Influence of the microsomal inducer and the incubation system on mutagenicity of complex mixtures

The mutagenicity of SRM 1649 and 1650 was tested in the presence of rat liver S9 mix which was induced by polychlorinated biphenyl (PCB) or by the combination of phenobarbital and 5,6-benzoflavone. The S9 mix induced by PCB activated benzo[a]pyrene strongly. The S9 mix induced by phenobarbital-5,6-benzoflavone activated the complex mixtures to approximately the same extent as that induced by PCB. This finding indicates that phenobarbital-5,6-benzoflavone instead of PCB may be suitable as an inducer under some conditions.The preincubation procedure for the mutagenicity test was performed by preincubating the test compound, S9 mix and bacteria for 20 min in a water bath. This procedure was as effective as the plate incorporation test.     

The use of the Salmonella/microsomal assay to determine mutagenicity in paired chemical mixtures.

The mutagenicities of two sets of chemicals acting singly and in pairwise combinations were determined by use of the Salmonella/microsomal assay. The first set consisted of the promutagens of benzo(a)pyrene and benzo(rst)pentaphene. The second set contained the direct-acting mutagens methyl-nitro-nitroso-guanidine and ethyl methane sulfonate. In the tests with the promutagens, the quantities of S-9 mix were varied over the range of 0.05 ml to 1.0 ml with increasing quantities of each chemical. The mutagenic responses or production of revertant colonies of the promutagens, acting singly and in pairwise combinations failed to show an additive effect. Excess quantities of S-9 mix appeared to inhibit partially or totally the mutagenic activity of each chemical, although for each particular dose there was an optimal quantity of S-9 mix to induce maximum activity. However, the direct-acting mutagens produced, individually, almost linear dose responses with increasing concentrations. In pairwise combinations, these chemicals also showed linear responses that closely approximated the theoretical additivity indicating that the mutagenicity of the mixtures was the sum of the activities of each component.     

Design and implementation of a collaborative study of the mutagenicity of complex mixtures in Salmonella typhimurium.

In 1987, the International Programme on Chemical Safety (IPCS) in collaboration with the U.S. Environmental Protection Agency (U.S. EPA) and the U.S. National Institute of Standards and Technology (U.S. NIST) initiated an international collaborative study of the mutagenicity of complex environmental mixtures in the Ames Salmonella typhimurium mutation assay. The objectives of this study were: (1) to estimate the inter- and intra-laboratory variability associated with the extraction of mixtures for bioassay, (2) to estimate the inter- and intra-laboratory variability associated with the Salmonella typhimurium bioassay when applied to complex mixtures, and (3) to determine whether standard reference complex mixtures would be useful in mutagenicity studies and to evaluate whether reference or certified mutagenicity values determined from this collaborative study should be reported. The complex mixtures used in this study were selected from standard reference materials (SRMs) which had previously been issued by the U.S. NIST as SRM 1597 (coal tar), SRM 1649 (diesel particulate matter) and SRM 1650 (urban air particulate matter) with certified values for polycyclic aromatic hydrocarbons. These SRM complex mixtures are available to scientists as reference standards for analytical chemistry research and are under consideration as SRMs for mutagenicity studies of complex environmental mixtures. This paper briefly describes the final study design, protocol, selection of the complex mixtures, and implementation of this international study.     

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

Influence of sex and inducer treatment on the high- and low-affinity forms of hepatic microsomal erythromycin N-demethylase in rats

To study the regulation of the multiple forms of erythromycin N-demethylase, we determined the influence of sex and inducer treatment on this mixed-function oxidase activity in adult Wistar rats injected intraperitoneally once daily for 4 days with dexamethasone, pregnenolone-16 alpha-carbonitrile, phenobarbital, or 2% Tween 80 (control). Based on the results from a computer curve-fitting procedure (ENZFITTER) as well as Eadie-Hofstee and Lineweaver-Burk plots, at least two forms of erythromycin N-demethylase were present in control, dexamethasone, pregnenolone-16 alpha-carbonitrile, and phenobarbital-treated male rats and in control and dexamethasone-treated female rats. Only a high-affinity form was apparent in pregnenolone-16 alpha-carbonitrile and phenobarbital-treated female rats. Therefore, more than one form of erythromycin N-demethylase exists in hepatic microsomes from adult rats, depending on sex and inducer treatment. As well, at a substrate concentration commonly used in the erythromycin N-demethylase assay, the relative contribution of the high- and low-affinity forms to the enzyme activity varies with sex and inducer treatment.     

Influence of the Uvr repair system on the mutagenicity of antiparasitic drugs

One amebicide (chloroquine diphosphate) and 2 anthelmintic compounds (niclosamide and pyrvinium pamoate) were found to be mutagenic for Salmonella typhimurium TA1537, TA1538, TA100 and TA98 Uvr- strains respectively. Drugs tested on homologous Uvr+ strains (TA1977, TA1978, UTH8414 and UTH8413) showed decreased mutagenic activity of the compounds. This indicates that premutational damage induced by the drugs was totally or partially repaired. Furthermore, results obtained in the present study suggest that niclosamide and pyrvinium pamoate induce premutational lesions by adduct formation, and that chloroquine diphosphate, known as an intercalating agent, behaves as an adduct-forming compound as regards its effects on Uvr- and Uvr+ S. typhimurium strains.     

Salmonella mutagenicity of three complex mixtures assayed with the microsuspension technique. A WHO/IPCS/CSCM study.

In a collaborative study on complex mixtures, three complex mixtures and two pure compounds were assayed with the Salmonella microsuspension technique. The two pure compounds were benzo[a]pyrene (BaP) and 1-nitropyrene (1-NP). The three complex mixtures were standard reference materials (SRMs) from the U.S. National Institute of Standards and Technology, SRM 1649, SRM 1650 and SRM 1597. The two samples SRM 1649, an urban dust particulate matter, and SRM 1650, a diesel particulate matter, were sonicated with dichloromethane. Sample SRM 1597 was an extract of a coal tar sample with a complex mixture of polycyclic aromatic hydrocarbons. The microsuspension assay was performed with Salmonella strains TA98 and TA100 according to Kado et al. (1983) with minor modifications (L?froth et al., 1988). The results showed that the microsuspension technique is a more sensitive assay than the plate incorporation method. Depending on sample, strain and metabolic condition the mutagenic responses were 3-37 times higher in the microsuspension assay than in the conventional plate incorporation assay. The microsuspension method is thus useful for environmental samples which are often available in only small amounts.     

Influence of oxygen on the microsomal electron transport system in Saccharomyces cerevisiae

The influence of oxygen on the level of microsomal electron transport chain components has been studied during the growth of Saccharomyces cerevisiae. Enzyme activities and cytochrome content were assayed in microsomal fractions prepared from a protoplast lysate free from mitochondrial contamination. It was found that the cytochrome P-450 and cytochrome b5 content, to get her with the NADPH-cytochrome (P-450)-reductase and NADH-cytochrome (b5)-reductase activities, were increased in the cells as the pO2 of the medium was decreasing. At the same time an increase in the membrane surface of the endoplasmic reticulum can be observed.     

Effect of pregnenolone-16alpha-carbonitrile, a microsomal enzyme inducer, on the regenerating rat liver.

PCN, a microsomal enzyme inducer, given orally (10 mg in 1 ml water twice daily for 5 days), increased liver weight and mitotic activity in intact as well as in partially hepatectomized rats. Electron microscopy revealed SER proliferation in the hepatocytes of animals treated with PCN alone. Accumulation of SER membranes was also evident in the liver cell cytoplasm of untreated, partially hepatectomized rats; it was however, more pronounced in the hepatocytes of partially hepatectomized animals given PCN. These results indicate that the steriod has a marked effect on the regeneration rat liver.     

Mutagenicity studies on complex environmental mixtures: selection of solvent system for extraction.

The mutagenic activities in the Salmonella/microsome assay of dichloromethane (DCM) and acetone extracts of complex environmental mixtures were compared. The particulate samples used in the IPCS collaborative study were Soxhlet-extracted twice with DCM followed by a third extraction with acetone. Compared with the mutagenic activity of the first extract, the third (acetone) extract of the urban particulate matter showed a relatively high mutagenic activity. In contrast to this the third extract of the diesel particulate matter contributed very little additional mutagenic activity. Furthermore, 10 filter samples of air particulates from a suburban airport area were collected for comparison of the extraction efficiency of DCM and acetone. Each sample was divided into two samples of identical size followed by extraction with acetone and DCM, respectively. No clear difference in the mutagenic activity of these extracts was observed in strains TA98 and TA98NR. It is concluded that for ambient air particulates (but not emission samples) acetone may extract some mutagenic compounds which are not extracted by DCM. The amount of these additional extractable compounds seems to depend on the composition of the sample. As DCM extracts are better suited for further fractionation and chemical analysis DCM is considered to be the best choice for a general solvent system for extraction of complex environmental mixtures.     

Results of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples--an air-particulate sample and a diesel-particulate sample--and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 17.5%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, +S9) to 6697 (TA100, +S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.     

Methyl iodide, a potent inducer of the adaptive response without appreciable mutagenicity in E. coli

Methyl iodide (MeI), a very weak mutagen, induced the adaptive response in E. coli to a similar extent to those induced by potently mutagenic methylating agents. MeI potentiated the mutagenicity of a methylating mutagen, N-methyl-N-nitrosourea, by its co-treatment. These results might give indication that MeI directly methylates O6-methylguanine-DNA methyltransferase resulting in induction of the adaptive response and depletion of the repair capacity of enzyme.     

Influence of incubation conditions on bacterial production estimates in an estuarine system

This study aimed to assess the influence of incubation conditions in the determination of bacterial production (BP). In order to achieve that goal, experimental setups were performed in situ and in the laboratory under both dark and light conditions. To test spatial and seasonal variations and the different natural light exposure of microorganisms, sampling was performed in two distinct zones of the estuary Ria de Aveiro (Portugal), typifying the marine and brackish water zones of the estuarine system. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments was used to monitor possible alterations in bacterial community composition induced by the incubation conditions. The results showed that BP determined in situ conditions significantly differed from in the laboratory. In the marine zone, a defined pattern of variation was detected, with consistent higher values of BP in laboratory dark conditions. This trend was not present in the brackish water zone. The seasonal and spatial variability of BP observed in field incubations was related to the physical–chemical proprieties of the water column, irradiance levels and the original community composition. The metabolic active profiles of bacteria were substantially different in the several incubation conditions, suggesting that methodological procedure influences the bacterial community composition, and the values of BP reported for aquatic ecosystems could be quite different from the real ones. In the light of these results, we suggest that BP determinations should be conducted under in situ conditions. However, due to execution limitations, BP needs to be frequently determined in the laboratory, and in this case, dark incubations provide more approximate values. This is the method routinely used, and although this incubation condition can cause stimulation of BP, the structure of the bacterial community is more similar to the one obtained with the in situ incubations.     

Studies on the incubation mixtures for the in vitro mutagenesis test with metabolic activation (microsomal assay). Behaviour of mixed function oxidase and lipid peroxidation in the presence of some xenobiotics.

The effect of some xenobiotics on microsomal mixed function oxidase and lipid peroxidation, in mice, in incubation mixtures for the in vitro mutagenesis test with metabolic activation was studied. Aniline 1 or 2 mM and aminopyrine 0.38 or 8.33 mM completely inhibited the lipid peroxidation with small protection of the monooxygenase. Styrene 50 or 100 mM inhibited to a lesser extent the lipid peroxidation with marked increase in the inactivation of the monooxygenase. By a technique based on successive additions of fresh microsomes it was possible to evaluate the part of the inactivation due to enzyme denaturation and that due to inhibition. EDTA 40 mM was not able to protect from inactivation in the presence of aniline 1mM. Data of this type could be utilized to obtain more reliable results of in vitro mutagenesis tests with metabolic activation by suitably managing the enzyme activity in the incubation mixtures in order to keep it as constant as possible.     

Studies on the mutagenicity of p-phenylenediamine in Salmonella typhimurium. Presence of PCB's in rat-liver microsomal fraction induced by Aroclor.

The mutagenicity of fresh solutions of p-phenylenediamine (PPD) and Aroclor 1254 was investigated. The histidine-requiring strains of Salmonella typhimurium were used in the absence and presence of uninduced and/or Aroclor-induced rat-liver homogenate. The presence of polychlorinated biphenyls (PCBs) was also examined by chromatographic methods in Aroclor-induced rat-liver homogenate. In the absence of metabolic activation, as well as in the presence of uninduced rat-liver homogenate, PPD was not mutagenic in the strains used. In the presence of Aroclor-induced S9 a twofold increase (or less) was observed in the number of revertant colonies over those of the controls in TA1538 and TA98. There was no increase in the number of revertant colonies over those of the controls when PPD was dissolved in NH4OH solution and the solution mixed with H2O2 before the addition of S9 mix. Aroclor 1254 was not mutagenic in TA1538 or TA98. However, the presence of PCBs in Aroclor-induced rat-liver homogenate (induced S9) was identified by gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) and gas--liquid chromatography/mass spectrometry (GC/MS).     

Influence of incubation period on the plasmid content of proliferating Nocardioides sp.

S. SANDHYA. 1996. Nocardioides sp., isolated for organic sulphur degradation, harbours catabolic plasmid pSB1 of molecular weight 34.2 kb. A correlation was noticed between the plasmid content and degradation of the organic sulphur compound dibenzothiophene (DBT). The maximum oxidation of DBT was only after 64 h, during which the content of plasmid DNA was also optimum.