The effect of liver postmitochondrial fraction concentration from Aroclor 1254-treated rats on promutagen activation in L5178Y cells

L5178Y/TK^+/− cells were treated with either N-acetyl-2-aminofluorene (AAF), 2-aminoanthracene (2A), benzanthracene (BA), benzo[a]pyrene (BP), 3-methylcholanthrene (3MCA), or dimethylnitrosamine (DMN) in the presence of varied concentrations (2.5–10% v/v) of liver S9 (9000 × g) postmitochondrial fraction from Aroclor 1254-dosed male CD rats. Consistent S9 concentration-dependent decreases in trifluorothymidine-resistant (TFT^r) mutant induction were noted following 3 h exposure of L5178Y/TK^+/− cells to either 2.5 μg/ml BP, 50 or 67 μg/ml AAF, 0.96 μg/ml 2A, 7.35 μg/ml BA, or 5.4 μg/ml 3MCA. The exception was DMN which yielded a moderate S9 concentration-dependent increase in TFT^r mutant induction when L5178Y/TK^+/− cells were treated for 3 h with 74.1 μg/ml DMN. Depending upon the promutagen being tested, these results suggest two different metabolic events: (1) activation via non-induced enzymes or other factors whose activities are totally S9 volume-dependent (DMN), and (2) deactivation via induced detoxification pathways, a sequence favored by increasing S9 concentration (all others). Utilization of a single “standard” (10%) concentration of liver S9 from Aroclor 1254-treated rats did not appear to provide optimal metabolic activation for 5 of these 6 promutagens.     

Effects of the hepatic S9 fraction from aroclor-1254-treated rats on the mutagenicity of benzo[a]pyrene and 2-aminoanthracene in the salmonella/microsome assay

The mutagenicity of benzo[a]pyrene and 2-aminoanthracene for Salmonella typhimurium TA98 in the plate-incorporation test was studied using liver S9 from untreated and Aroclor-1254-treated rats. The induction of liver S9 protein, arylhydrocarbon hydroxylase (AHH), and cytochrome P448/450 was followed with time. There was no change in protein concentrations with induction; AHH and cytochrome levels were increased at 1, 3, 5 and 7 days post Aroclor treatment. Benzo[a]pyrene mutagenicity was enhanced with Aroclor treatment while 2-aminoanthracene mutagenicity was depressed. The benzo[a]pyrene mutagenicity showed a positive correlation with the levels of AHH and cytochrome on the plate; 2-aminoanthracene showed a negative correlation with activity in induced samples.     

Hamster hepatocyte-mediated activation of procarcinogens to mutagens in the L5178Y/TK mutation assay

Eight procarcinogens including three nitrosamines, three polycyclic hydrocarbons, and two aromatic amines were tested for mutagenic potential at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells co-cultivated with viable hamster hepatocytes. All eight chemicals produced substantial mutagenic activity as indicated by increased trifluorothymidine resistance in L5178Y cells treated in the presence of hepatocytes. Mutagenic responses to benzo[a]pyrene, 3-methyl-cholanthrene, N-nitrosodiethylamine, and N-nitrosodipropylamine first increased, then plateaued within the range of mutagen concentrations tested, while consistent dose-dependent increases in mutant frequencies were observed following 2-aminoanthracene, 2-aminofluorene, or N-nitrosodimethylamine treatments. The relatively flat portions of the mutant frequency curves for benzo[a]pyrene and 3-methylcholanthrene coincided with maximum chemical solubility as obvious from visible or microscopically detectable precipitate. These hamster cells readily facilitated the metabolism of 1,2-benzanthracene to a detectable mutagen and were especially competent in the activation of the two aromatic amines. Thus, cultured hamster hepatocytes can activate a variety of chemical carcinogens including polycyclic hydrocarbons to mutagens in a whole cell-mediated in vitro assay using L5178Y/TK^+/? cells as the target organism.     

Mixtures of polycyclic aromatic compounds inhibit mutagenesis in the Salmonella/microsome assay by inhibition of metabolic activation

We observed that complex mixtures of aromatic compounds isolated from a coal-derived oil suppressed the mutagenic activity of the indirect mutagens benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 2-aminofluorene, and 2-acetylaminofluorene as measured in the Salmonella/microsome mutagenicity assay, using strain TA98 and metabolic activation with Aroclor-induced rat-liver S9 or microsomes. The mixture also inhibited S9-dependent benzo[a]pyrene metabolism and covalent binding to DNA in a cell-free system. The mixture did not suppress the activity of either the direct acting mutagens 2-nitrofluorene and benzo[a]pyrene diol-epoxide, or of the indirect mutagen N-hydroxy-2-acetylaminofluorene which requires a microsomal deacetylase for metabolic activation. Spectrophotometric measurements showed that components of the mixture bound to microsomal cytochrome P-450. The mixture did not inhibit microsomal NADPH-cytochrome c (P-450) reductase. These observations show that the mixtures inhibited metabolic activation by the microsomal monooxygenase system, probably by binding of unidentified components to cytochrome P-450. The resulting inhibition of mutagenesis may have implications for risk estimates for the mixtures we examined as well as for other types of complex mixtures for which similar inhibitory effects have been observed.     

Metabolic activation of selected aromatic amines and polycyclic hydrocarbons by isolated subcellular fractions of Drosophila melanogaster

The applicability of microsomal preparations from Drosophilamelanogaster as the metabolic factor in the Salmonella mutagenicity assay with strains TA98 and TA100 was evaluated. Isolated cellular fractions (S27) from PB-pretreated flies activated N-acetyl-2-aminofluorene (2-AAF), N-hydroxy-N-aceyl-2-aminofluorene (N-OH-AAF), benzo[a]pyrene (BP), 9,10-dimethylanthracene (DA) and 2 -naphythylamine (NA)_into mutagenic metabolites, 7,-12-Dimethylbenz[a]-anthracene (DMBA) was ineffective under the conditions of the test.This study was performed in an effort to determine optimal conditions for activating, by Drosophila enzymes, aromatic amines and polycyclic hydrocarbons, with 2-AAF and BP as model mutagens. The following alterations improved the sensitivity of this combined Salmonella/Drosophila assay. (1) Incubation of the plates at 25°C for 1 night instead of permanent exposure at 37°C. (2) Isolation of S27 fractions instead of the conventional S9, because 9000 × g was not sufficient tio spin down Drosphila mitochondria.     

Activation and detoxication of promutagens by toadfish (Opsanus tau) hepatic postmitochondrial fractions in the Salmonella assay

Three groups of experiments were conducted to characterize the hepatic postmitochondrial fraction (S9) from the oyster toadfish (Opsanus tau) as an activation system for promutagens in the Salmonella assay and to provide an initial evaluation of the extent to which data from standard in vitro assays with mammalian activation systems are predictive of possible genotoxic effects in this marine fish. In the first group of experiments the effects of increasing the concentration of S9 from untreated and 3-methylcholanthrene (MC)- or Aroclor 1254 (AC)-pretreated toadfish and Sprague-Dawley rats on the mutagenicities of different concentrations of 2-aminoanthracene (2AA) and benzo[a]pyrene (BAP) were examined in Salmonella (TA98) plate assays. The maximum levels of 2AA mutagenicity attained by S9 from untreated (UI S9) toadfish and rats were comparable, but UI S9 from toadfish was moreeffective than UI S9 from rats in mediating BAP mutagenicity. MC pretreatment decreased maximum levels of 2AA mutagenicity and increased maximum levels of BAP mutagenicity mediated by S9 from both species. MC pretreatment also altered the pattern of dependence of 2AA mutagenicity on the concentration of S9 protein for S9 from both species. A similar alteration in the pattern of dependence of BAP mutagenicity on the concentration of S9 protein was also observed with S9 from MC-pretreated toadfish. Although AC pretreatment of rats effected changes in the mutagenicities of both test chemicals similar to those effected by MC pretreatment, AC pretreatment of toadfish effected little or no change in the mutagenicities of either test chemical. The changes in the pattern of dependence of 2AA and BAP mutagenicities on the concentration of S9 protein effected by MC pretreatment of toadfish were confirmed in a separate group of experiments. A third group of experiments was designed to examine the effects of α-naphthoflavone (ANF) on the mutagenicities of 2AA and BAP mediated by UI and MC S9 from toadfish. Although ANF did not affect the 2AA mutagenicity mediated by UI S9, a significant decrease in 2AA mutagenicity and a significant increase in BAP mutagenicity mediated by MC S9 and a significant decrease in BAP mutagenicity mediated by UI S9 were observed. These results indicate that 2AA and BAP are effectively activated by toadfish S9 and that, as in rats, these two test chemicals are activated and/or detoxicated by different cytochrome P-450-dependent pathways. These results also support the contention that cytochrome P-450-dependent detoxication pathways can be an important determinant of the mutagenic potency of some promutagens in vitro.     

Effect of induction by DDT on metabolism and mutagenicity of benzo[a]pyrene

Liver microsomal enzymes are essential for the detection of benzo[a]pyrene (B[a]P)-mediated mutagenesis in the Salmonella/mammalian microsome mutagenicity test and, furthermore, this mutagenicity is considerably enhanced by induction of hepatic enzymes involved with drug metabolism. Although Aroclor 1254 is most commonly used for induction of S9 enzymes, DDT is also capable of this induction. This paper reports a comparison of liver S9 fraction induced by the two agents: there is a marked difference in their concentration optima for metabolism of B[a]P; greater numbers of revertant colonies are seen with Aroclor-induced S9, which is optimal at a concentration of 10% (v/v), whereas DDT-induced S9 is optimal at 2.5% (v/v); Aroclor induces aryl hydrocarbon hydroxylase (AHH), cytochrome P-450 and epoxide hydrase while DDT induces only AHH, to about half the level detected in the Aroclor-induced S9 fraction. A comparison of metabolite distribution for Aroclor- and DDT-induced hepatic microsomes reveals quantitative differences only. DDT-induced microsomes yield a greater proportion of B[a]P-4,5-oxide and its metabolic product B[a]P-4,5-dihydrodiol than do Aroclor-induced microsomes. Time course studies on the mutagen half-life measured on the agar plate provides good evidence that metabolites responsible for mutagenicity were different for each inducer.     

Benz[j]aceanthrylene: A novel polycyclic aromatic hydrocarbon with bacterial mutagenic activity

Initial studies on the mutagenicity and metabolism of a novel cyclopenta-PAH, benz[j]aceanthrylene, are reported in the Salmonella bacterial system. The spectrum of activity of benz[j]aceanthrylene over the 5 Ames tester strains is similar to that of benzo[a]pyrene, and the dose-response curves for strain TA98 are comparable. Like other biologically active PAH, benz[j]aceanthrylene is a frame-shift mutagen requiring metabolic activation. An interesting feature of the S9 dependence of activity is the low concentration (≅10-fold smaller than for benzo[a]pyrene) at which optimal activity is observed. The 1,2-dihydro-1,2-diol (product of metabolism of the cyclopenta-ring) appears to be the predominant metabolite, and implicates the 1,2-oxide as the ultimate mutagenic species.     

Effects of the hepatic S9 fraction from aroclor-1254-treated rats on the mutagenicity of benzo[alpha]pyrene and 2-aminoanthracene in the Salmonella/microsome assay.

The mutagenicity of benzo[alpha]pyrene and 2-aminoanthracene for Salmonella typhimurium TA98 in the plate-incorporation test was studied using liver S9 from untreated and aroclor-1254-treated rats. The induction of liver S9 protein, arylhydrocarbon hydroxylase (AHH), and cytochrome P448/450 was followed with time. There was no change in protein concentrations with induction; AHH and cytochrome levels were increased at 1, 3, 5 and 7 days post Aroclor treatment. Benzo[alpha]pyrene mutagenicity was enhanced with Aroclor treatment while 2-aminoanthracene mutagenicity was depressed. The benzo[alpha]pyrene mutagenicity showed a positive correlation with the levels of AHH and cytochrome on the plate; 2-aminoanthracene showed a negative correlation with activity in induced samples.     

Metabolic activation and deactivation of mutagens by preparations of human lung parenchyma and bronchial tree

69 S12 preparations of human lung (43 of peripheral lung parenchyma and 26 bronchial preparations) were assayed for their ability to activate procarcinogens to mutagenic metabolites in the Ames test or to lower the mutagenic response of direct-acting compounds. No sample activated polycyclic aromatic hydrocarbons, such as 3-methylcholanthrene, benzo[a]pyrene or its metabolites 3-hydroxy-B(a)P and B(a)P-trans-7,8-diol, and only borderline effects were observedwith cigarette-smoke condensates. Conversely, some activating ability was detected in the case of 2-aminofluorene and of cyclophosphamide. The same samples produced a slight decrease of mutagenicity of epichlorohydrin and ICR-191 and a more pronounced loss of activity of sodium dichromate and 4-nitroquinoline N-oxide.     

Enzymatic characterization of the polynuclear aromatic hydrocarbons activating rat-liver preparations used in the mutagenicity test of Ames

Stability studies were performed on the mono-oxygenase system involved, in particular, in the activation of polynuclear aromatic hydrocarbons (PAHs) present in rat-liver preparations used in the Ames mutagenicity test. The results indicated a good stability of the spectral response of the cytochrome-P-450 system, but a much lower stability of its enzymatic activities measured with various substrates, thus showing the inadequacy of the spectral response to characterize the PAH mono-oxygenase activity of the preparations. Epoxide hydrolase activity was found to be stable. Various mono-oxygenase activities were measured in preparations induced with phenobarbital, 3-methylcholanthrene or Aroclor 1254. The activities of two enzymes, benzo[a]pyrene hydroxylase and ethoxyresorufin-O-dealkylase, were found suitable to characterize the capacity of the preparations to metabolize PAH to mutagens. The efficiency of the same preparations to promote the mutagenicity of benzo[a]pyrene and aflatoxin B1 in the Ames test was determined. There was an excellent general correlation between the efficiencies for mutagenic activation of the preparations and the two enzymatic activities mentioned above. Determination of ethoxyresorufin-O-dealkylase (or benzo[a]pyrene hydroxylase) and benzo[a]pyrene 4,5-oxide hydrolase activities is proposed for characterizing the rat-liver preparations used in the Ames test.     

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

The Salmonella/mammalian microsome mutagenicity test: comparison of human and rat livers as activating systems

The mutagenicity of several test compounds was verified by the Salmonella/microsome mutagenicity test (Ames test), using both human liver and rat liver (untreated or pretreated with Aroclor 1254) S9 under identical experimental conditions. Aflatoxin B1, 3-methylcholanthrene, and cigarette-smoke condensate were less mutagenic in the presence of human-liver S9 than in the presence of rat-liver S9 (particularly after treatment with Aroclor 1254). The opposite was observed with 2-aminonanthracene and to a lesser degree with 2-aminofluorene; correlation studies indicate that the two compounds were activated by the same or by very similar enzymes, probably cytochrome P-450s. These results clearly indicate that human-liver S9, as an activating system, behaves differently than rat-liver S9; therefore, it may constitute a useful, additional tool for the study of mutagenicity and probably, carcinogenicity in man.     

DNA--benzo[a]pyrene adducts formed in a Salmonella typhimurium mutagenesis assay system.

The DNA adducts formed in Salmonella typhimurium when bacteria are incubated with radioactive benzo[a]pyrene and liver microsomal enzymes from several sources has been investigated. When enzyme preparations from Aroclor I254 or 3-methylcholanthrene induced C57BL/6N (B6) mice were used to mediate activation, the predominant product was an adduct between the 10 position of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the N-2 position of deoxyguanosine. Similar results were obtained with human liver and with Aroclor-induced rat-liver enzyme preparations. This adduct is also the major DNA product previously found when human tissues or certain rodent cells were incubated with benzo[a]pyrene. On the other hand, when activation of benzo[a]pyrene was mediated by a phenobarbital-induced B6 mouse-liver enzyme preparation, the extent of binding was quite low and the profile of DNA adducts in S. typhimurium DNA was quite different. Thus, under appropriate conditions, the activation and DNA binding of benzo[a]pyrene inthe microsome mediated S. typhimurium mutagenesis assay generally resembles that seen in intact mammalian cells. Caution must be exercised, however, in the choice of microsome-activation systems.     

Chemical models for cytochrome P450 as a biomimetic metabolic activation system in mutation assays

DNA damage is a critical factor in carcinogenesis. The Ames assay is a short-term test that screens for DNA-damaging agents. To be detected in the assay, most carcinogens require oxidation by cytochrome P450, a component of the liver homogenate preparation (S9 mix) that is traditionally used to metabolize promutagens to an active form in vitro. A combination of iron(III) porphyrin plus an oxidant activates many promutagens by mimicking cytochrome P450 metabolism. We previously reported that the mutagenicity of the N-nitrosodialkylamines was detected following reaction with tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (Fe(F(5)P)Cl) plus tert-butyl hydroperoxide (t-BuOOH), which yielded the same alcohols and aldehydes as the enzymatic reaction. In the present study, to extend the scope of biomimetic models, we tested the mutagenicity of other carcinogens exposed to chemical oxidation systems.We investigated the optimal assay conditions for the models in Salmonella typhimurium TA1538, a strain sensitive to frame-shift mutagens. We activated 2-aminofluorene (AF), benzo[a]pyrene (B[a]P), a tryptophane pyrolysate 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), and 2-acetylaminofluorene (AAF) with Fe(F(5)P)Cl plus an oxidant-t-BuOOH, m-chloroperoxybenzoic acid (mCPBA), or magnesium monoperoxyphthalate (MPPT)-and we noted the effect of three solvents-acetonitrile (CH(3)CN),1,4-dioxane, and N,N-dimethylformamide (DMF)-on AF activation.All the promutagens became mutagenic in the presence of Fe(F(5)P)Cl plus an oxidant, with the effectiveness of the oxidant varying with the chemical. Aromatic amines, for example, showed the strongest mutagenicity with t-BuOOH whereas polycyclic hydrocarbons showed the strongest mutagenicity with mCPBA. All the promutagens were mutagenic in the presence of Fe(F(5)P)Cl plus MPPT. For AF activation, the order of effectiveness of the solvents was CH(3)CN>1,4-dioxane>DMF. The results suggested that these systems would serve as useful models for microsomal activating systems.     

Non-enzymic activation of polycyclic aromatic hydrocarbons as mutagens.

Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ⁶⁰Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo[a]anthracene, benzanthrone, benozo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose-responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dobenzo[a,h]anthracene, picene, and triphenylene negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ⁶⁰Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

The dosing determines mutagenicity of hydrophobic compounds in the Ames II assay with metabolic transformation: Passive dosing versus solvent spiking

The Ames II bacterial mutagenicity assay is a new version of the standard Ames test for screening chemicals for genotoxic activity. However, the use of plastic micro-titer plates has drawbacks in the case of testing hydrophobic mutagens, since sorptive and other losses make it difficult to control and define the exposure concentrations, and they reduce availability for bacterial uptake or to the S9 enzymes. With passive dosing, a biocompatible polymer such as silicone is loaded with the test compound and acts as a partitioning source. It compensates for any losses and results in stable freely dissolved concentrations. Passive dosing using silicone O-rings was applied in the Ames II assay to measure PAH mutagenicity in strains TA98 and TAMix – a mixture of six different bacterial strains detecting six different base-pair substitutions – after metabolic activation by S9. Initially, 10 PAHs were tested with passive dosing from saturated O-rings, aiming at levels in the test medium close to aqueous solubility. Fluoranthene, pyrene and benzo(a)pyrene were mutagenic in both TA98 and TAMix, whereas benz(a)anthracene was mutagenic in TA98 only. The concentration-dependent mutagenic activity of benzo(a)pyrene was then compared for passive dosing and solvent spiking. With spiking, nominal concentrations greatly exceeded aqueous solubility before mutagenicity was observed, due to sorptive losses and limiting dissolution kinetics. In contrast, the passive dosing concentration-response curves were more reproducible, and shifted towards lower concentrations by several orders of magnitude. This study raises fundamental questions about how to introduce hydrophobic test substances in the Ames II assay with biotransformation, since the measured mutagenicity not only depends on the compound potency but also on its supply, sorption and consumption during the assay.     

Liver, lung and kidney homogenates used as an activation system in mutagenicity studies of airborne particles and of expectorate and urine samples from exposed workers in a coke plant

A comparison was made between lung and kidney homogenates on the one hand and liver S9 from rats on the other hand in order to compare their ability to activate promutagens. The Salmonella reversion assay was used on extracts of airborne particles from the top of coke oven batteries, and of expectorate and urine samples from exposed workers in the same coke plant. The contents of benzo[a]anthracene and benzo[a]pyrene in the different test solutions were measured by high-resolution gas chromatography/mass spectrometry. Both mutagens were detected in the filter extract and in the expectorates from the exposed workers but not in the expectorates from the control groups or in the urine samples. The liver S9 gave significantly higher mutagenicity than lung and kidney activation with both filter samples and expectorate and urine samples.