Aroclor 1254-induced Rat Liver Homogenate Research Articles

Evaluation of mutagenic and antimutagenic activities of neem (Azadirachta indica) seed oil in the in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test

The possible mutagenic and antimutagenic activity of neem oil (NO) and its DMSO extract (NDE) were, examined in the Ames Salmonella/microsome mutagenicity test and the mouse bone marrow micronucleus assay. Eight different strains of Salmonella typhimurium were, used to study the genotoxicity of neem oil both in the presence and absence of Aroclor-1254 induced rat liver homogenate (S9). Two-dose treatment protocol was, employed to study the cytogenetic activity in micronucleus assay. Similarly, the antimutagenic activity of neem oil and NDE was studied against mitomycin (MMC) and 7,12-dimethylbenz[a]anthracene (DMBA) in the above two test systems. Neem oil was non-mutagenic in all the eight tester strains of Salmonella typhimurium both in the presence and absence of S9 mix. In the present study, there was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in neem oil treated groups over the negative control (DMSO) group of animals, indicating the non-clastogenic activity of neem oil in the micronucleus test. Neem oil showed good antimutagenic activity against DMBA induced mutagenicity compared to its DMSO extract. However, neem oil showed comparatively less antimutagenicity against MMC in the Ames assay. In vivo anticlastogenic assays shows that neem oil exhibited better activity against DMBA induced clastogenicity. These results indicate non-mutagenic activity of neem oil and significant antimutagenic activity of neem oil suggesting its pharmacological importance for the prevention of cancer.

Studies of the Toxicological Potential of Tripeptides (L-Valyl-L-prolyl-L-proline and L-lsoleucyl-L-prolyl-L-proline): IX. Evaluation of the Mutagenic Potential of Synthesized L-Valyl-L-prolyl-L-proline in the Salmonella-Escherichia coli/Microsome, Incorporation Assay

The objective of this study was to assess the mutagenic potential of a synthesized tripeptide, L-valyl-L-prolyl-L-proline (VPP), to induce mutational changes in Salmonella typhimurium LT2 strains TA1535, TA1537, TA98, and TA100, and Escherichia coli strain WP2uvrA in the classical Ames test protocol. Bacteria were exposed to plate concentrations of VPP of 0, 156.2, 312.5, 625, 1250, 2500, and 5,000 microg/plate in distilled water, in the presence and absence of Aroclor 1254-induced rat liver homogenate preparation (S9). Positive-control agents included sodium azide (TA100 and TA1535); 2-aminoanthracene (TA98, TA100, TA1535, TA1537, and WP2uvrA); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); and N-ethyl-N'-nitro-N-nitrosoguanidine (WP2uvrA) in DMSO. Incubations were conducted at 37 degrees C for about 48 h then revertant colonies were counted. All positive-control agents were consistently and unequivocally positive, but there was no evidence that VPP induced increases in the incidences of revertant colonies in any bacterial strain with and without metabolic activation. These findings were replicated in a second, confirmatory test performed with and without S9. The results of the experiments revealed no treatment-associated changes in the incidence of revertant colonies in any bacterial strain tested. These results support a conclusion that, under the experimental conditions described, there is no evidence that VPP possesses mutagenic potential.

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4′-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6β-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4′-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6β-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6β-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.

The use of initiated cells as a test system for the detection of inhibitors of gap junctional intercellular communication.

The effects of five non-mutagenic carcinogens--Aroclor 1260, benzoyl peroxide (BP), phenobarbital (PB), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,1'-(2,2,2-trichloroethylidene)bis[4-chlorobenzene] (DDT)--on gap junctional intercellular communication (GJIC) were tested in a cell line consisting of initiated cells (3PC). Four agents suspected of tumor promotion activity--o-anisidine, clofibrate, L-ethionone and d-limonene--were also tested for their effects on GJIC. Finally sodium fluoride (NaF), whose carcinogenic property is still unclear, was tested for its effects on GJIC in the 3PC cell line. Four of the five selected tumor promoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC between these initiated epidermal cells. The four non-mutagenic carcinogens with tumor-promoting activity in vivo (o-anisidine, clofibrate, L-ethionine and d-limonene) all inhibited GJIC, whereas NaF had no effect. Seven compounds (o-anisidine, Aroclor 1260, BP, DDT, L-ethionine, d-limonene and TPA) had a dose-dependent as well as time-dependent inhibitory effect on GJIC. Under the experimental conditions used, clofibrate showed only a dose-related inhibition of GJIC. PB showed no inhibitory effect on GJIC in the 3PC cell line. In order to determine the role of biotransformation in the tumor-promoting activity of PB, its effect on GJIC was also examined in the presence of an Aroclor 1254-induced rat liver homogenate (S9 mix) and in the hepatoma cell line HepG2. In the presence of rat liver homogenate PB decreased GJIC in the 3PC cell line, whereas in the HepG2 cells PB showed a time- and dose-dependent inhibitory effect. To study the potential differences in susceptibility of cells representing different stages in the process of tumor formation, the effect of the selected tumor promoters on GJIC was also investigated in primary mouse keratinocytes and in a mouse skin carcinoma-derived cell line (CA3/7). Primary keratinocytes were sometimes more (BP and clofibrate) and sometimes less sensitive (ethionine and limonene) for inhibitory effects on GJIC compared to the effects in the cell line 3PC. Except for TPA and anisidin, GJIC between the CA3/7 cells was less affected by the selected agents compared to the 3PC cell line. These results show that, during the process of tumor formation the susceptibility of cells to inhibition of GJIC by tumor promoters is variable. Overall the CA3/7 cells are less sensitive compared to 3PC cells. The susceptibility of primary keratinocytes is variable compared to 3PC cells, depending on the agent used. These results also show that GJIC is a valid parameter for testing the tumor-promoting activity of compounds. Finally, this study demonstrates that mouse keratinocyte cell lines could serve as an in vitro model for the detection of non-mutagenic carcinogens with diverse target organs in vivo. For this use the cell line consisting of initiated cells (3PC) is more sensitive than the carcinoma-derived cell line CA3/7.

Mutagenicity evaluation of riot control agent o-chlorobenzylidene malononitrile (CS) in the Ames Salmonella/microsome test.

o-Chlorobenzylidene malononitrile (CS), a riot control agent, was evaluated for its possible mutagenic activity in the Ames Salmonella/mammalian microsome mutagenicity test. Five histidine-deficient (His-) mutant tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104--were used. The liquid preincubation procedure was used with metabolic activation (presence of S9 mixture) and without metabolic activation (absence of S9 mixture). For the experiments with metabolic activation, three different concentrations of S9 fraction (supernatant of Aroclor 1254-induced rat liver homogenate at 9000 g)--5%, 15% and 30% in S9 mixture--were used. Along with mutagenic activity, CS was also evaluated for cytotoxic activity in all the five tester strains of Salmonella typhimurium, both in the presence and absence of S9 mixture. The mutagenic and cytotoxic activities of CS were assessed by counting the His+ revertant colonies and by counting the microcolonies (His-, auxotrophs in the background lawn), respectively, and the respective mean values were compared with the relative negative (solvent) control. A dose range of 12.5-800 micrograms plate-1 for CS did not induce a mutagenic response either in the presence or absence of S9 mix. No change in the negative mutagenic response of CS has been observed even in the presence of an elevated level of S9 fraction in the S9 mix. A dose of 200 micrograms plate-1 for CS was found to be cytotoxic by decreasing the surviving cells as well as His+ revertant colonies; however, the effect was reduced in the presence of an elevated level of S9 fraction in the S9 mix.

Genotoxicity studies on selected organosilicon compounds: In vitro assays

A series of 12 organosilicon compounds representing potential intermediates in the synthesis and degradation of polydimethylsiloxanes were evaluated for genotoxic potential with a battery of in vitro assays. Microbial assays included the Ames bacterial reverse mutation in Salmonella, mitotic gene conversion in Saccharomyces cerevisiae D4 and DNA repair in E. Coli pol A +/−. These assays were conducted with and without a metabolic activation system containing Aroclor 1254-induced rat-liver homogenate. Forward gene mutation, sister-chromatid exchange, DNA alkaline elution and chromosome aberration potential were evaluated in mouse lymphoma L5178Y tissue culture cells. The tissue culture assays were performed with and without metabolic activation mixture utilizing uninduced mouse-liver S-9. The use of this enzyme preparation was felt to more closely mimic the actual in vivo situation and to be more compatible with mouse cells employed in the assay. No evidence of gene mutation was observed. However, six of the 12 compounds evaluated demonstrated potential in vitro clastogenic (chromosome damaging) activity.

In vitro antimutagenicity of curcumin against environmental mutagens

The effects of curcumin, the yellow pigment of the spice, turmeric ( Curcuma longa) on the mutagenicity of several environmental mutagens were investigated in the Salmonella/microsome test with or without Aroclor 1254-induced rat-liver homogenate (S-9 mix). With Salmonella typhimurium strain TA98 in the presence of S-9 mix, curcumin inhibited the mutagenicity of bidi and cigarette smoke condensates, tobacco and masheri extracts, benzo[α]pyrne and dimethyl benzo[α]anthracene in a dose-dependent manner. Curcumin did not influence the mutagenicity without S-9 mix of sodium azide, monoacetylhydrazine and streptozocin in strain TA100 nor of 4-nitrophenylenediamine in strain TA98. Our observations indicate that curcumin may alter the metabolic activation and detoxification of mutagens.

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (>500 μg/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.

An In vitro test for teratogens: Its practical application

An in vitro test for teratogens has been used successfully for more than 3 years. The method involves exposing undifferentiated rat embryo midbrain and limb cells to test compounds and observing the effect on subsequent cell differentiation. Experience of using the test has confirmed the accuracy of prediction (> 90%) suggested by a blind trial. The test has been used at the early stages of pharmacological evaluation in the selection of non-teratogenic pharmaceuticals and 250 compounds are tested on average each year. Maternal metabolism is modelled by the inclusion of Aroclor 1254-induced rat liver homogenate plus cofactors (S-9 mix). The concentrations of S-9 mix (50–100 μl/ml culture medium) conventionally used in the Ames bacterial mutagenicity test are toxic to rat embryo cells, but greatly reduced concentrations (3–5 μl/ml) are not toxic but are still able to activate pro-teratogens such as cyclophosphamide. However, most potentially teratogenic compounds tested are toxic in the absence of active preparations of drug-metabolizing enzymes. The conclusion that most teratogens are direct acting may be premature, since evidence has been found for drug-metabolizing activity in the embryo cells themselves.

Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells.

Seven promutagens belonging to two chemical classes--polycyclic aromatic hydrocarbons (PAHs) (benzo[a]pyrene, dimethyl benz[a]anthracene, 3-methylcholanthrene, fluoranthene) and alkyl nitrosamines (dimethyl, diethyl, and dibutyl nitrosamine)--were studied in Chinese hamster ovary (CHO) cells. Findings of practical importance in the use of Aroclor 1254-induced rat liver homogenate (S9) in the CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay were made. Our novel findings are (1) the inclusion of CaCl2 in the S9 cofactor mixture dramatically decreased the cytotoxicity of S9, and (2) different S9 optimum concentrations were observed for structurally similar promutagens. The inclusion of CaCl2 in the S9 cofactor mixture and the testing of each chemical of unknown S9 requirement at several S9 concentrations are therefore recommended for assaying promutagens in the CHO/HGPRT mutation assay.