Diethyldithiocarbamate suppresses the plant activation of aromatic amines into mutagens by inhibiting tobacco cell peroxidase.

Diethyldithiocarbamate is an antimutagen and repressed the activation of promutagens by plant systems. Earlier work implicated the involvement of tobacco cell (TX1) peroxidases in the plant cell activation of aromatic amines. We now present data that diethyldithiocarbamate represses the activation of 2-aminofluorene and m-phenylenediamine by inhibiting intracellular TX1 peroxidases under in vivo conditions. Concentrations of diethyldithiocarbamate that caused a 50% repression of TX1 cell activation of 2-aminofluorene and m-phenylenediamine also induced a 50% inhibition of TX1 cell peroxidase activity. Diethyldithiocarbamate in a concentration range between 25 and 500 microM directly inhibited peroxidase activity in TX1 cell homogenates in a concentration-dependent manner. Similar results were observed with purified horseradish peroxidase. The kinetics of peroxidase activity were studied in homogenates from control cells and cells treated with 750 microM and 25 mM diethyldithiocarbamate. There was no significant difference among the Km values among the three groups with a mean (+/- standard error) Km of 2.58 +/- 0.23 mM. However, the Vmax differed from 4.02 to 2.12 nmoles tetraguaiacol/min/micrograms protein, in the control and in the 25 mM diethyldithiocarbamate treatment group, respectively. These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.     

Plant cells at different stages in their growth curve differentially activate promutagens

Mutagenic activity of the promutagens 2-aminofluorene (2AF) and a contaminant of 4-nitro-o-phenylenediamine (NOP-X) was followed in Ames Salmonella strain TA98 following metabolism by cotton and carrot cell suspension cultures using the plant cell/microbe coincubation assay. Both cell lines were capable of activating each chemical. However, activation capacities of the cell lines differed relative to their respective stage of growth when used. For 2AF activation early-log phase cotton cells and mid-log phase carrot cells proved superior while mid-log phase cotton cells and stationary phase carrot cells proved superior for NOP-X activation. These data indicate that the phase of the growth cycle at which plant cells are harvested can significantly affect their promutagen activation potential.     

Plant metabolic activation of 1,2-dibromoethane (EDB) to a mutagen of greater potency

The results presented in this paper suggest that EDB is capable of being converted to a mutagen by two reaction pathways, one involving a “breakdown” product in vitro and the other involving metabolic activation by plants. Associated with this study the methodology for in vitro qualitatively detecting promutagens which are converted by plant metabolism to active mutagens is described.     

Drosophila wing-spot test: improved detectability of genotoxicity of polycyclic aromatic hydrocarbons

The somatic mutation and recombination tests (SMART) using eyes or wings in the fruit fly Drosophila melanogaster are flexible and sensitive systems for the detection of genotoxicity of individual chemical compounds and complex mixtures. It is of special interest that adults and larvae possess cytochrome P-450-dependent activation systems able to metabolize most promutagens, e.g., nitrosamines, aflatoxins, pyrrolizidine alkaloids, safrole, etc. The polycyclic aromatic hydrocarbons (PAHs) represent a class of promutagens poorly detectable in Drosophila genotoxicity systems. Therefore, new tester strains for the wing-spot test were constructed by introducing chromosomes 1 and 2 from a wild-type strain with increased cytochrome P-450-dependent metabolism linked to a gene on chromosome 2. Previous investigations with the new strains showed their increased detection capability for diethylnitrosamine. Comparative tests with the 3 PAHs benzo[a]pyrene, benz[a]anthracene and 7,12-dimethylbenz[a]anthracene demonstrate, in a reproducible way, that with the new strains all 3 can be detected as active genotoxic compounds. The dose-response curves for all compounds show a plateau with higher exposures. This is interpreted as indicative of a saturation of the cytochrome P-450-dependent activation systems.     

Activation of promutagenic chemicals by Streptomyces griseus containing cytochrome P-450soy

Streptomyces griseus cells containing cytochrome P-450soy oxidize a diverse array of xenobiotic compounds. This metabolic capability was exploited for activation of promutagenic chemicals such as polycyclic aromatic hydrocarbons, aromatic amines and small aliphatics in a modified Salmonella/Ames plate incorporation assay using tester strains TA98 and TA1538. In this assay promutagens such as 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, benzidine, 2-acetylaminofluorene, 2-aminoanthracene, 2,4-diaminotoluene, 4-aminobiphenyl, benzo(a)pyrene, chloropicrin and N-nitrosodimethylamine were oxidized to mutagenic metabolites by S. griseus intact cells which mutated Salmonella tester strains (TA98 and TA1538). S. griseus failed to activate 7,12-dimethylbenzanthracene and 4-chloro-2-nitroaniline. In parallel tests performed with rat liver homogenate (S9), N-nitrosodimethylamine was not activated.     

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.     

Promutagen activation by Vicia faba: an assay based on the induction of sister-chromatid exchanges in Chinese hamster ovary cells

Plant activation of promutagens was studied using Vicia faba S10 (in vitro activation) and the extracts prepared from promutagen-treated roots of Vicia faba (in vivo activation). The induction of sister-chromatid exchanges in Chinese hamster ovary cells was used as an endpoint to evaluate the cytogenetic effects of promutagens activated by Vicia faba. Cyclophosphamide and ethyl alcohol were activated both by Vicia S10 and by the Vicia extracts, and their activation resulted in an increase in SCEs. Benzo[a]pyrene, 2-aminofluorene, and maleic hydrazide were not activated. Aniline was activated, but without effect on the induction of SCEs. The activation capacity in vitro and in vivo of Vicia faba was not very pronounced, except for the activation of ethyl alcohol, when compared with that of rat-liver S9, and showed differences in activation for the 6 chemical agents tested.     

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.     

The plant activation of m-phenylenediamine by Tradescantia clone 03 and clone 4430 cells in liquid suspension culture

In this study, we expanded the use of the genus Tradescantia to investigate the plant activation of promutagens and further refine the methodology of the plant cell/microbe coincubation assay. Liquid suspension cell cultures of Tradescantia clone 03 and Tradescantia clone 4430 were used to activate the promutagen m-phenylenediamine into a mutagenic compound which was detected by Salmonella typhimurium strain TA98 in the plant cell/microbe coincubation assay. Optimum treatment parameters were established for both plant cell lines. Optimum was defined as the lowest concentration or shortest time period that provided consistently positive results and high rates of revertants. Preliminary experiments with both cell lines defined 2.5 mumoles m-phenylenediamine per plate as the optimum concentration to be used in the determination of the optimal coincubation period and the optimal concentration of plant cells. These experiments also determined the optimal physiological stage at which both clones should be used in the coincubation assay. Differences were found in the optimal of coincubation (1h for clone 03, 2 h for clone 4430) and growth stage (mid-log for clone 03, mid- to late-log for clone 4430). Similar activation responses were seen for both clones when the concentration of plant cells (mg/ml) was varied. Under optimized conditions, clone 03 cells demonstrated an approximately 10% higher activation response than clone 4430.     

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.     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Interspecies homology of cytochrome P-450: toxicological significance of cytochrome P-450 cross-reactive with anti-rat P-448-H antibodies in liver microsomes from dogs, monkeys and humans

The mutagenic activation of various promutagens by liver microsomes from dogs, monkeys and humans was investigated. Dog liver microsomes efficiently catalyzed the mutagenic activation of Trp-P-2 and Glu-P-1 followed by IQ and AAF. Monkey liver microsomes were most active in the activation of IQ followed by Glu-P-1, AAF and Trp-P-2. Although there were remarkable individual differences, human liver microsomes were found to be most active in the mutagenic activation of IQ followed by Trp-P-2, Glu-P-1 and AAF. Antibodies against rat P-448-H inhibited the mutagenic activation of Glu-P-1, Trp-P-2 and IQ in rat and dog liver microsomes, and Glu-P-1 and Trp-P-2 in monkey liver microsomes. The activation of Glu-P-1 and IQ in human liver microsomes was also strongly inhibited by anti-P-448-H antibodies. The amounts of cytochrome P-450 cross-reactive with anti-P-448-H antibodies in human liver microsomes highly correlated with the capacity to activate Glu-P-1, Trp-P-2 and IQ but not AAF.     

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.     

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.     

Detection of photomutagens in natural and synthetic fuels

The contribution of non-ionizing radiation to synfuel-related skin carcinogenesis is largely unknown. We have employed a modification of the Salmonella histidine reversion test system to detect photomutation by various fuel substances. For photomutation testing we washed and resuspended cells in buffer, irradiated with 'visible' light in the presence of test substance, and removed aliquots after various light exposures for assay by the plate incorporation method. We have assayed photomutagenicity and microsome-mediated mutagenicity of a crude petroleum, a shale oil, and coal hydrogenation process intermediates. Photomutagenicity was studied using one concentration of each oil; peak revertants per plate for most oils tested were relatively similar to revertants per plate with microsomal activation of the same oil at the corresponding concentration. The shale oil was an exception to this pattern: with light activation, peak revertant numbers per plate were approximately 10 times the value observed with microsomal activation. The samples tested are not assumed to be representative of all petroleums, shale oils or coal oils. Our results do suggest that environmental radiation may be a significant factor in synfuel-related skin carcinogenesis and that photomutagens may be different from enzyme-activatable promutagens.     

Consumption of tea modulates the urinary excretion of mutagens in rats treated with IQ. Role of caffeine.

The present study was undertaken to investigate whether the consumption of green tea and black tea influences the excretion of mutagens and promutagens in rats treated orally with the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Rats were maintained on aqueous extracts (2.5%, w/v) of green tea, black tea or decaffeinated black tea as their sole drinking liquid. After 4 weeks, the animals received, by gastric intubation, a single dose of IQ (5 mg/kg), and urine was collected for 48 h. Direct and indirect mutagenicity, in the presence of an activation system derived from Aroclor 1254-treated rats, was determined in the urine samples using the Ames mutagenicity assay. Consumption of green tea and black tea, but not of decaffeinated black tea, markedly decreased the urinary excretion of mutagens and promutagens. In a further study, supplementation of decaffeinated black tea with caffeine suppressed the excretion of mutagens and promutagens in the urine of rats pretreated with IQ. It is concluded that both green tea and black tea modulate the bioactivation and metabolism of IQ, and that caffeine is largely responsible for this effect.     

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.     

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.     

Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.     

An overview of bioactivation of chemical carcinogens

Most environmental carcinogens require metabolic activation to reactive intermediates and are mutagenic in appropriate test systems. During the last decade, the cDNAs of numerous xenobiotic-metabolizing enzymes have been cloned. The individually expressed enzymes were used to study their substrate specificities and their inhibition by other compounds. Various enzymes were expressed directly in target cells of in vitro mutagenicity tests. This is illustrated in the present study for rat and human sulphotransferases (SULTs) expressed in Salmonella typhimurium TA1538. Numerous compounds were mutagenic in the new test system. Some of these promutagens were activated by several different SULT forms, whereas many other promutagens were activated with high selectivity by a specific enzyme form, but not by genetically closely related forms from the same species (e.g. allelic variants) or orthologous enzymes from other species. Similar findings have been made using recombinant test systems for specific forms of other classes of enzymes (e.g. cytochromes P450). This high selectivity in activation (and inactivation) may explain some organotropisms as well as species and inter-individual differences in the action of carcinogens. Many carcinogen-metabolizing enzymes are induced or inhibited by other xenobiotics. Such interactions can be exploited for chemo-prevention, which however may be carcinogen- and tissue-dependent.