Mouse S9 Research Articles

Metabolism of the new nonbenzodiazepine anxiolytic agent, RWJ-51204, in mouse, rat, dog, monkey and human hepatic S9 fractions, and in rats, dogs and humans

The in vitro and in vivo metabolism of the nonbenzodiazepine anxiolytic agent, RWJ-51204 was investigated after incubation with mice, rat, dog, monkey, and human hepatic S9 fractions in the presence of NADPH-generating system, and a single oral dose administration to rats (100 mg/kg), dogs (5 mg/kg), and humans (2.5 mg/subject). Plasma and red blood cells (2 h, rat) and urine samples (0-24 h, rat, dog and human) were obtained postdose. Unchanged RWJ-51204 (39-93% of the sample in vitro; < or =5% of the sample in vivo) plus 14 metabolites were profiled, quantified and tentatively identified on the basis of API-MS and MS/MS data, and by comparison of synthetic samples. The in vitro and in vivo metabolic pathways for RWJ-51204 are proposed, and the metabolite formations are via the following five pathways: 1. phenyl oxidation, 2. pyrido-oxidation, 3. N-deethoxymethylation, 4. dehydration, and 5. glucuronidation. Pathway 1 formed 4-hydroxy-2-fluoro-phenyl-RWJ-51204 (M1, 7-24% in vitro; 5-60% in vivo) in major amounts, OH-benzimidazole-RWJ-51204 (M2, 5-8% in vitro and in vivo) and diOH-phenyl-RWJ-51204 (< or =5-16% in vitro and in vivo); in conjunction with pathway 5 produced M1 glucuronide (60% in rat & dog; 17% in human), M2 glucuronide (16% in human). Pathways 2-4 formed minor/trace oxidized, and dehydrated metabolites. RWJ-51204 is extensively metabolized in vitro (except dog) and in vivo in rats, dogs and humans.

Mutagenicity of 4-Aminobiphenyl and 4-Acetylaminobiphenyl in Salmonella typhimurium Strains Expressing Different Levels of N-Acetyltransferase

4-Aminobiphenyl (4-ABP), an aromatic amine present in tobacco smoke, is an animal and human carcinogen. 4-ABP can undergo several biotransformation reactions to yield DNA-binding species. The role of acetylation in the biotransformation of 4-ABP to reactive intermediates was investigated by determining mutagenicity in Salmonella typhimurium strains expressing various levels of acetyltransferases (NAT/OAT). Strain YG1029, which has multiple copies of the NAT/OAT gene, was the most sensitive to 4-ABP. With rat S9 activation, 4-ABP (5 μg/plate) induced 789 ± 98 revertants/plate. At that concentration, an average of 200 revertants/plate was seen in both TA100, which has a single copy of the NAT/OAT gene, and in TA100/1,8DNP6, which is NAT/OAT deficient. This pattern was also present when the bacteria were exposed to the acetylated derivative, 4-acetylaminobiphenyl (4-AABP). At 10 μg/plate, 4-AABP induced 855 ± 47 revertants/plate in YG1029 while 169 ± 39 and 149 ± 28 revertants/plate were observed in strains TA100 and TA100/1,8DNP6, respectively. The mutagenic profiles of 4-ABP and 4-AABP observed with the mouse S9 activating system were similar to that seen with the rat. These data establish a correlation between increased bacterial NAT/OAT activity and increased mutagenicity of 4-ABP. Results with both 4-ABP and 4-AABP support acetylation of the oxygen to be a key step in activation.

Chromosomal aberrations in mouse lymphocytes exposed in vitro and in vivo to benzidine and 5 related aromatic amines

Mouse lymphocytes were exposed in vitro for 2 h or in vivo for 24 h to benzidine and related aromatic amines to test for chromosome aberrations (CA) and mitotic indices. Uninduced mouse S9 was used to activate the amines for the in vitro tests to be consistent with the in vivo tests. Contrary to a previous report, no difference could be established in the genotoxicity of benzidine following activation with uninduced S9 compared to induced S9. There were concentration related increases in CA for benzidine and all the amines in vitro except for 4,4′-diaminostilbene which exhibited the greatest cellular toxicity towards cultured lymphocytes. Benzidine and its derivatives showed significant increases in CA in vivo compared to its negative control. The CA values for 4-aminostilbene were significantly higher than the other amines in both in vivo and in vitro studies. These genotoxicity results for 4-aminostilbene are consistent with our previous report of the pronounced CA effects in murine bone-marrow cells but would not be predicted from Salmonella mutagenicity tests.

Effect of four plant phenols, β-carotene and α-tocopherol on 3(H)benzopyrene-DNA interaction in vitro in the presence of rat and mouse liver postmitochondrial fraction

The modulatory effect of β-carotene, α-tocopherol and the four plant phenols eugenol, hydroxychavicol, curcumin and catechin on mouse and rat liver postmitochondrial fraction (S9 mix)-mediated 3(H)benzopyrene (B(a)P)-DNA interaction in vitro was studied. All the plant phenolics significantly inhibited 3(H)B(a)P-DNA interaction in the presence of both mouse and rat liver S9 fractions. In contrast, α-tocopherol proved to be ineffective in the presence of both types of S9 mix, whereas β-Carotene inhibited only mouse S9 mix-mediated 3(H)B(a)P-DNA interaction.

Modified metabolism of a carcinogen, 3-amino-1-methy-5 H-pyrido[4,3- b]indole (Trp-P-2), by liver S9 from Schistosoma japonicum-infected mice

schitosoma japonicum infection has been associated with an increased incidence of liver and colorectal cancers in humans. To explore the mechanisms underlying this association, we investigated the carcinogen-metabolizing properties of liver S9 preparations from S. japonicum-infected mice and compared then with those of S9 from uninfected animals. When the carcinogen 3-amino-1-methyl-5 H-pyridol[4,3- b]indole (Trp-P-2) was incubated with these S9s and the products were analyzed by high-performance liquid chromatography, we observed that the S9 from infected mice had a lower ability to convert Trp-P-2 into 30-hydroxyamino-1-methyl-5 H-pyridol[4,3- b]indole (Trp-2(NHOH)), an activated form of promutagenic Trp-P-2, than the S9 from uninfected mice. We found that both of these S9 preparations have a high ability to reduce Trp-P-2(NHOH) into Trp-P-2; however, the infected-mouse S9 showed a significantly greater reducing power than the control S9. This difference appears to be responsible for the observed lower mutagen-activating potential of the infected mouse S9. These results suggest that hepatic enzyme activities of S. japonicum-infected mice are quantitatively different from those of normal mice.

In vitro and in vivo genotoxicity of 1,3-butadiene and metabolites.

1,3-Butadiene and two major genotoxic metabolites 3,4-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB) were used as model compounds to determine if genetic toxicity findings in animal and human cells can aid in extrapolating animal toxicity data to man. Sister chromatid exchange (SCE) and micronucleus induction results indicated 1,3-butadiene was genotoxic in the bone marrow of the mouse but not the rat. This paralleled the chronic bioassays which showed mice to be more susceptible than rats to 1,3-butadiene carcinogenicity. However, 1,3-butadiene did not induce unscheduled DNA synthesis (UDS) in the rat or mouse hepatocytes following in vivo exposure. Likewise, UDS in rat and mouse hepatocytes in vitro was not induced by EB or DEB. Salmonella typhimurium gene mutation (Ames) tests of 1,3-butadiene using strains TA1535, TA97, TA98, and TA100 and employing rat, mouse, and human liver S9 metabolic systems were barely 2-fold above background only in strain TA1535 at 30% 1,3-butadiene in air with induced and uninduced rat S9 and mouse S9 (uninduced). 1,3-Butadiene was negative in in vitro SCE studies in human whole blood lymphocytes cultures treated in the presence of rat, mouse, or human liver S9 metabolic activation. In general, 1,3-butadiene is genotoxic in vivo but is a weak in vitro genotoxin.

Mutagenicity in v79 cells does not correlate with carcinogenity in small rodents for 12 aromatic amines

The aim of this investigation was to study the correlation between carcinogenicity in small rodents and mutagenic potency of aromatic amines, as measured by the induction of 6-thioguanine resistance in V79 Chinese hamster cells. It has been previously shown that the carcinogenic potency of these compounds is not correlated to their ability to induce DNA breakage, SCEs, or point mutations in bacteria, but a correlation exists with autoradiographic DNA repair test (in primary hepatocyte cultures). Twelve aromatic amines were tested and the rat liver S9 fraction was routinely incorporated in the mutation assay; mouse liver and hamster liver S9 fractions were also used as metabolizing systems. The comparison of the ranks of mutagenic and oncogenic potencies by means of the Spearman test shows no correlation between carcinogenicity and V79 cell mutagenicity of the tested aromatic amines. There was a generally low mutagenicity seen for aromatic amines in V79 cells. In some cases this could be attributed to an insufficient metabolic activation by rat S9. For example, benzidine, which was inactive when assayed in the presence of rat S9, became mutagenic when in the presence of mouse S9. On the other hand, hamster S9, which has been shown to be the best activating system for 2-acetylaminofluorene in the Ames test, did not activate this compound in V79 cells. Inadequate metabolic activation of the standard system (rat S9) used in this work could explain the low mutagenicity and the lack of correlation observed between mutagenicity and carcinogenicity. A second possibility is that point mutation is not the essential end point for the initiating activity of aromatic amines during the carcinogenic process. A third possibility is that the activity of some aromatic amines is not restricted to the initiation step in carcinogenesis. Chronic treatments with the sublethal doses often result in significant promoting activities, which could mask efficiently the initiating potential of the same chemicals.

Dependence on intact cells for the in vitro activation of dimethylnitrosamine to an immunosuppressive form

The in vitro activation of dimethylnitrosamine (DMN) to an immunosuppressive form was studied utilizing liver-enzyme fractions and intact hepatocytes. The N-demethylation of DMN by mouse S9 and microsome preparations was confirmed by determination of formaldehyde generation. S9 fractions from both phenobarbital(PB)- and isopropanol(iso)-pretreated mice displayed significantly greater demethylase activity than uninduced S9 fractions. However, when incubated with spleen cells, neither S9 preparation was capable of activating DMN to a form capable of suppressing antibody responses by recovered spleen cells. In contrast, the positive control, cyclophosphamide, was activated to a markedly immunosuppressive form. S9 fractions failed to activate DMN to an immunosuppressive form regardless of S9 concentration, time of preincubation, or rocking speed. Liver microsomes from PB-pretreated mice displayed significantly greater N-demethylase activity than S9 fractions yet were unable to activate DMN to an immunosuppressive form. In contrast, the addition of DMN to mixed cultures of mouse hepatocytes and mouse spleen cells resulted in activation of DMN and marked suppression of antibody responses. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. Unlike the metabolism of DMN to a mutagenic form, the in vitro activation of DMN to an immunosuppressive form was therefore dependent on intact cells. Furthermore, the activation by intact hepatocytes was shown to be dependent on cell-cell contact or close proximity of activating and target cells.

Retinol (vitamin A) inhibits the mutagenicity of o-aminoazotoluene activated by liver microsomes from several species in the Ames test

Retinol (vitamin A) has earlier been shown to inhibit the mutagenicity of o-aminoazotoluene (OAAT) in the Salmonella/microsome assay when OAAT is activated with S9 from Sprague-Dawley rats. The resuts presented in this paper confirm this and also show that S9 from mice, hamsters and gerbils activates OAAT to mutagenic metabolites detected by Salmonella typhimurium TA100. However, S9 from rabbits is inactive. The S9 fraction from rabbits also shows a low aryl hydrocarbon hydroxylase (AHH) activity. The AHH activity or protein content of the microsomal fraction cannot be used to predict the activating capacity of S9 from the other species. Retinol, added in vitro, inhibits the mutagenic effect of OAAT activated by mouse, gerbil or hamster S9. The strongest inhibition is observed with hamster S9 while the inhibition of mouse and gerbil S9 is lower but still higher than in the rat.

Comparison of S9 fractions from rats, mice, and Chinese hamsters to metabolize dimethylnitrosamine and diethylnitrosamine to intermediates that induce sister-chromatid exchanges in V79 cells

This study was designed to compare the ability of hepatic S9 preparations from rats, mice and Chinese hamsters to activate dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) into intermediates capable of causing sister-chromatid exchanges (SCEs) in V79 cells. The results indicated that the hepatic preparations from the species studied differed in their ability to activate DMN while in the case of DEN no species difference in bioactivation was observed. For DMN, a dose-dependent increase of SCEs was seen in cultures treated with various concentrations plus rat, mouse or hamster S9 mix and the mouse S9 mix was the most effective activator of DMN and this was followed by the rat and Chinese hamster S9 mix with little difference noted for the latter 2 species. For DEN, an increase of SCE was seen in cultures treated with various doses plus S9 mix from any of the species studied but unlike DMN, the SCE rate did not increase in a significant dose-dependent manner and no species difference in bioactivation was observed.

Effects of plant-derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food

The ability of 3 plant flavonoids (morin, myricetin and quercetin) and 4 polyphenolic acids (caffeic acid, chlorogenic acid, ellagic acid and ferulic acid) to inhibit the genotoxic effects of a number of cooked-food mutagens (IQ, MeIQ, MeIQx, Trp-P-1 and Trp-P-2), was investigated in a bacterial mutation assay using Salmonella typhimurium TA98 as indicator and hepatic S9 mixes from either SWR mice or Syrian hamster as metabolic activating systems. Although the polyphenolic acids failed to have an effect, the flavonoids generally inhibited IQ, MeIQ, MeIQx and Trp-P-1 induced mutagenesis in a dose-dependent manner, irrespective of the source of S9. This was not the case with Trp-P-2 where the flavonoids were only observed to inhibit when SWR mouse S9 but not Syrian hamster S9 was used. Of the 3 compounds, myricetin and quercetin were superior to morin in their inhibitory capacity.

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by (i) exposure level, (ii) organ distribution of (DNA) dose, and (iii) genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenicity potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria ( E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG > ENU ⩾ DES > DEN ≅ EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations. The results also indicated that O 6-ethylguanine was the DNA adduct most probably responsible for mutagenesis both in the bacteria and the mammalian cells. Comparative mutagenesis experiments performed with the E. coli cells in vitro with and without mammalian liver preparations (mouse S9) and in animal-mediated assays, in which the E. coli indicators were present in livers of treated mice, gave the following results: The addition of mouse liver S9 allows the detection of activating (DEN) or deactivating (ENNG) effects of mammalian metabolism, but the ranking order of mutagenic potency remained the same, namely ENNG > ENU > DES > EMS ≅ DEN. In the animal-mediated assays, on the other hand, the order of mutagenic potency was quite different, namely, DEN > ENU > ENNG > EMS. This latter order of activity is similar to the order of carcinogenicity of these compounds in rodents, and probably also reflects the degree of DNA alkylation in liver cells of the treated animals. These and other earlier results indicate that the relative ranking order of mutagenic potency observed in vitro, regardless whether it was obtained with bacterial or mammalian cells, is not representative of the situation in vivo. Although the addition in in vitro assays of mammalian liver preparations allows the detection of those compounds which are likely to be activated or deactivated in vivo, it appears that the magnitude of the effect is not adequately represented in the in vitro assays. In search for rapid methods for estimating genotoxic factors in various organs in vivo, use was made of the DNA repair host-mediated assay. In this test, repairable DNA damage is measured in pairs of E. coli K12 cells which differ by their ability of repairing lethal DNA damage, and which can be recovered from stomach, intestine, colon, liver, spleen, lungs, kidneys, testes and the blood of treated rodents (mice). Preliminary experiments using a variety of differently acting compounds have indicated that this assay procedure may become useful in rapidly assessing the presence and persistence of genotoxic factors in various organs of mice; its general usefulness as genotoxic dose monitor in vivo is presently being investigated.

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisole O-demethylase ( p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures. In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD). Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH. At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1- b]thiazole) in the D 7 strain of Saccharomyces cerevisiae.

NADPH-regenerating system: influences of microsomal mono-oxygenase stability during incubations for the liver microsomal assay with rat and mouse S9 fractions

NADPH-regenerating system: influences of microsomal mono-oxygenase stability during incubations for the liver microsomal assay with rat and mouse S9 fractions

Genetic effects of procarbazine in the yeast Saccharomyces cerevisiae, strain D4.

Procarbazine ( PCZ ) was tested for its ability to induce mitotic gene conversions at the ade and trp loci of Saccharomyces cerevisiae, strain D4. The influence of the following factors was examined: growth phase of the yeast cells (log vs stationary phase), pH of the treatment solution, and addition of mouse S9 fractions prepared from different organs. The drug was found more toxic and mutagenic at low doses (up to 25 mg/ml) for log phase cells, and scarcely toxic but highly mutagenic, even at high doses, for stationary phase cells. PCZ activity was reduced by acidic pH, and suppressed by S9 mix. Gene conversions were also analyzed in the intrasanguineous host-mediated assay performed in mice orally administered with PCZ . In such conditions PCZ was ineffective in stimulating mitotic gene conversions, probably owing to its inactivation in the acidic environment of the gastroenteric tract.

A comparison of the mutagenicity and macromolecular binding of the carcinogens Michler's ketone and reduced Michler's ketone

The mutagenicity of 4,4′-bis(dimethylamino)benzophenone (Michler's ketone, MK, CAS No. 90–94-8) and 4,4′-methylenebis( N, N-dimethyl)benzamine (reduced Michler's ketone, RMK, CAS No. 101-61-1) for Salmonella typhimurium strain TA100 was compared using activated 9000 × g (S9) liver supernatants from 2 animal species. RMK, but not MK, was mutagenic when incubated with phenobarbital-induced B6D2F 1 mouse or Osborne-Mendel rat-liver S9. The mutagenic response for RMK was linear at doses from 1 to 33 μg/plate. A higher percentage of RMK and MK became irreversibly bound to mouse-liver macromolecules than to rat-liver macromolecules when incubated at 37°C in the presence of reduced nicotinamide adenine dinucleotide phosphate.

Promutagen activation by rodent-liver postmitochondrial fractions in the L5178Y/TK cell mutation assay

Individual S9 microsomal fractions prepared from normal livers of 8 rodent species or strains and from 1 rat strain pretreated with Aroclor 1254, were used to metabolize the promutagens N-acetyl-2-aminofluorene, 1,2--benzanthracene, to metabolize the promutagens N-acetyl-2-aminofluorene, 1,2-benzanthracene, benzo[ a]pyrene, and 3-methylcholanthrene to active forms during 3-h co-incubation in the presence of L5178Y/TK +/− cells. The 8 compatible S9 preparations all converted each of the 4 chemical carcinogens into active mutagens with varied efficiencies except for the Aroclor-induced rat S9/benzanthracene combination which produced only weak activity. Aroclor induction did not notably enhance the mutagenicity of benzo[ a]pyrene or 3-methylcholanthrene beyond that activity mediated by the other non-induced preparations. Syrian hamster S9 and, to a lesser degree, C57BL/6J mouse S9 were exceptionally active in converting N-acetyl-2-aminofluorene to toxic and mutagenic metabolites. One source of Swiss mouse liver (Blu : Ha ICR) provided the most active S9 when tested with the 3 polycyclic aromatic hydrocarbons. In general, mutagenicity and cytotoxicity were roughly correlated within S9 + promutagen combinations. Almost all of the methylcholanthrene metabolizing activity was lost by the 12th week when Aroclor-induced rat S9 was held at −20°C, yet this activity remained constant when similar S9 was stored at −80°C for 14 weeks. Surprisingly, some S9 sources including the induced rat-liver preparation converted anthracene to a weak or border-line mutagen. The activation of both 1,2-benzanthracene and anthracene may be linked within each species or strain although Aroclor induction enhanced anthracene mutagenicity yet attenuated the mutagenicity of 1,2-benzanthracene. Collectively, these data underscore the current inchoate state of development for S9 coupled somatic cell mutation assays.