S9 Activation System Research Articles

Effects of cellular differentiation in human primary bronchial epithelial cells: Metabolism of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone

Many of the toxicants in tobacco smoke undergo biotransformation in the lungs of smokers, both to reactive and to detoxified derivatives. Human air-liquid-interface (ALI) airway tissue models have emerged as an advanced in vitro model for evaluating the toxicity of inhaled substances; however, the metabolic potential of these cultures has not been evaluated extensively. In this study, we compared the metabolic activities of an ALI tissue model to the undifferentiated normal human primary bronchial epithelial (NHBE) cells from which it was derived. Measurement of the basal levels of gene expression for 84 phase I drug metabolism enzymes indicated that most genes were upregulated in ALI cultures compared to NHBE cells. Furthermore, the enzymatic activities of three cytochrome P450s involved in the bioactivation of tobacco-specific nitrosamines were higher in the ALI cultures, and the bioactivation of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), as measured by the formation of two of its major metabolites, i.e., keto acid and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was significantly greater in the ALI cultures. Finally, NNK was a direct-acting genotoxicant in the ALI cultures, while the genotoxicity of NNK was detected in NHBE cells only in the presence of an exogenous liver S9 activation system. Taken together, our findings demonstrate the greater metabolic potential of well-differentiated ALI cultures than primary NHBE cells, supporting the potential use of ALI airway cultures as an alternative in vitro model for evaluating inhaled toxicants that require metabolic transformation.

Determination of mutagenic potencies of aqueous extracts of Thermopsis turcica by Ames test

Thermopsis turcica is an endemic species of Turkey popularly known as Eber Sarısı or Piyam; it is a member of the Thermopsis genus, which contains plants considered to be medicinal throughout the world. In this study, the mutagenic effect of aqueous extracts obtained from the leaves, root, stem, and flowers of T. turcica were evaluated by the Ames test, using Salmonella typhimurium TA97, TA98, TA100, and TA102 strains with and without an S9 activation system. The results obtained were analyzed statistically using SPSS for Windows, and the Mann-Whitney test was performed for comparisons. The results showed that all of the aqueous extracts of T. turcica were mutagenic for S. typhimurium TA98 with S9 mix, and, at a dosage of 3750 µg/plate, the leaf extracts were also found to be mutagenic for S. typhimurium TA102 with or without S9 mix. The results further demonstrated that, in relation to the concentration, leaf extracts of T. turcica increased the frequency of reverse mutation in S. typhimurium TA102 with S9 mix.

Strategy for genotoxicity testing—Metabolic considerations

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens “missed” in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or “major” metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.

PAHs and Mutagenicity of Inhalable and Respirable Diesel Particulate Matter in Santiago, Chile

Delayed human effects such as cancer could be a consequence of chronic exposure, over long periods of time, to inhalable (PM10) and respirable (PM2.5) particles containing environmental carcinogen mixtures. Air pollution in Santiago, Chile, is a major public health problem due to the high levels of regulated pollutants such as PM10, CO, and ozone. In this work, we studied the levels of polycyclic aromatic hydrocarbons (PAHs) onto PM10 and PM2.5 collected in diesel revision plants, in an urban area with a high flow of buses and trucks and in a rural area not exposed to diesel emissions. The PM10 average levels in diesel emission plants were higher than the Chilean PM10 standard and higher than those found in the urban and rural areas. In the urban area the PM10 average levels were lower than the Chilean PM10 standard, although some 24 h levels surpassed the levels established to decree preemergency or emergency and in some cases were higher than the levels reported by all the official monitoring stations. The levels of total PAHs onto PM10 were higher in the diesel plants than in the urban area and rural area, but the levels of six carcinogenic PAHs were similar in the diesel plants to those found in the urban area. Organic extracts from PM10 collected in diesel plants in Salmonella typhimurium TA98 in the presence and in the absence of an S9 activation system were significantly higher than in the urban area and rural area. Mutagenic activity with and without S9 in diesel plants and the urban area showed that indirect (PAHs) and direct (nitro-PAHs) mutagenic compounds are present in organic extracts from PM10. In conclusion, these results showed that in Santiago diesel emission particles were highly mutagenic and contain carcinogenic PAHs. This might represent a risk for long-term respiratory effects in Santiago's inhabitants.

Effects of black tea theafulvins on aflatoxin B(1) mutagenesis in the Ames test.

Black tea theafulvins, a fraction of thearubigins isolated from black tea aqueous infusions, potentiated the mutagenic activity of the mycotoxin aflatoxin B(1) in the Ames test, in the presence of a hepatic S9 activation system derived from Aroclor 1254-treated rats. In contrast, when the S9 activation system was replaced with isolated microsomes, theafulvins suppressed the mutagenicity of the mycotoxin. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, incorporation of the theafulvins into the activation system reduced the mutagenic activity, whereas if it was added before termination of microsomal activity a potentiation of mutagenic response was observed. In in vitro studies, theafulvins inhibited epoxide hydrolase and glutathione S-transferase activities in a concentration-dependent manner. Finally, the mutagenicity of aflatoxin B(1) was much more pronounced in bacteria that were pre-exposed to theafulvins but from which they were subsequently washed off. It may be inferred from the above studies that the genotoxic synergy between aflatoxin B(1) and black tea theafulvins does not occur during the bioactivation of the carcinogen, but may partly be due to decreased deactivation of the reactive intermediate, aflatoxin B(1) 8,9-oxide, by conjugation with glutathione.

Evidence for site-specific bioactivation of alachlor in the olfactory mucosa of the Long-Evans rat.

Alachlor (2-chloro-2',6'-diethyl-N-[methoxymethyl]-acetanilide) is a restricted-use chloracetanilide herbicide which has been shown previously to produce a dose-dependent incidence of olfactory mucosal tumors in rats following chronic dietary exposure. However, the mechanism of alachlor carcinogenicity is poorly understood. Alachlor was administered i.p. to male Long-Evans rats for up to 28 days at doses that are carcinogenic in chronic studies in order to study olfactory lesion development and alterations in cell proliferation. Neither treatment-related olfactory mucosal lesions nor regenerative cell proliferation, as assessed with BrdU labeling, was detected. In vitro genotoxicity studies using Salmonella typhimurium strain TA100 showed that alachlor was non-mutagenic in the absence of metabolic activation. When pre-incubated with an olfactory mucosal S9 activation system, alachlor induced a weak, dose-dependent mutagenic response at 500-1250 micrograms/plate, with toxicity at higher doses. In contrast, an S9 activation system derived from nasal respiratory mucosa, the tissue physically juxtaposed with the olfactory mucosa but reportedly not susceptible to alachlor-induced tumors, did not produce a mutagenic response for alachlor or the positive control. Thus, this result suggested site-specificity of alachlor activation consistent with the target site of carcinogenicity. The mutagenicity of alachlor to Salmonella, in the presence of an olfactory mucosal-activating system, was confirmed by a limited positive response in the mouse lymphoma assay. Here there were increases in small colony mutants (indicative of chromosomal effects) as well as large colony mutants (which reflect gene mutations). This study suggests that target tissue bioactivation of alachlor results in the formation of one or more mutagenic metabolite(s), which may be critical in alachlor-induced nasal tumorigenesis.

Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays.

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[ a]pyrene: Studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen

Mechanisms of the antimutagenic action of chlorophyllin (CHL) towards benzo[ a]pyrene (BP) were studied in vitro. In the Salmonella assay, CHL inhibited the mutagenic activity of BP in the presence of an S9 activation system and was particularly effective against the direct-acting ultimate carcinogen, benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Spectral studies indicated that the time-dependent hydrolysis of BPDE to tetrols was augmented in the presence of CHL concentrations on the order of 5 μM. Dose-related inhibition of several cytochrome P450-dependent enzyme activities was observed upon addition of CHL to in vitro incubations. Spectral changes for the interaction between CHL and cytochrome P450 indicated that CHL does not bind to the active site of the enzyme, but exerts its inhibitory effect indirectly. This was achieved by inhibiting NADPH-cytochrome P450 reductase ( K i ∼ 120 μM with cytochrome c as substrate), and did not involve lowering of the effective substrate concentration by complex formation with the procarcinogen. It is concluded that the in vitro antimutagenic activity of CHL towards BP involves accelerated degradation of the ultimate carcinogen, with inhibition of carcinogen activation occurring only at high CHL concentrations. The latter mechanism is unlikely to occur in vivo following p.o. administration due to the limited uptake of CHL from the gut, but tissue concentrations may be sufficiently high to cause degradation of BPDE.

Effects of sterigmatocystin and T-2 toxin on the induction of unscheduled DNA synthesis in primary cultures of human gastric epithelial cells.

Primary cultures of human gastric epithelial cells were tested for induction of unscheduled DNA synthesis (UDS) by sterigmatocystin (ST) and T-2 toxin. Autoradiographic results indicated that ST (10(-6)-10(-4)M) induced UDS in the presence of S9 activation system. The repair rates were 24-91% (net grains > or = 3) and 2-71% (net grains > or = 5). T-2 toxin did not induce UDS in this study.

Effects of methrpyrilene measured in mitochondria isolated from naive and methapyrilene-treated rat and mouse hepatocytes

Methapyrilene (MP) is a rat-specific liver carcinogen that alters mitochondrial number and morphology both in vivo and in vitro. This biological phenomenon may be due to the effects of MP on mitochondrial function. To test this hypothesis, studies were conducted to examine the effects of MP on DNA and protein synthesis and respiration in isolated mitochondria. DNA and protein synthesis activities were measured using [ 3H]thymidine and [ 3H]leucine incorporation. Mouse liver mitochondria were also examined for comparison since no tumor formation or alterations in mitochondrial morphology have been associated with MP treatment in mice. A signicant decrease in basal DNA and protein synthesis levels was observed in mitochondria isolated from rats and mice following in vivo MP treatment. This effect could not be reproduced when mitochondria were exposed to 0 or 100 μ m MP following isolation, despite the presence of an S9 activation system. Electron microscopic examinations were performed on isolated rat mitochondria and revealed morphologic differences between mitochondria from naive and MP-treated rats. Although significant differences in State 3 and State 4 respiratory rates were noted, the respiratory control ratio, ADP O ratio, and uncoupler-stimulated respiratory rates were unaffected. Results demonstrate that: (1) MP irreversibly depresses DNA and protein synthesis in a majority of mitochondria, despite only localized morphologic changes; (2) these changes are not reflected by a decrease in respiratory function; and (3) depression of DNA and protein synthesis does not correlate with carcinogenic susceptibility.

Chromosome damaging effects of pan masala

Effects of aqueous extracts of a popular brand of pan masala with and without tobacco (PM-T and PM) were studied for short duration treatment employing an in vitro system. Metabolic activation with S9 mix was also included. Frequency of all the three cytogenetic endpoints viz., chromosome aberration (CA); sister chromatid exchange (SCE) and % micronucleated cells (% MNC) were found to be elevated significantly in a dose-dependent manner in cultures without metabolic activation. However, addition of S9 activation system resulted in supression of chromosomal damage. Our findings indicate that pan masalas contain water soluble direct acting mutagens.

Mutagenicity of 4-nitrodiphenyl thioether-derived products and their potential metabolites

4-Nitrodiphenyl thioether (4-NO 2TE) and its various potential metabolites 4-nitroso- (4-NOTE), 4-hydroxylamino- (4-NHOHTE), 4-hydroxyacetylamino- [4-N(OH)ACTE], 4-acetoxy-acetylamino- [4-N(OAc)ACTE], 4-amino- (4-ATE) and 4-acetamido- (4-AATE) were tested for their mutagenicity toward Salmonella typhimurium TA98 in the presence and absence of S9 activation system. 4-NO 2TE, 4-NOTE, 4-NHOHTE and 4-N(OAc)ACTE were direct-acting mutagens with 4-NHOHTE having the highest activity. With S9 mix, the mutagenicity of 4NO 2TE almost doubled and that of 4-NOTE and 4-N(OAc)ACTE decreased. In TA98NR, 4-NOTE induced higher mutations, while with 4-NHOHTE the number of revertants decreased. Both 4-NHOHTE and 4-N(OAc)ACTE showed decreased mutagenicity in TA98/1,8-DNP 6. 4-ATE, 4-AATE and 4-N(OH)ACTE expressed mutagenicity only with S9 mix. 4-Nitrodiphenyl sulphoxide (4-NO 2SO) and 4-nitrodiphenyl sulphone (4-NO 2SO 2), the sulphur-oxidised products of 4-NO 2TE, were non-mutagenic as such and after addition of S9 activation system

Mechanism of the in vitro antimutagenic action of retinol

The antimutagenic action of retinoids against three amino-imidazoazaarene pre-carcinogens, i.e. 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), was investigated using the Ames test and hepatic activation systems derived from rats pretreated with Aroclor 1254. Both retinol and retinal, when incorporated into the S9 activation system, gave rise to a concentration-dependent decrease in the mutagenicity of all three mutagens, retinol being generally the more effective. Retinol suppressed the mutagenic activity of IQ even when isolated microsomes were used as activation systems. Moreover, retinol gave rise to a concentration-dependent inhibition of the microsomal dealkylations of pentoxy- and benzyloxy- and, especially, ethoxy-resorufin, but had no effect on the NADPH-dependent reduction of cytochrome c. Exposure of the bacteria to retinol with subsequent removal of the vitamin did not influence the mutagenicity of IQ. It is concluded that retinoids suppress the mutagenicity of aminoimidazoazaarenes and this is achieved through inhibition of their cytochrome P450-dependent metabolic activation. Retinol is a non-selective in vitro inhibitor of the hepatic cytochrome P450-dependent mixed function oxidase system as predicted by a computer graphic analysis of its molecular shape.

Exposure to Aflatoxins in Denmark

AbstractHighly sensitive immunoassays have been developed to quantitate human exposure to aflatoxins. Using a competetive ELISA, one or more substances recognized by a monoclonal antibody against aflatoxin Bl were detected in urine samples collected in Denmark. The concentration of the urinary alfatoxin-like substance was equivalent to 0-8 ug aflatoxin Bl/day/adult. The amount of urinary aflatoxin-like substance depends on the food ingested 24-48 hrs prior to urine collection, and is increased when the diet include beer, dairy products and meat. High pressure liquid chromatography analysis of the antigenic material concentrated on an affinity column revealed the presence of several antigenic compounds, that are different from the known aflatoxin metabolites. The major component is cytotoxic to Salmonella thypimurium (3 ng equivalent AFB/plate) when tested without the S9 activation system. It is suggested, that the antigenic material is produced by enzymatic degradation of animal proteins from animals fed ...

A method for the efficient degradation of melphalan into nonmutagenic products

A new chemical method has been developed for the destruction of an antineoplastic agent melphalane in laboratory waste, based on its oxidation by potassium permanganate in sodium hydroxide medium. To monitor the efficiency of destruction, analytical methods based on UV spectrophotometry, differential pulse voltammetry on a glassy carbon rotating disk electrode, and HPLC coupled with UV spectrophotometry have been developed. It has been demonstrated that the proposed method of destruction is highly efficient (more than 99.8% destruction) and the corresponding degradation products were found to be nonmutagenic to Salmonella typhimurium strains TA98, TA100, and TA1535, in both the presence and the absence of a rat liver S9 activation system.

Mutagenic activity of fluorides in mouse lymphoma cells

The L5178Y mouse lymphoma cell forward-mutation assay was used to test for the mutagenic activity of sodium and potassium fluoride at the thymidine kinase locus. Mutants were detected by colony formation in soft agar in the presence of trifluorothymidine. Mutagenic and toxic responses were observed in the concentration range of 300–600 μg/ml with both sodium and potassium fluoride. Approximately 3-fold increases in mutant frequency were observed for concentrations in the 500–700 μg/ml range that reduced the relative total growth to approximately 10% in the absence or presence of a rat-liver S9 activation system. A sample of 30% sodium fluoride-70% sodium bifluoride (NaHF 2) induced a similar mutagenic response but was more toxic with respect to the fluoride concentration. A specificity for fluoride ions in causing mutagenesis was indicated by the fast that much higher concentrations of sodium or potassium chloride were necessary to cause toxicity and increases in the mutant frequency. The possible involvement of chromosomal changes was signaled by the predominant increase in the small colony class of mutants.

Destruction of Aromatic Amines in Laboratory Wastes Through Oxidation with Potassium Permanganate/Sulfuric Acid into Non-mutagenic Derivatives

Nine aromatic amines, i.e., benzidine; o-tolidine; o-dianisidine; 3,3′-dichlorobenzidine; 4-aminobiphenyl; 1- and 2-naphthylamine; 4,4′-methylene bis(2-chloroaniline) and m-toluenediamine, were oxidized with potassium permanganate/sulfuric acid. Experimental conditions for complete degradation of these aromatic amines are described. The disappearance of the parent compound through oxidation was measured using HPLC coupled with UV spectrophotometry. The corresponding degradation products were found to be non-mutagenic to Salmonella typhimurium strains TA100, TA98 and TA97, both in the presence and absence of a rat liver S9 activation system. A collaborative study, involving 11 laboratories, has shown the applicability and the reproducibility of this degradation method.

Destruction of aromatic amines in laboratory wastes through oxidation with potassium permanganate/sulfuric acid into non-mutagenic derivatives.

Nine aromatic amines, i.e., benzidine; o-tolidine; o-dianisidine; 3,3'-dichlorobenzidine; 4-aminobiphenyl; 1- and 2-naphthylamine; 4,4'-methylene bis(2-chloroaniline) and m-toluenediamine, were oxidized with potassium permanganate/sulfuric acid. Experimental conditions for complete degradation of these aromatic amines are described. The disappearance of the parent compound through oxidation was measured using HPLC coupled with UV spectrophotometry. The corresponding degradation products were found to be non-mutagenic to Salmonella typhimurium strains TA100, TA98 and TA97, both in the presence and absence of a rat liver S9 activation system. A collaborative study, involving 11 laboratories, has shown the applicability and the reproducibility of this degradation method.

Erythrocytes-mediated metabolic activation of cyclophosphamide in yeast mutagenicity test

The degree of conversion of cyclophosphamide (CP) into mutagenic intermediates was studied using mouse erythrocytes as the metabolic activation system. The amount of mutagenic intermediates produced was measured indirectly in terms of induced frequencies of mitotic recombination, mitotic gene conversion, and reverse mutation in the diploid D7 strain of Saccharomyces cerevisiae. In the absence of S9 microsomal fraction or erythrocytes, CP did not induce any genetic response. In the presence of erythrocytes, on the other hand, CP clearly induced increases in the three genetic endpoints. The responses, however, were lower than those observed with the S9 activation system. The activating principle seems to be the oxyhemoglobin. In fact, neither p-nitroanisole O-demethylase activity nor genotoxic responses performed with red-blood cells from uninduced and PB-induced mice indicate that (possible) water-soluble forms of cytochrome P-450 were responsible for the activation of CP by erythrocytes.

The role of DNA-repair processes in N-nitrosopyrrolidine-induced mutagenesis.

The cytotoxic and mutagenic effects of increasing concentrations of N-nitrosopyrrolidine (NPYR) were studied using various DNA repair mutants of Escherichia coli together with rat-liver S9 activation system. Irrespective of which strain was used, the cytotoxic effects of NPYR were similar to those observed in the parent strain. Mutagenicity studies revealed that the uvrA- derivative was more mutable than its repair proficient parent. These observations suggest that NPYR reacts with DNA to generate bulky lesions, which although potentially mutagenic, do not contribute significantly to cell-killing. Subsequent experiments with the metabolic inhibitor SKF 525A revealed that this compound only partially inhibited the mutagenic activity of NPYR, suggesting that although hepatic mixed function oxidase enzymes may participate in NPYR activation other pathways of metabolism are also involved.