The process of promutagen biotransformation studied by the Ames test. II. The extent of the manifestation of the mutagenic action of nitrosomorpholine, diethylnitrosamine and cyclophosphane using various subfractions of rat liver homogenate

The data are presented on involvement of components of microsomal and cytosolic subfractions composing the S-9 fraction of rat liver homogenate in processes leading to formation of active metabolites of nitrosomorpholine (NM), diethyl nitrosoamine (DENA) and cyclophosphane (CP) promutagens and their detoxication resulting from the reaction with glutathione (G-SH) added to the system. It is established that the process of metabolic activation is only connected with microsomal subfraction, while reactions of the first phase of CP and DENA metabolism take place when both microsomal and cytosolic subfractions are added. Decrease in the effect of all promutagens studied under the action of G-SH was observed after microsomal and cytosolic subfractions of the S-9 fraction were introduced into the activating mixture. Various values of dependence of the metabolic activation level and the extent of decrease in the mutagenic action, upon addition of G-SH, on the protein content in microsomal and cytosolic subfractions were obtained.     

Study of promutagen biotransformation in the Ames test. III. The role of conjugation with glucuronic acid and sulfate in the modification of mutagenic effect of nitrosomorpholine, diethylnitrosamine and cyclophosphamide

The role of reactions of conjugation with uridine diphosphoglucuronic acid (UDPGA) and with 3-phosphoadenosine-5-phosphosulfate (PAPS) in modification of the mutagenic effect of diethyl nitrosamine (DENA), nitrosomorpholine (NM) and cyclophosphane (CP) was studied by the Ames test. It was shown that adding UDPGA to the activating mixture significantly decreased the level of the mutagenic effect of DENA, NM and CP on bacteria Salmonella typhimurium TA 1950, when S9 and microsomal fractions of rat liver homogenate were used. Adding PAPS to the activating mixture when S9 and cytosole fractions were used, did not affect mutagenic action of DENA on S. typhimurium TA 1950 and TA 1535, enhancing the mutagenic effect of CP on TA 1535, with no such influence on TA 1950. Introduction of PAPS into the activating mixture elevated the mutagenic effect of NM on both bacterial strains using S9 fraction but not cytosole fraction.     

Study of promutagen biotransformation in the Ames test. IV. The effect of transplanted tumors on the biotransformation of various antineoplastic agents

The effect of transplantation of rat tumours Jensen sarcoma, sarcoma 45, sarcoma M-1, as well as of inoculation of rat normal connective tissue on the processes of biotransformation of antitumour preparations cyclophosphane (CP), thiophosphamide, prospidine and of model compound nitrosomorpholine (NM) was studied. The study was accomplished by means of the Ames test with indicator bacterial strain Salmonella typhimurium TA 1950 in relation to the reactions of the 1st and the 2nd phases of xenobiotics metabolism. It was shown that the presence of tumours leads to inhibition of both metabolic activation processes of the promutagens NM and CP and the conjugation reactions of genetically active metabolites of these compounds with reduced glutathione. Genetic danger is supposed to be increased during application of antitumour preparations, the mutagenic activity of which is due to the activity of their metabolites. It is noted that the most essential effect on biotransformation processes of NM and CP was exhibited by sarcoma M-1, the most important changes of the biotransformation processes of promutagens being observed in the initial period of pathologic process, i.e. on the 3rd day after inoculation. Transplantation of the normal connective tissue of rats had no effect on reactions of both the 1st and the 2nd phase of metabolism of the promutagens studied.     

Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.     

Comparative in vivo mutagenicity testing by SCE and micronucleus induction in mouse bone marrow

Summary The treatment of mice with repeated injections of BUdR and FUdR allows for the demonstration of differentially stained metaphases from bone marrow after FPG (fluorescence plus Giemsa; Perry and Wolff, 1974) treatment. Thus, it is possible to determine the number of SCE's under in vivo conditions, which appears as a very promising system for mutagenicity testing. We studied the response of this system in comparison to the micronucleus test using six mutagenic agents: triaziquone, cyclophosphamide (CP), dimethylphenyltriazene (PDMT), methylnitronitrosoguandine (MNNG), dimethylnitrosamine (DMNA), and diethylnitrosamine (DENA). With the exception of MNNG and DENA, all these agents induce both, SCE and micronuclei, MNNG and DENA being ineffective in both systems. The most potent SCE-inducing agent was triaziquone, followed by PDMT, CP, and DMNA. The quantitative comparison indicates that SCE are induced at 1/10–1/100 of the concentrations which are required for the detection of micronuclei.     

Differential responses of N-nitrosoamines and aromatic amines in the plant cell/microbe coincubation assay

The plant cell/microbe coincubation assay is based on employing living tobacco cells in suspension culture as the activating system for promutagens and the Ames/Salmonella cells as the genetic indicator system. In contrast to aromatic amines(e.g. 2-aminofluorene andm-phenylenediamine) that were previously reported to be activated to products mutagenic in theS. typhimurium strains TA98 or YG1024 by tobacco cells, promutagenic N-nitrosoamines (N-nitrosodimethylamine, N-nitroso-morpholine, N-nitrosopiperidine, N-nitrosomethyl-2-hydroxypropylamine) were not activated to product(s) mutagenic inS. typhimurium TA 100.     

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.     

Comparison of the cytogenetic effects of cyclophosphamide on monkey lymphocytes in vivo and in vitro

The comparative in vivo and in vitro study of chromosomal aberrations and SCE induced by cyclophosphamide (CP) in macaca rhesus lymphocytes was performed. The dose of mutagenic exposure for quantitative estimation of effects was determined as a product of concentration of alkylating CP metabolites on the exposure time. The mutagenic effect caused by the same doses of CP (CP metabolites) appeared similar in vivo and in vitro. This suggests that the results obtained in adequate in vitro mutagen-testing experiments may be quantitatively extrapolated for the in vivo conditions.     

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.     

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.     

Cytogenetic action of the pesticides captan and benomyl in a lymphocyte culture of human peripheral blood in the absence and presence of a system of metabolic activation

Results of the investigation of mutagenic effects of fungicides captan and benomyl in the culture of human peripheral lymphocytes in vitro with and without metabolic activation showed that captan did not manifest cytogenetic activity in any experimental variants, while benomyl induced colchicine-like and weak clastogenic effect in the concentration 10 micrograms/ml in the presence of microsomal activating mixture only.     

The detection of promutagen activation by extracts of cells expressing cytochrome P450IA2 cDNA: preincubation dramatically increases revertant yield in the Ames test.

Two slightly different protocols, the plate incorporation method and the preincubation method, are used in the Ames Salmonella mutagen test. Using a preincubation method, we recently demonstrated efficient activation of a number of food-derived promutagens by extracts of mammalian cells expressing cDNAs of rat-liver cytochrome P450IA2 and of a P450IA2-IA1 hybrid. We report here that, for 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 1-aminoanthracene and several other promutagens, preincubation dramatically increased the number of revertant colonies in the Ames test when extracts of cytochrome P450IA2-containing transfected cells or low concentrations of rat-liver extracts were used as the source of activating enzymes. At higher concentrations of rat-liver extract protein, the effect of preincubation was less pronounced. The effect of preincubation was not due to the low protein concentrations in the assays since increasing the total protein concentration did not abolish the requirement for preincubation for the detection of MeIQ activation at low concentrations of rat-liver extract. In experiments where P450IA2 synthesized in transfected cells in culture is used to study promutagen activation, the plate incorporation protocol may seriously underestimate the capacity of cell extracts to activate promutagens. Thus, interlaboratory comparisons become difficult and unnecessarily large quantities of cell extract protein may be needed to detect promutagen activation. Whenever Ames test assays are carried out under conditions where P450 concentration limits revertant yield, it would be prudent to examine both the preincubation and plate incorporation protocol.     

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.     

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37 degrees C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50-80% of the activity of these compounds observed in the standard assay. Morever, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.     

Cumene hydroperoxide and yeast cytochrome P-450: spectral interactions and effect on the genetic activity of promutagens.

Cells of $\text{Saccharomyces}\text{cerevisiae}$, harvested from log phase cultures, contain cytochrome P-450 and are capable of activating promutagens to products that are genetically active in the same cell. The effect of cumene hydroperoxide, a compound known to support cytochrome P-450-mediated reactions, on the activation of a variety of the promutagens was investigated. In all cases the genetic activity of the promutagens was increased. With dimethyl-nitrosamine as the promutagen, the increased rate of gene conversion was linear for at least 1 hr. Yeast cytochrome P-450 was stable in intact cells in the presence of cumene hydroperoxide. However, in microsomal preparations the cytochrome was rapidly destroyed. When cumene hydroperoxide was added to a suspension of intact yeast cells, a spectrum with a Soret maximum at 455 nm — indicative of an interaction with cytochrome P-450 — was observed.     

The effect of changes in CYP2E1 activity in the liver on toxicity and carcinogenicity of diethylnitrosamine in mice

In this work, the biological effects of diethylnitrosamine (DENA) have been studied under controlled conditions of its metabolism in mice of different ages. The results indicate that the general toxic and hepatocarcinogenic effects of DENA are mostly due to the parent compound, whereas the alkylating metabolites cause hepatic cell damage. Our findings cast doubt on the conventional understanding of the exclusive role of mutagenic activation in the carcinogenic action of chemicals.     

The detection of promutagen activation by extracts of cells expressing cytochrome P450IA2 cDNA: preincubation dramatically increases revertant yield in the Ames test

Two slightly different protocols, the plate incorporation method and the preincubation method, are used in the Ames Salmonella mutagen test. Using a preincubation method, we recently demonstrated efficient activation of a number of food-derived promutagens by extracts of mammalian cells expressing cDNAs of rat-liver cytochrome P450IA2 and of a P450IA2-IA1 hybrid. We report here that, for 2-amino-3,4-dimethylimidazo[4,5-ƒ]quinoline (MeIQ), 1-aminoanthracene and several other promutagens, preincubation dramatically increased the number of revertant colonies in the Ames test when extracts of cytochrome P450IA2-containing transfected cells or low concentrations of rat-liver extracts were used as the source of activating enzymes. At higher concentrations of rat-liver extract protein, the effect of preincubation was less pronounced. The effect of preincubation was not due to the low protein concentrations in the assays since increasing the total protein concentration did not abolish the requirement for preincubation for the detection of MeIQ activation at low concentrations of rat-liver extract. In experiments where P450IA2 synthesized in transfected cells in culture is used to study promutagen activation, the plate incorporation protocol may seriously underestimate the capacity of cell extracts to activate promutagens. Thus, interlaboratory comparisons become difficult and unnecessarily large quantities of cell extract protein may be needed to detect promutagen activation. Whenever Ames test assays are carried out under conditions where P450 concentration limits revertant yield, it would be prudent to examine both the preincubation and plate incorporation protocol.     

Effect of mutagenic metabolic activation of N-nitrosomorpholine on microorganisms]

Almost similar sensitivity is demonstrated of different methods of studying mutagenic metabolic activation of N-nitrosomorpholine (NM) in Salmonella typhimurium TA 1950 (host-mediated assay and the system of metabolic activation with rat liver homogenate). The role of correlation of certain ingredients of NM metabolic activation system with homogenates (homogenate and cofactor NADPH concentrations) is studied. The fact of influence of microsome protein activator (phenobarbital) on the NM mutagen activation effect is established.     

Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity.

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho-anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N-acetyltransferase (YG1029). p-Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o-anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p-anisidine was greater than with o-anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p-anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N-acetyltransferase enzymes play an important role in their metabolism to mutagenic species.     

In vitro detection of indirect-acting genotoxins in the comet assay using Hep G2 cells

The induction of DNA damage by four known promutagens (cyclophosphamide (CP), benzo(a)pyrene (BP), dimethylbenz(a)anthracene and 2-acetylaminofluorene (2AAF) was investigated on Hep G2 using the alkaline single cell electroporesis (SCGE) test, most often referred as the "comet assay". After a 3-day incubation, lysed cells embedded in agarose were electrophoresed under alkaline conditions, dyed with a SYBRgold fluorogen and analysed by the Komet software. Among the comet parameters provided by the image analysis program, statistical analysis did not identify any in particular that could best represent the DNA damages. All promutagens, when compared with the control, caused a statistically significant increase in DNA migration as determined by different parameters such as Olive tail moment, tail extent moment, tail/head or tail length. The data demonstrated the ability and the sensitivity of the comet assay when performed on Hep G2 in the detection of DNA damage induced by promutagens, and its suitability in mutagenicity testing in in vitro short-term assays.