Some aspects of the detection of potential mutagenic agents in Drosophila.

The Drosophila system is a valuable test for detecting and characterizing mutagenic agents. Tester strains are available or can be synthesized for determining almost all types of genetical change ranging from gene mutations to chromosome rearrangements in a great variety of cell types of both sexes. Metabolic activation of all groups of indirect mutagens tested so far (aryldialkyltriazenes, cyclophosphamides, nitrosamines, azo-, hydrazo- and azoxyalkanes, aflatoxins, and polycyclic hydrocarbons; about 35 representatives in all), gives strong although indirect support for the considerable metabolizing ability of Drosophila. This capability would be expected from comprehensive biochemical data on bioactivation of foreign compounds in other insects. From a comparison of which types of genetical change are induced at high, low and threshold concentrations, it appears that lethal tests remain the most reliable method for any screening program. Mutagenic agents such as diethylnitrosamine, hycanthone and certain triazenes, which are highly efficient in the induction of recessive lethals (gene mutations and/or deficiencies), would not have been detected in Drosophila if chromosome breakage were the only indicator for mutagenic activity. Moreover, for several mono- and polyfunctional agents, the lowest dose which is still genetically active was definitely lowest for recessive lethals when compared with dominant lethals, chromosome rearrangements or loss. If a new mutagen is discovered by a screening procedure using Drosophila, an accurate picture of its ability to cause either or both gene mutations and chromosome aberrations can be drawn. Such work will be valuable in helping to clarify similar problems in mammalian systems. For instance, it was important to learn that mutagens of the nitrosamine type apparently fail to produce breakage events in Drosophila. Similarly, three cyclophosphamides appeared not to have chromosome breaking ability. However, from a more detailed study, in which a series of concentrations was used, it became obvious that a penetration effect or, more likely, a rate-limiting factor in bioactivation, was the cause of the negative results obtained with these agents.     

Relative mutagenic potencies determined in the micronucleus test

Data from two publications which include dose-response data were subject to model fitting using a Poisson distribution and a linear dose-response function. In Swiss mice, acetylaminofluorene was the weakest mutagen tested. Aflatoxin B₁, 3-methylcholanthrene and dimethylnitrosamine were 66, 14, and 140 times as potent, respectively. In hamsters aflatoxin B₁ was inactive while 3-methylcholanthrene and dimethylnitrosamine were 21 and 246 times as potent as acetylaminofluorene. EMS was the weakest mutagen tested by Matter & Grauwiler with colchicine and TEM being 232 and 3294 times as potent. Trenimon, cyclophosphamide and MMS were best approximated by a quadratic function due to their pronounced bone marrow toxicity. Neither MNNG nor caffeine were active in this test.     

The mutagenic effect of benzene, toluene and xylene studied by the SCE technique.

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

Exposure to mutagenic airborne particulate in a rubber manufacturing plant

Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer. Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions. The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes. The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant. Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas. Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding). The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation. Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS). The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples. No mutagenic activity was present in S. typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer. HPLC analysis revealed very low concentrations of PAHs. GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate. We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations. PAHs were not involved in indoor pollution. GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes. These substances are potential carcinogen though their mutagen properties have not been clearly determined.     

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A_L). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59⁻ and CD59⁺ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.     

Chemical characterization of a dye processing plant effluent--identification of the mutagenic components

This work shows the chemical characterization of a dye processing plant effluent that was contributing to the mutagenicity previously detected in the Cristais river, S?o Paulo, Brazil, that had an impact on the quality of the related drinking water. The mutagenic dyes Disperse Blue 373, Disperse Orange 37 and Disperse Violet 93, components of a Black Dye Commercial Product (BDCP) frequently used by the facility, were detected by thin layer chromatography (TLC). The blue and orange dyes were quantified by high performance liquid chromatography (HPLC/DAD) in a raw and treated effluent samples and their contribution to the mutagenicity was calculated based on the potency of each dye for the Salmonella YG1041. In the presence of S9 the Disperse Blue 373 accounted for 2.3% of the mutagenic activity of the raw and 71.5% of the treated effluent. In the absence of S9 the Disperse Blue 373 accounted for 1.3% of the mutagenic activity of the raw and 1.5% of the treated effluent. For the Disperse Orange 37, in the presence of S9, it contributed for 0.5% of the mutagenicity of the raw and 6% of the treated effluent. In the absence of S9; 11.5% and 4.4% of the raw and treated effluent mutagenicity, respectively. The contribution of the Disperse Violet 93 was not evaluated because this compound could not be quantified by HPLC/DAD. Mutagenic and/or carcinogenic aromatic amines were also preliminary detected using gas chromatograph/mass spectrometry in both raw and treated and are probably accounting for part of the observed mutagenicity. The effluent treatment applied by the industry does not seem to remove completely the mutagenic compounds. The Salmonella/microsome assay coupled with TLC analysis seems to be an important tool to monitor the efficiency of azo dye processing plant effluent treatments.     

Studies on novel D-ring substituted steroidal pyrazolines as potential anticancer agents

An efficient and facile synthesis of 17-pyrazolinyl derivatives of pregnenolone and their evaluation as potential anticancer agents against various human cancer cell lines are reported. The scheme involves the transformation of the starting pregnenolone acetate into pregnenolone, conversion of pregnenolone to the corresponding benzylidine derivatives and finally the conversion of this derivative to the stable steroidal 17-pyrazoline. Various compounds 4b, 4c, 4e, 4f, 4h and 4j showed significant cytotoxic activity especially against HT-29, HCT-15, 502713 cell lines.     

Human lymphocytes as target cells in a metabolizing test system in vitro for detecting potential mutagens.

A metabolizing test system is presented that comprises mouse liver microsomes for metabolic activation of the test compound, NADPH as co-factors, dimethylnitrosamine (DMN) as model mutagen and human peripheral lymphocytes as target cells. Lymphocytes were cultured in RPMI 1640 medium in the presence of phytohemagglutinin for 48 h before being used in the system. Incubation of the lymphocytes for 45 min in the metabolically active system reduced thymidine incorporation by 50%, whereas in the metabolically inactive system without NADPH thymidine incorporation was reduced by 15%. The latter reduction was due to the cytotoxic effects of the mouse liver microsomes and of DMN. When the lymphocytes were cultured for another 24 h after exposure to the metabolizing system and DMN, significant increased formation of chromosomal aberrations such as gaps were observed, cross-overs and breaks being less frequent.     

Salmonella typhimurium as a test system for detecting the mutagenic activity of environmental pollutants. I. The mutagenic action of heavy metal salts in in vivo and in vitro systems without metabolic activation]

The work presents the data on mutagenic effects of heavy metal salts (Zn and Cd) on Salmonella typhimurium test strains using mutagenicity test in vitro without metabolic activation and host-mediated assay. The techniques used enabled to determine also the types of mutations arising from the exposure to ZnCl2 and CdCl2.     

化工厂生产废水致突变性研究

本文就合肥某化工厂生产用水、氯乙烯工段生产废水、化工厂总排污渠污水、南淝河化工厂下游水、化工厂深井水及阴性对照双蒸水进行了有机浓集物的致突变性研究,Ａｍｅｓ试验结果表明：南淝河水在化工厂用于生产之间已受到致突变性污染,ＴＡ９８＋Ｓ９和ＴＡ９８－Ｓ９均出现阳性,化工厂总排污渠污水ＴＡ９８＋Ｓ９呈阳性且有增强趋势,氯乙烯工段生产废水和南淝河化工厂下游水ＴＡ９８＋Ｓ９也呈阳性。深井水和双蒸馏水ＴＡ９８＋     

Evaluation of the mutagenic potential of yangambin and of the hydroalcoholic extract of Ocotea duckei by the Ames test

Ocotea duckei Vattimo is a plant popularly known as "louro-de-cheiro" found in the northeast of Brazil. Traditional medicinal uses of this plant are not known, but recent pharmacological studies with the isolated major constituent yangambin have shown various qualities: platelet activating factor (PAF) antagonist, protective effects against cardiovascular collapse and anaphylactic shock, anti-allergic properties, analgesic activity, and depressant effect in the central nervous system. In this work, the Ames test was used to evaluate the mutagenic potential of the hydroalcoholic extract of O. duckei leaves and of yangambin. Using TA97a, TA98, TA100, TA102 and TA1535 strains of Salmonella typhimurium, positive results were obtained only with the hydroalcoholic extract, with or without metabolic activation. Yangambin was not mutagenic, which is of interest due to its pharmacological properties.