was reduced as the added chlorine was increased. Ether extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) and chloral, 1,3-dichloro-2-propanone, 1,2,3-trichloro-1-propene, tetrachloroethylene and 1,1,1,3,3-pentachloro-2-propanone were identified as mutagenic compounds.

Evaluation of the SOS chromotest for the detection of antimutagens.

The SOS chromotest was applied for the detection of antimutagens. To raise SOS induction, the bacteria were treated with the mutagens, UV, 4-nitroquinoline N-oxide (4NQO), N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), or benzo[a]pyrene (B[a]p). The inhibitory effects of L-ascorbic acid, glutathione, vanillin, 5-fluorouracil (5-FU), 5-chlorouracil (5-CU), cobaltous chloride, sodium selenite and sodium arsenite, which are known as antimutagens, were investigated with their addition either simultaneously or post treatment time. It became clear that the SOS chromotest was very useful for the detection of antimutagens.

Enhancement of the mutagenicity of Trp-P-1, Trp-P-2 and benzo[a]pyrene by bupleuri radix extract.

Boiling-water extract of Korean-Saiko (Bupleuri Radix, from South Korea, Bupleurum falcatum L.) enhanced the mutagenic activity of Trp-P-1, Trp-P-2 and benzo[a]pyrene with S9mix. The boiling-water extract was fractionated with ether and then n-BuOH. Both the ether and the n-BuOH fractions also enhanced mutagenicity of Trp-P-1, respectively. The n-BuOH fraction was separated into seven fractions by silica gel chromatography and the chloroform eluate had the strongest enhancing effect on the mutagenic activity of Trp-P-1 with S9mix. The chloroform eluate fraction was further separated into five spots by thin-layer chromatography. Two of the spots had the strongest enhancing effect on the mutagenic activity of Trp-P-1. Since saikosaponin is a well known component in Bupleuri Radix, the effects of its existence were tested and saikosaponins a and c were found. The enhancement activity of saikosaponin a was very weak. The effective components are now being studied.

Mechanism of antimutagenicity of aquatic plant extracts against benzo[a]pyrene in the Salmonella assay.

The mechanism of antimutagenicity of water extracts of grass-wrack pondweed (Potamogeton oxyphylus Miquel), curled pondweed (Potamogeton crispus L.) and smartweed (Polygonum hydropiper L.) towards benzo[a]pyrene mutagenicity in Salmonella typhimurium was investigated. The antimutagenic components in the aquatic plants were water-soluble, heat-resistant and had a high molecular weight; chlorophyll did not play an important role.

Inhibitory action of peony root extract on the mutagenicity of benzo[a]pyrene.

The inhibitory effects of peony root extract on the mutagenicity of benzo[a]pyrene (B [a]p) have been investigated in the Salmonella typhimurium reversion test. Four kinds of experiments were performed: direct chemical reaction (1) between peony root extract and B [a]p, and (2) between peony root extract and active metabolite(s) of B [a]p, (3) inhibition of metabolic processes of B[a]p with S9 mix, and (4) inhibition of activation on mutagenicity. Peony root extract interfered with the action of enzymes in the S9 mix, and inactivated the activity of B[a]p metabolites. The bio-antimutagenic effect was assayed by reversion in Salmonella typhimurium TA98 and TA100.

Mechanism of resistance to benzalkonium chloride by Pseudomonas aeruginosa.

The mechanisms of resistance of Pseudomonas aeruginosa to benzalkonium chloride (BC) were studied. The effluence of cell components was observed in susceptible P. aeruginosa by electron microscopy, but resistant P. aeruginosa seemed to be undamaged. No marked changes in cell surface potential between Escherichia coli NIHJC-2 and a spheroplast strain were found. The contents of phospholipids (PL) and fatty and neutral lipids (FNL) in the cell walls of resistant P. aeruginosa were higher than those in the cell walls of susceptible P. aeruginosa. The amounts of BC adsorbed to PL and FNL of cell walls of BC-resistant P. aeruginosa were lower than those for BC-susceptible P. aeruginosa. Fifteen species of cellular fatty acids were identified by capillary gas chromatography and gas chromatography-mass spectrometry. The ability of BC to permeate the cell wall was reduced because of the increase in cellular fatty acids. These results suggested that the resistance of P. aeruginosa to BC is mainly a result of increased in the contents of PL and FNL. In resistant P. aeruginosa, the decrease in the amount of BC adsorbed is likely to be the result of increases in the contents of PL and FNL.

Differential inhibitory effects of various herb extracts on the activities of reverse transcriptase and various deoxyribonucleic acid (DNA) polymerases.

Forty preparations of the extracts from 28 kinds of Asian herbs were tested for ability to inhibit the activities of murine retroviral reverse transcriptase and human deoxyribonucleic acid (DNA) polymerases. Among the 40 extracts, 35 inhibited reverse transcriptase activity and 29 inhibited DNA polymerase alpha activity. The inhibitory potencies of these extracts were expressed as the 50% inhibition concentrations (IC50), at which the enzyme activities were inhibited by 50%. Very strong inhibitions were observed with the extracts from Millettia pachycarpa (Leguminosae) and Mallotus apelta (Euphorbiaceae) as shown by their low IC50 values for reverse transcriptase (0.4-0.5 micrograms/ml) and DNA polymerase alpha (0.9-1.4 micrograms/ml). Enzyme kinetic analysis revealed that the mode of inhibition of reverse transcriptase by these two extracts was competitive with respect to the template.primer [poly(rA).oligo(dT)] and noncompetitive with respect to deoxythymidine triphosphate (dTTP) substrate. Besides reverse transcriptase and DNA polymerase alpha, DNA polymerase I and ribonucleic acid (RNA) polymerase from E. coli were inhibited by these two extracts. These results indicate that the herb extracts contain as yet unidentified substance(s) which inhibit the activities of reverse transcriptase and cellular DNA polymerases.

The mutagenicity of refuse leachate from a municipal incinerator.

The mutagenicity of refuse leachate from a municipal incinerator was studied by liquid rec-assay and Ames assay. Volatile components were found to be negative, and nonvolatile components positive, in the Ames assay and the leachate was found to have DNA-damaging capacity in the liquid rec-assay with S-9 mix. PAHs derived from tobacco ash and carbonyl compounds generated by the putrefaction of foods were confirmed to be main contributors to DNA-damaging capacity and mutagenicity in refuse leachate.

Effects of medicinal plant extracts from Chinese herbal medicines on the mutagenic activity of benzo[a]pyrene.

The effects of medicinal plants on the mutagenicity of benzo[a]pyrene were studied with Salmonella typhimurium tester strains. The chosen medicinal plants are very frequently used as Chinese herbal medicines. Each medicinal plant was extracted with hot water, which is similar to the method used in Chinese medicinal treatment. Cinnamomi cortex, Rhei rhizoma, Scutellariae radix and Rehmanniae radix were found to decrease the mutagenic activity of benzo[a]pyrene. Atractylodis rhizoma also reduced the mutagenicity of benzo[a]pyrene, but this was not certain, because it showed a killing effect on the cell survival test. Bupleuri radix and Aurantii nobilis pericarpium had an enhancing effect, but then neither of these extracts is itself mutagenic. Each medicinal plant extract showed a different effect on the mutagenicity of benzo[a]pyrene. These effects were classified into 5 types: (I) decreasing effect, (II) killing effect, (III) enhancing effect, (IV) enhancing and decreasing effect and (V) inactive.

The evaluation of genotoxic activities of disinfectants and their metabolites by umu test.

The genotoxic potential of 6 disinfectants and their 9 metabolites was investigated by umu test. In the tested disinfectants, glutaraldehyde showed positive genotoxicity independent of metabolic activation system and acrinol was positive only in the presence of S9 mixture. Alkylaminoethylglycine, benzalkonium chloride, chlorhexidine digluconate and methylrosaniline chloride were negative in the presence or absence of S9 mixture. In some metabolites of benzalkonium chloride, chlorhexidine digluconate or glutaraldehyde, only pyrogallol showed positive genotoxicity in the absence of S9 mixture and the activity was not affected by the metabolic activation system. Aniline, p-chloroacetoanilide, p-chloroaniline, p-chlorophenol, decabutyldimethylamine, glutaric acid, phenol and pyrocatechol did not induce umu gene expression independently of the presence of S9 mixture. The results in the umu test of these compounds were compared with their findings in the liquid rec-assay and Ames test. The umu test is a more useful and simplified method for the detection of genotoxicity of the compounds with killing effects on tester bacteria.

Distribution of silicones in water, sediment and fish in Japanese rivers.

A new analytical method for assaying polyorganosiloxanes (silicones) in environmental samples is presented. The method utilizes petroleum for solvent extraction of the sample (water, sediment, biological tissue) together with inductively coupled plasma detection of the extracted organic silicones. The detection limit for silicones in the final methyl isobutyl ketone sample/extract is approximately 0.01 ppm, and the method is applied to samples from various Japanese rivers to quantify silicones in several environmental materials. Silicones are reported from river waters (up to approximately 50 ppb), riverine sediments (up to 6 ppm), and as an extractable component of fish tissue (up to approximately 0.9 ppm).

Possible methylation of inorganic mercury by silicones in the environment.

Organosiloxanes (silicones) are being released to the environment in increasing quantities, and there are questions concerning the reactivity of silicones as methylating agents under environmental conditions. Specifically, the interaction of inorganic mercury and polydimethylsiloxanes, and other silicones, to give methylmercury is important in this content. To this end a series of laboratory experiments, in which inorganic mercury, short and long chain silicones, cyclic siloxanes, oxidized silicone gums, and industrial formulations containing silicones, were conducted. In all cases, there was evidence of the production of methylmercury, a finding of potential significance. Analyses for methylmercury and related silicon-containing degradation products were conducted using a combination of gas chromatography, mass spectrometry, and head-space gas analysis.

Isolation of dinitropyrene in emission gas from a municipal incinerator and its formation by a photochemical reaction.

In a previous paper, direct-acting mutagenicity was reported in emission gas from incomplete municipal incineration using Salmonella typhimurium TA-98 and TA-100. This paper reports the detection of dinitropyrene (DNP) as a direct-acting mutagen using nitrogen selective gas chromatography. The gas-phase photochemical reaction of pyrene with nitrogen dioxide was examined in a quartz vessel with various reaction times and temperatures. 1-Nitropyrene (1-NP) was readily formed from pyrene in the absence of light irradiation and at low temperature, but DNP was not formed under similar conditions. DNP formation was catalyzed by nitric acid. The formation of DNP is dependent on light irradiation, temperature and HNO3 as catalyst. In a combustion source these factors affect the formation of DNP.