Activation of the food-derived mutagen 2-amino-3-methylimidazo[4, 5-f]quinoline by human-liver microsomes

The ability of human-liver microsomes to metabolically activate the food-derived heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and the model mutagen, 2-aminofluorene (AF), has been investigated using Salmonella typhimurium TA98. In 6 subjects tested the number of revertants produced by 0.1 micrograms IQ per mg microsomal protein varied from 11, 830 +/- 320 to 42, 830 +/- 290 (mean +/- SD). With the same livers and a dose of 10 micrograms AF per plate the number of revertants varied from 15,770 +/- 1600 to 29,380 +/- 810 per mg microsomal protein. Metyrapone and alpha-naphthoflavone caused differential inhibition of the mutagenesis of both IQ and AF indicating the involvement of different forms of cytochrome P450 in the metabolic activation of these amines in human-liver microsomes. In presence of human-liver microsomes IQ produced no detectable increase in mutations at the hypoxanthine phosphoribosyl transferase locus in lymphocytes and caused no increase in micronuclei formation at realistic exposure levels.     

Chemical basis for photomutagenicity in synthetic fuels and correlation with carcinogenicity

Photomutagens (chemicals that enhance the mutagenicity of non-ionizing radiation) have been detected in experimental coal- and oil shale-derived synthetic fuel samples using Salmonella typhimurium strain TA98 and fluorescent light. In this study, photomutagenic activity was measured among distillation and chemical class fractions from a blend of direct coal liquefaction process materials. Photomutagenicity increased with increasing boiling point and was concentrated in fractions enriched in neutral polycyclic aromatic compounds (neutral PACs). The photomutagenic activities of the materials tested correlate well with the previously reported tumorigenic activities of the same samples on mouse skin, but correlate poorly with the previously reported mutagenic activities of the same samples in the Salmonella/mammalian-microsome test (using strain TA98), in which neutral PAC-enriched fractions were not active. These data suggest that relatively high boiling neutral PACs are important chemical photomutagens in synthetic fuels and suggest the potential use of the photomutation assay as an improved, relatively simple, inexpensive and short-term bioassay for detecting carcinogens as mutagens in complex mixtures such as synthetic fuels.     

Microbial utilization of raw and hydrogenated shale oils.

The biodegradability of raw and hydrogenated shale oils prepared by a retort process were studied under psychrophilic and mesophilic conditions. Changes in bacterial numbers and the chemical composition of the oils were monitored using a plate count and chromatographic techniques respectively. Raw shale oil was found to be relatively resistant to microbial attack whereas hydrogenated shale oil was readily utilized for microbial growth. Populations enriched on raw shale oil had a reduced ability to use hydrogenated shale oil under similar conditions. Gram-negative rods predominated in all enrichment populations. It is recommended that to facilitate clean up of shale oil spills, raw shale oil be reduced at the extraction site before transport.     

Metabolic activation of 10-aza-substituted benzo[a]pyrene by cytochrome P450 1A2 in human liver microsomes

We previously reported that 10-azabenzo[a]pyrene (10-azaBaP), a 10-aza-analog of BaP and an environmental carcinogen, showed greater mutagenicity than BaP in the Ames test using pooled human liver S9. To investigate the cytochrome P450 (CYP) isoform involved in the activation of 10-azaBaP to the genotoxic form, the mutagenicity of 10-azaBaP using nine individual donors' and pooled human liver microsome preparations was compared with each CYP activity. Induced revertants by 2.5 nmol per plate 10-azaBaP with 0.5 mg per plate human liver microsomal protein showed a large inter-individual variation (42-fold) among the nine donors. The number of induced revertants highly correlated with the CYP1A2-selective catalytic activity from each microsome preparation, and no correlation was observed with other CYP isoform-selective catalytic activities. Moreover, recombinant human CYP1A2 contributed to the mutagenicity of 10-azaBaP more markedly than recombinant human CYP1A1. These results suggest that CYP1A2 may be the principal enzyme responsible for the metabolic activation of 10-azaBaP in human liver microsomes. With regard to the proposal that BaP may be activated by human CYP1A1, our results suggest that the nitrogen-substitution at position-10 of BaP may cause the CYP enzyme-specificity in metabolic activation to change from CYP1A1 to CYP1A2.     

Analytical and biological analyses of test materials from the synthetic fuel technologies. I. Mutagenicity of crude oils determined by the Salmonella typhimurium/microsomal activation system.

We have assayed the mutagenicity of crude industrial products and effluents with the Salmonella/microsomal activation system. Test materials (crude products from coal-conversion processes and natural crude oils) were initially fractionated into primary classes by liquid--liquid extraction and then further fractionated by column chromatography. Prescreening was accomplished over a wide concentration range with the Ames tester strains. Active fractions (mainly the neutral fractions containing polycyclic aromatic hydrocarbons and certain basic fractions) can be identified, and dose--response relationships can be established. Standard values are expressed as revertants/mg of the test material assayed with frameshift strain TA98 including metabolic activation with rat-liver preparations. Total mutagenic activity of synthetic fuel samples was consistently higher than that of natural crude "controls." Activities of subfractions are roughly additive and presumably reflect the mutagenic potential of the whole test material. These results are being extended to other genetic assays. Chemical identification is carried out along with the bioassays.     

Effect of phenolic antioxidants on the mutagenicity of aflatoxin B1.

The Ames assay employing Salmonella typhimurium TA100 and TA98 was used to investigate potential interactions between aflatoxin B1 (AFB1) and the phenolic antioxidants butylated hydroxytoluene, butylated hydroxyanisole, and propyl gallate. AFB1 doses were within the linear response range, and the antioxidants were used at levels of 0 to 50 micrograms per plate. All three antioxidants were nonmutagenic in either bacterial tester strain, with or without the hepatic S-9 enzyme preparation; toxic effects were observed at doses higher than 20 micrograms per plate. Butylated hydroxytoluene and butylated hydroxyanisole substantially increased AFB1-induced mutagenesis in the two tester strains with microsomal activation. The addition of 5 to 20 micrograms of butylated hydroxytoluene or hydroxyanisole to 5 to 20 ng of AFB1 per plate caused more than a twofold increase in the number of His+ revertants. Addition of propyl gallate resulted in only a moderate increase in the number of revertants. Whereas several anticarcinogenic and antimutagenic effects by phenolic antioxidants have been reported, particularly in studies with polycyclic aromatic hydrocarbons, the enhancement of mutagenic potency of AFB1 by these compounds suggests a specificity with respect to the chemical nature of AFB1.     

Estimation of the dermal carcinogenic activity of petroleum fractions using a modified Ames assay

The Ames Salmonella/microsomal activation mutagenesis assay has been adapted to improve sensitivity to complex hydrocarbon mixtures produced by the refining of petroleum. Extraction of oil samples with dimethyl sulfoxide produces aqueous-compatible solutions that more easily interact with the tester bacteria. These extracts, therefore, produce higher revertant values than do equivalent volumes of oil delivered neat or dissolved in organic solvent. Parallel increases in the liver microsomal S-9 concentration further improve the sensitivity of the assay, allowing detection of mutagenicity in otherwise inactive samples. The effect of increased microsomal fraction from rodent liver is apparently attributable to the higher levels of activating enzymes rather than to the concomitant increase in the overall hydrophobicity of the test system. The modified assay has been used to rank thirteen petroleum-derived oils and a corn oil control for relative mutagenic activity. This ranking closely correlates (r = 0.97) with potency rankings of the same samples previously determined from dermal carcinogenicity bioassays.Abbreviations DMSO dimethyl sulfoxide - S-9 Microsomal fraction from rodent liver - 2-AA 2-aminoanthracene - BaP benzo(a)pyrene - NADP nicotinamide adenine dinucleotide phosphate - DMF dimethyl formamide - EGDE ethylene glycol dimethyl ether     

Mutagenic potential of toluidine blue evaluated in the Ames test.

Toluidine blue is a vital, metachromatic thiazine dye which is used as an adjunct in clinical examination for the early detection of asymptomatic recurrent or secondary primary carcinoma in individuals who are at high risk for developing oral cancer. Because available data on the mutagenicity of toluidine blue was limited and contradictory, this study was conducted to evaluate the mutagenic potential of toluidine blue in the in vitro Ames Salmonella test. Tester strains TA97a, TA98, TA100 and TA102 were used. Toluidine blue was tested at concentrations of 0.1, 1.0, 10, 50, 100, 250 and 500 micrograms/plate, with and without S9 microsomal activation, and positive and negative controls were included. Results from tests without S9 showed a significant increase (p less than 0.05) in number of revertants in TA102 and in TA97a with 50 and 100 micrograms toluidine blue/plate, respectively. In tests with S9 activation, doses of toluidine blue ranging from 10 to 250 micrograms/plate induced dose-related increases in the number of revertants in all 4 strains. The results of this study indicate that toluidine blue has a mutagenic effect in the Ames test.     

Predicting carcinogenicity of petroleum distillation fractions using a modified Salmonella mutagenicity assay

The Ames Salmonella/microsomal activation mutagenesis assay has been modified to improve sensitivity and reproducibility to complex mixtures derived from the refining and processing of petroleum. Oil samples were dissolved in cyclohexane and subsequently extracted with dimethyl sulfoxide to produce aqueous compatible solutions which readily interact with tester bacteria. Also, the liver homogenate (S-9) and NADP cofactor concentrations were increased and hamster rather than rat liver S-9 was used. The initial slope of the dose response curve relating mutagenicity (revertants per plate) to the dose of extract added was used as an index of mutagenic activity, this slope was obtained through a computerized curve fitting procedure. The modified assay was used to rank 18 oil samples for mutagenic activity, this ranking correlates highly (r = 0.92) with potency rankings of the same samples previously determined from dermal carcinogenicity bioassays. Sensitivity and reproducibility of the assay are sufficient to permit routine use for detecting potential carcinogenic activity of individual refinery streams and blends which contain components boiling above 500°F.Abbreviations API American Petroleum Institute - B[a]P benzo[a]pyrene - DMSO dimethyl sulfoxide - NADP nicotinamide adenine dinucleotide phosphate - PAH polycyclic aromatic hydrocarbon - S-9 microsomal fraction from rat liver     

Absence of mutagenic and antimutagenic activities of fluoride in Ames salmonella assays

The mutagenicity of fluoride (as sodium fluoride, NaF) was investigated with Ames Salmonella/microsome assays in strains of TA97a, TA98, TA100, TA102 and TA1535. The concentrations of NaF tested ranged from 0.44 to 4421 micrograms/plate (0.1 to 1000 ppm F), both with and without microsome activation. In addition, the suggested antimutagenic effect of fluoride was evaluated with known mutagens at various concentrations of NaF (0.44-442.2 micrograms/plate, 0.1-100 ppm F). The data showed that NaF, in amounts from 0.44 to 442.2 micrograms/plate (0.1-100 ppm F), failed to significantly increase the number of the revertants over the number observed in the solvent (distilled deionized water) controls. Increases of NaF to, and beyond, 1100 micrograms/plate (250 ppm F) resulted in a toxic effect and a reduction of the revertants to various degrees among the strains. NaF in the presence of known mutagens did not significantly decrease the number of the revertants. The results of this study indicate that NaF does not have mutagenic or antimutagenic effects in the strains tested with Ames Salmonella assays.     

A quantitative comparison of a percentile rule with a 2-fold rule for assessing mutagenicity in the Ames assay

Using a model based on the bivariate normal density function, this paper compares the effectiveness of two commonly employed decision rules for assessing mutagenicity in the standard Ames Salmonella assay. The 2-fold method, which considers a compound significantly mutagenic if its mean number of revertants per plate at any dose is equal to or greater than twice the mean number of revertants per plate in the concurrent control, may be a poor indicator of significant mutagenesis. In the percentile method, the frequency of induced mutations for the test compound is tested against the 95th percentile of the accumulated historical data for the spontaneous mutation frequency. As judged by the higher probability of declaring a compound mutagenic that elevates the reversion rate above background, the percentile rule is more reliable than the 2-fold method.     

Evaluating the relationship of metabolic activation system concentrations and chemical dose concentrations for the Salmonella spiral and plate assays

A factorial experimental design was used within this study to evaluate the influence of multiple metabolic activation system concentrations on the dose-response exhibited by promutagens (indirect-acting mutagens) in the Salmonella spiral and plate assays. The mutagenic activity of the three compounds used spanned three orders of magnitude. The mutagenic activity of the compounds ranged from 10 to 100 revertants/micrograms for acetylaminofluorene (2AAF) to more than 1000 revertants/micrograms for 2-aminoanthracene (2AA). Benzo [a] pyrene (BaP) activity was within an intermediate range (100-1000 revertants/micrograms). During a single experiment, a mutagen was tested in TA100 at 13 doses plus a negative control dose. Each dose was tested at 10 S9 concentrations. The S9 concentrations ranged from 0.1 mg protein/plate to 4 mg protein/plate in the standard plate assay and from 0.25 to 4.90 mg-equivalents in the spiral assay. The spiral Salmonella assay, an automated version of the standard assay, generates dose-response data from a concentration gradient on a single agar plate, thereby providing a straightforward approach to this type of study. This study demonstrates not only that even small differences in S9 concentrations can affect the measurement of mutagenic potency but that S9/compound interactions cannot be generalized through the use of interaction studies. This study also shows that spiral assay data and plate assay data for promutagens cannot be compared directly unless the S9 concentrations for all chemical doses are also comparable.     

Effects of the formaldehyde releasing preservatives dimethylol urea and diazolidinyl urea in several short-term genotoxicity tests

The two formaldehyde (FA)-releasers dimethylol urea (DMU) and diazolidinyl urea (DZU) are widely used as preservatives or additives. They were tested for genotoxicity in three short-term test systems, i.e. in the Salmonella typhimurium mutagenicity assay, in the in vitro micronucleus test with V79 Chinese hamster cells and in the in vitro tubulin assembly assay using isolated tubulin from pig brains. The polymerization products obtained in the tubulin assembly assay were examined additionally by electron microscopy.In the S. typhimurium mutagenicity assay with the pre-incubation assay both FA-releasers tested show a clear and concentration-dependent increase in the number of revertants in strains TA98, TA100 and TA102 with and without metabolic activation (rat liver S9 mix). In all cases, a biologically relevant increase in the number of revertants was achieved within the concentration range tested (DZU: 0.04-1.8 micromol per plate, DMU: 0.21-8.33 micromol per plate). FA was tested at 0.06-2.5 micromol per plate and lead to similar effects.Both compounds induce the formation of micronuclei (concentration range tested: DZU: 2.5-50 micromol/l, DMU: 3.3-333 micromol/l). However, DMU shows a comparatively weaker effect exclusively in the absence of the metabolizing enzymes. By contrast, DZU yields a distinct increase of the micronucleus rate in the absence and in the presence of S9. In addition, DZU predominantly causes an increase of large micronuclei, which suggests that this compound has a marked aneugenic potential. Cytotoxic effects accompany the clastogenic effects of both DMU and DZU.The examination of DMU and DZU in view of a possible aneugenic potential in the tubulin assembly assay yielded the following results: DMU at concentrations up to 10 mmol/l did not influence the formation of microtubuli, whereas DZU inhibited this process completely at 3 mmol/l. FA at 6 mmol/l completely inhibited the tubulin assembly. These results could clearly be confirmed by electron microscopy examination. The different potential of the two compounds with respect to the inhibition of tubulin formation is apparently due to a significant difference in the degree of FA release.According to these results, both compounds have to be considered as genotoxic in vitro. On account of these data and because of the widespread use of these two compounds in various products used in daily life, a reevaluation of the risk associated with these compounds seems to be necessary.     

The anti-mutagenic and antioxidant effects of bile pigments in the Ames Salmonella test

The aim of this study was to explore the potential pro- and anti-mutagenic effects of endogenous bile pigments unconjugated bilirubin (BR), biliverdin (BV) and a synthetic, water soluble conjugate, bilirubin ditaurate (BRT) in the Ames Salmonella test. The bile pigments were tested over a wide concentration range (0.01-2 micromol/plate) in the presence of three bacterial strains (TA98, TA100, TA102). A variety of mutagens including benzo[alpha]pyrene (B[alpha]P), 2,4,7 trinitrofluorenone (TNFone), 2-aminofluorene (2-AF), sodium azide (NaN(3)) and tertiary-butyl hydroperoxide (t-BuOOH), were used to promote the formation of mutant revertants. Tests were conducted with (B[alpha]P, 2-AF, t-BuOOH) and without (TNFone, NaN(3), t-BuOOH) metabolic activation incorporating the addition of the microsomal liver preparation, S9. The bile pigments alone did not induce mutagenicity in any of the strains tested (p>0.05). Anti-mutagenic effects of the bile pigments were observed in the presence of all mutagens except for NaN(3) and the anti-mutagenic effects appeared independent of the strain tested. For TNFone induced genotoxicity, the order of effectiveness was BR> or =BRT>BV. However, the order was BV> or =BRT> or =BR for 2-AF. Antioxidant testing in the TA102 strain revealed bile pigments could effectively inhibit the genotoxic effect of t-BuOOH induced oxidative stress. The apparent antioxidant and anti-mutagenic behaviour of bile pigments further suggests their presence in biological systems is of possible physiological importance.     

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation

In order to determine the usefulness of selected chemicals as potential reference materials for calibrating the Salmonella assay, two laboratories tested a series of Salmonella mutagens that require exogenous activation. When the variance for individual substances within a bioassay is sufficiently low and the rankings of those substances are of acceptable consistency, they can later be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The purpose of this project, therefore, was to evaluate the variability in the mutagenic response of potential reference chemicals that require exogenous metabolic activation in the standard plate-incorporation Salmonella mutagenicity assay, and to develop ranking criteria for mutagenic activity based on these data. Ten indirect-acting mutagens were tested in two laboratories using Salmonella typhimurium TA100 and an Aroclor-induced rat liver S9. Each laboratory conducted four definitive testing rounds. A different batch of S9 was utilized for every two rounds. Of the 10 chemicals tested only 2-anthramine had a mean slope value greater than 1000 revertants/micrograms. Three chemicals had slope values between 1000 and 100; and five chemicals had slope values between 100 and 10. The remaining compound, 9,10-dimethyl-1,2-benz[a]anthracene, could not be placed into a single category because it had slope values on either side of 100 revertants per mg. Coefficients of variance were low (i.e., below 25% in most cases). The low variability achieved in this study may be accounted for by two parameters of the study. First, based on Claxton et al. (1991a) and the S9 optimization for three compounds, the amount of S9 was calibrated to a set amount of protein per plate (1.1 mg/plate). Secondly, the 10 test doses were placed in the initial, linear, nontoxic portion of the dose-response curves. The use of ten closely spaced, nontoxic doses allowed for a more accurate estimate of the slope.     

The hair-dye reagent 2-(2',4'-diaminophenoxy)ethanol is mutagenic to Salmonella typhimurium

A new hair-dye ingredient, 2-(2',4'-diaminophenoxy)ethanol (2,4-DAPE), was described as being devoid of any genotoxic activity on the basis of a multi-laboratory study. Since 2,4-DAPE is a close analogue of 2,4-diaminoanisole (2,4-DAA), which is mutagenic and carcinogenic, we tested this claim by assaying 2,4-DAPE for bacterial mutagenicity. Two samples of 2,4-DAPE X 2HCl were synthesized by reduction of the corresponding dinitrophenoxyethanol and identity and purity were established by elemental analysis, NMR spectrometry, mass-spectrometry, UV-spectrophotometry, TLC and HPLC. Fresh aqueous solutions of 2,4-DAPE X 2HCl were assayed in several separate plate tests using S. typhimurium TA1538, TA97, TA98 and TA100, and E. coli WP2uvrA (pKM101), 3 plates per dose and 0%, 4%, 10% and 30% Aroclor 1254-induced rat-liver S9 in S9 mixes. We obtained negative results in TA100 and E. coli. Reproducible, statistically significant dose-related increases in revertants (up to 14 times the background) were obtained in frame-shift mutants of S. typhimurium in the dose range 10-80 micrograms per plate. Mutagenicity was S9-dependent, significant increases in revertants being obtained only with 50 microliter per plate or more of S9. 2,4-DAPE induced significant mutagenic effects at doses of less than 1 micrograms per ml in TA1538 and TA98 in fluctuation tests using 2% S9 in the S9 mix. In plate tests, 2,4-DAPE was less mutagenic (by a factor of about 8) than 2,4-DAA, which gave the highest mutant yields with 20 microliter S9 per plate (4% S9 in the S9 mix). 2,4-DAPE obtained commercially was about 8 times more mutagenic than our sample of 2,4-DAPE. After purification, the commercial product, now chromatographically identical with our own sample, gave plate-test results close to those obtained for our samples of 2,4-DAPE. A review of the published reports (in which 2,4-DAPE was claimed to be inactive in a variety of short-term tests) revealed: (a) the use of protocols for bacterial mutagenicity testing which, in the light of our own results, were probably too limited in scope, especially in the choice of conditions for metabolic activation; (b) insufficient information on the identification and purity of the samples of 2,4-DAPE tested in the published collaborative study.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10μg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB₁, FB₂, and FB₃ against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB₁ is known to be responsible for the hepatocarcinogenicity of the fungus in rats.     

Conversion of tris(8-quinolinolato-N1, O8) aluminum to 8-hydroxyquinoline and activity in bacterial reverse mutation assays

Tris(8-quinolinolato-N1, O8) aluminum (AlQ), an aluminum chelate of 8-hydroxyquinoline (8OHQ) is an important charge transfer molecule in semiconducting imaging devices. This study was conducted to evaluate AlQ and 8OHQ for the ability to induce reverse mutations, either in the presence or absence of mammalian microsomal enzymes, and to determine if AlQ decomposes or is metabolized to 8OHQ under assay conditions. The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA (pKM101). The assays were conducted in the presence and absence of S9. AlQ doses were 1-1000 microg per plate while 8OHQ doses were 0.947-947 microg per plate to maintain molar equivalency. Stability studies were carried out for 4h at 37 degrees C under conditions designed to mimic mutation assays. Samples were analyzed by HPLC and LC/MS to tentatively identify potential metabolites of AlQ and 8OHQ. The results of the bacterial mutagenicity assay indicate that in the presence of S9, both AlQ and 8OHQ, caused increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA (pKM101). No increases were observed with any of the remaining tester strain/activation condition combinations. The stability study showed that AlQ degrades readily to 8OHQ under standard mutagenicity test conditions, and the positive test result with AlQ is due to the bioactivation of 8OHQ. In the presence of S9, 8OHQ is metabolized to one detectable product with molecular weight indicative of a one-oxygen insertion. 8OHQ N-oxide and 2,8-quinolinediol were ruled out as possible metabolites; 8OHQ epoxides and other quinolinediols were neither confirmed nor ruled out. Bacterial mutagenicity tests have not been shown to predict in vivo effects of 8OHQ; these assays are similarly expected to be poorly predictive of in vivo genotoxic and carcinogenic potential of AlQ.     

苍术挥发油的提取及其抑菌活性研究

采用水蒸气蒸馏法、微波萃取法和索氏提取法3种方法提取苍术挥发油。平板法涂布研究了3种苍术挥发油对3种细菌和4种真菌的最低抑菌浓度(MIC),滤纸片固相扩散法研究了苍术挥发油对供试菌体的抑菌活性。结果表明,3种方法提取的苍术挥发油对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、青霉、黑曲霉、黄曲霉的MIC分别为:水蒸气蒸馏法为5.00、150.00、150.00、5.00、5.00、5.00、20.00 mL/L;索氏提取法的为10.00、150.00、200.00、20.00、5.00、60.00、40.00 mL/L;微波萃取法的为10.00、150.00、150.00、20.00、20.00、20.00、20.00 mL/L。3种苍术挥发油对供试细菌和真菌都具有相当强的抑菌活性,且浓度越高效果越好。抑菌实验表明3种方法提取的苍术挥发油对金黄色葡萄球菌、酵母、青霉、黑曲霉、黄曲霉的抑菌圈直径都比对大肠杆菌、枯草芽孢杆菌的抑菌圈直径大。不同提取方法得到的苍术挥发油对同一种菌的最低抑制浓度和抑菌效果不相同,同一种方法提取的苍术挥发油对不同菌的最低抑制浓度和抑菌效果也不相同。