Lack of tumor-promoting effects of flavonoids: studies on rat liver preneoplastic foci and on in vivo and in vitro gap junctional intercellular communication.

Possible tumor-promoting activity of four flavonoids, quercetin (QC), tangeretin (TG), flavone (FO), and flavanone (FN), was examined in a rat liver short-term carcinogenesis assay as well as with in vivo and in vitro assays of inhibition of gap junctional intercellular communication (GJIC). Rat hepatocarcinogenesis was induced by aflatoxin B1 treatment followed by a selection phase (2-acetylaminofluorene treatment and partial hepatectomy), then treatment with or without test chemicals (in vivo studies of antipromotion were not performed). Using glutathione S-transferase placental form (GST-P)-positive foci, we compared the effects of flavonoids (at 1,000 ppm in the diet) with the effects of phenobarbital (PB) on the occurrence of liver preneoplastic lesions. In addition, we studied the effects of flavonoids on GJIC in the livers derived from these experiments and in two types of cultured cells. No significant difference in the number and area of GST-P-positive foci was found after one or three months of treatment between any flavonoid group and control group. In the positive control group, PB markedly increased the numbers and areas of preneoplastic lesions at three months. Whereas PB also decreased by 60% the average size of lucifer yellow dye spread in slices of liver parenchyma free of preneoplastic lesions among the different flavonoids, only TG decreased the dye transfer in vivo: by 30% at one month and 50% at three months. With the dye transfer assay applied to a rat liver epithelial cell line (REL) and the Chinese hamster V79 metabolic cooperation assay, none of the tested flavonoids (< or = 25 microM) inhibited GJIC. Conversely, protective properties were seen for some of the compounds in antipromotion in vitro studies, because TG and FN enhanced the dye transfer in REL cells and FO, TG, and QC partly prevented the inhibition of metabolic cooperation by 12-O-tetradecanoylphorbol-13-acetate. Thus, taken together, our results suggest that QC, FO, and FN do not show tumor-promoting activity. Concerning TG, some discrepancies in the in vivo data are observed. Some of them (GJIC inhibition in liver slices) are probably more relevant to promotion of hepatocarcinogenesis.

Structure-activity relationships within various series of p-phenylenediamine derivatives.

The mutagenicity of 20 p-phenylenediamine derivatives has been investigated in Salmonella typhimurium. Tests were performed in the presence and in the absence of Aroclor 1254-induced liver S9 fractions derived from male Wistar rats. Among five series of compounds tested, nitro-p-phenylenediamines with substituents at the C6 position (4-amino-3-nitro-6-methylaniline; 4-amino-3-nitro-6-methoxyaniline; 4-amino-3-nitro-6-fluoroaniline; 4-amino-3-nitro-6-chloroaniline; and 4-amino-3-nitro-isopropylaniline) were the most mutagenic. In all cases, the compounds were less mutagenic in the absence of S9 than in its presence, but three of the five compounds (the methoxy, fluoro, and chloro derivatives) were still mutagenic without the metabolic activation system. In contrast to the mutagenicity of the C6-substituted compounds, the mutagenicity of analogues with substituents on the C5 position (4-amino-3-nitro-5-beta-hydroxypropylaniline; 4-amino-3-nitro-5-isopropylaniline; 4-amino-3-nitro-5-methylaniline; and 4-amino-3-nitro-5-beta-hydroxyethylaniline) was abolished or reduced. A dramatic reduction in mutagenic activity was also achieved when two methyl groups, instead of one, were added to 4-amino-3-nitroaniline. For example, 4-amino-3-nitro-5,6-dimethylaniline and 4-amino-3-nitro-2,5-dimethylaniline were only weakly mutagenic, and 4-amino-3-nitro-2,6-dimethylaniline as 4-amino-2-6-dimethylaniline; 4-amino-5,6-dimethylaniline; was nonmutagenic. Monocyclic compounds such as 4-amino-2,6-dimethylaniline; 4-amino-5,6-dimethylaniline; 4-amino-2-methoxy-3,5-dimethylaniline, and 4-amino-2,3,5,6-tetramethylaniline were all nonmutagenic in Salmonella typhimurium. The compound 4-amino-2,5-dimethylaniline was weakly mutagenic or nonmutagenic, whereas 4-amino-2,5-dimethoxyaniline was mutagenic. It appears that the mutagenic activity or inactivity of these compounds depends on both the chemical groups present and their positions in the molecule. In this context, it seems that the presence of the NO2 group and the nature of the substituent groups at the C5 and C6 positions on the benzene ring are crucial factors in determining the mutagenicity of these compounds.

Studies on the modulating effects of retinoic acid and retinol acetate using dye transfer and metabolic cooperation assays.

The effects of retinoic acid and retinol acetate on gap junctional communication were examined in two in vitro tests. Rat liver epithelial cell line IAR 203 was used for dye transfer assays, and hamster lung fibroblast V79 cells were used for metabolic cooperation assays. A reversible dose-dependent inhibition of dye transfer was detected after a 1-hr treatment with retinoic acid or retinol acetate at concentrations ranging from 10 to 50 microM. On the other hand, enhancement of dye transfer was observed after a 24-hr treatment with retinoic acid at 0.1 microM. A dose-dependent inhibition of metabolic cooperation was obtained with retinoic acid at noncytotoxic concentrations ranging from 5 to 50 microM. Retinoids and TPA (1 ng/ml) acted synergistically in their inhibition of cell communication. Thus, the assays appear to be complementary: the dye transfer assay was useful in studying the time course and the reversibility of the inhibition or enhancement of dye transfer, whereas the metabolic cooperation assay was effective in quantifying the inhibitory effect of TPA or retinoids and interactions between them.

Mutagenicity of structurally related phenylazo-3-pyridines in Salmonella typhimurium.

Studies were conducted to explore structure-activity relationships for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and nine structurally related compounds in Salmonella typhimurium tester strains TA1535, TA100, TA1537, TA1538, TA98. Each compound was tested for mutagenicity at five or more concentrations that varied from 10-5000 micrograms/plate. We used the standard plate test and the investigations were carried out both in the absence and presence of Aroclor-1254-induced rat-liver homogenate and the components of the NADPH-generating system. Negative response was observed for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and five of its analogues (4'-N,N-diethylamino-1' phenylazo-3-pyridine; 4'-N,N-di-(beta-hydroxyethylamino)-1' phenylazo-3-pyridine; 4'-N-methylamino sulfonic acid-1'-phenylazo-3-pyridine; 4'-N,N-dimethylamino-6'-acetamido-1' phenylazo-3-pyridine, and 4'-N,N-di-(beta-hydroxyethylamino)-6'-methyl-l' phenylazo-3-pyridine). When S9 induced by Aroclor-1254 was present, the compound 4'-N,N-dimethylamino-6-methoxy-1' phenylazo-3-pyridine exhibited mutagenic activity in the two strains TA1538 and TA98. The compound 4',6'-diamino-3-methyl-1'-phenylazo-3-pyridine was also mutagenic, both in the presence and in the absence of S9 mix. The two compounds 4'-N,N-dimethylamino-6-butoxy-1'-phenylazo-3-pyridine and 4'N,N-di-(beta-hydroxyethylamino)-1'-phenylazo-3-[6-N,N-di-(beta- hydroxyethylamino) pyridine were either weakly mutagenic or nonmutagenic. On the basis of these data, it is concluded that the mutagenicity of phenylazo-3-pyridines, like monocyclic aromatic amines and azo dyes, is influenced by the nature of the substituent chemical groups and their positions in the molecular structure of the compounds.

Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5.

Effects of p-aminobenzoic acid (PABA) and of 4-[(2-oxo-3-bornylidene)methyl]-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems. A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci. The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination. The induction of forward mutations was measured in Chinese hamster V79 cells. Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5. V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5. PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations. At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.

Comparative studies on the lethal, mutagenic, and recombinogenic effects of ultraviolet -A, -B, -C, and visible light with and without 8-methoxypsoralen in Saccharomyces cerevisiae.

Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8-MOP) with UV-A or visible light were studied in the haploid strain XV185-14C and diploid strain D5 of Saccharomyces cerevisiae. The induction of his+, lys+, and hom+ reverse mutations was measured in strain XV185-14C. In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over. UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain. UV-C was more mutagenic and recombinogenic than UV-B. UV-A or visible light alone did not induce genotoxic effects at the doses tested. However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185-14C, although mutagenic activity was less than that of UV-B. Visible light plus 8-MOP also induced genotoxic effects in strain XV185-14C. In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses. Visible light plus 8-MOP was also genetically active in strain D5. The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.

Evaluation of the mutagenicity of azo dyes in Salmonella typhimurium: a study of structure-activity relationships.

In order to explore structure-activity relationships, 4,4'-diaminoazobenzene and four structurally related azo dyes were tested for their ability to induce gene mutations in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98. Only 4,4'-diaminoazobenzene and 4,4'-N(beta-hydroxyethylamino)azobenzene were found to be active in the two frameshift strains TA1538 and TA98. Further tests were performed in strain TA98, both in the presence and in the absence of Aroclor 1254-induced rat or hamster liver S9 preparations. The amount of S9 used per plate was 50, 100, 150 or 300 microliters, which corresponds to 10, 20, 30 or 60% of S9 in S9 mix. 4,4'-Diaminoazobenzene was found to be mutagenic, and its mutagenicity depended on the percentage of S9 in S9 mix and the type of S9 fraction used. 4,4'-N-(beta-Hydroxyethylamino)azobenzene was less mutagenic than 4,4'-diaminoazobenzene, indicating a reduction in mutagenicity associated with the beta-hydroxyalkyl substituents. The other three azo dyes [4'-methyl-4-N,N-di(beta-hydroxyethylamino) azobenzene; 4'-amino-6-methyl-4-N,N-di(beta-hydroxyethylamino)azobenzene; and 4'-N(beta-hydroxyethyl-amino)4-N,N-di(beta-hydroxyethylamino)azobe nzene] were inactive, both in the presence and in the absence of the metabolic activation system. The use of the preincubation test did not alter the observed positive or negative response of these compounds. The importance of this finding is that the non-mutagenicity or decreased mutagenicity of these four compounds is predictable on the basis of their chemical structures. These azo dyes, like the non-mutagenic members of series of monocyclic aromatic amines, contain large substituents on one or both of the amino groups of the parent compound, in this case 4,4'-diaminoazobenzene. From our earlier data and the experiments discussed in this paper, we conclude that the study of structure--activity relationships can provide useful information for the prediction and interpretation of mutagenic responses.

Studies on the mutagenicity of aniline in association with norharman in the Salmonella/mammalian microscome assay.

Synopsis The mutagenicity of aniline was investigated in association with norharman using Aroclor 1254-induced and uninduced liver S9 fractions. The non-mutagenicity of aniline in the Salmonella/mammalian microsome test was also re-examined under various experimental conditions. Aniline caused no increase in the number of revertants per plate in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 at concentrations of up to 5000 mug/plate. S9 preparations from rat liver, hamster liver, rat spleen, or hamster spleen were all ineffective in activating aniline to a mutagen in tests using various concentrations of S9 in S9 mix. In contrast to the results with aniline alone, mixtures of aniline and norharman were mutagenic in strain TA1538 in the presence of S9 fractions from liver of rats pretreated with Aroclor 1254. The observed mutagenicity decreased as a function of the concentration of S9 in S9 mix. In the presence of uninduced liver S9 preparations, the mutagenicity of the mixture of the two compounds was slight (three-fold).

Relationships between structure and mutagenic activity of environmental chemicals.

This review analyzes relationships between chemical structure and biological activity for several series of compounds. Its focus is on mutagenicity and carcinogenicity and the predictability of these properties on the basis of the chemical structure. Examples are selected from monocyclic aromatic amines, benzidine derivatives, aminoazobenzene derivatives, nitrofurans, aflatoxins, and sterigmatocystins. Results from mutagenicity tests in Salmonella typhimurium are summarized, and their correlation with carcinogenicity is discussed. The review is concluded with generalizations on the usefulness of studies on relationships between chemical structure and mutagenic activity.

Comparisons of mutation induction by six monocyclic aromatic amines in Salmonella typhimurium tester strains TA97, TA1537, and TA1538.

The mutagenicity of six monocyclic aromatic amines (2,4-diaminoanisole, 2,4-diaminoethoxybenzene, 2,4-diaminopropoxybenzene, m-phenylenediamine, 2,4-diaminotoluene, and nitro-p-phenylenediamine) was investigated in Salmonella typhimurium strains TA97, TA1537, and TA1538 in the presence of two different amounts of Aroclor 1254-induced S9 preparations. Strain TA1538 was found to be the most responsive of the three strains with this group of compounds. Regarding the other strains, TA1537 responded to three of the compounds better than strain TA97, if one calculates responsiveness as the fold-increase in numbers of revertants per plate. However, if one calculates the number of revertants per nanomole or compares the number of induced revertants per plate, TA97 was more responsive than TA1537 for all six compounds. Comparisons of mutagenesis from tests involving strain TA97 are complicated by the large variations in spontaneous mutation frequencies in this strain. The amount of S9 per plate is another important variable in tests of monocyclic aromatic amines; in general, more revertants are detected when the S9 mix contains 10% S9 than when it contains 4% S9. Nevertheless, in all our tests of 2,4-diaminoanisole, 2,4-diaminoethoxybenzene, and 2,4-diaminopropoxybenzene, the same relationship between chemical structure and mutagenic activity was observed. In all three strains, the mutagenic responses become less when the alkoxy substituent on the molecule becomes larger.

Mutagenicity evaluation of nitroanilines and nitroaminophenols in Salmonella typhimurium.

Synopsis In our studies of structure-activity relationships, three nitroanilines and nine nitroaminophenols were tested for their ability to induce mutations in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The compounds m-nitroaniline, 4-nitro-2-aminophenol, and 3-nitro-6-aminophenol were active in two or more of these strains. The compounds o-nitroaniline, 2-nitro-3-aminophenol, 3-nitro-4-aminophenol, 4-nitro-3-aminophenol, 3-nitro-2-aminophenol, 2-nitro-6-aminophenol, 2-nitro-4-aminophenol and 2-nitro-5-aminophenol were inactive, both in the presence and in the absence of S9 mix. The compound p-nitroaniline was also inactive in all tests with the possible exception of that in strain TA98 in the presence of S9 mix, where it was either very weakly mutagenic or non-mutagenic. The mutagenic activity or inactivity of these compounds appears to be dependent on the positions of the amino, nitro, and hydroxy groups in their chemical structures. Etude en mutagénèse des nitroanilines et nitroaminophénols sur Salmonella typhimurium.

Mutagenicity of structurally related aromatic amines in the Salmonella/mammalian microsome test with various S-9 fractions.

The related monocyclic aromatic amines 2,4-diaminoanisole (DAA), 2,4-diaminopropoxybenzene (DAPB) and 2,4-diaminobutoxybenzene (DABB) were tested for mutagenic activity in Salmonella typhimurium strain TA 1538, using S-9 fractions from livers, kidneys and spleens of male Wistar rats for metabolic activation. In the presence of an uninduced liver S-9 fraction, DAA was weakly mutagenic (a two- or threefold increase), and the other compounds were negative. Uninduced S-9 preparations from kidney, spleen or a mixture of kidney, spleen and liver homogenates did not activate any of the compounds. On the other hand, S-9 fractions from rat liver induced with Aroclor 1254 or with a combination of phenobarbital and 5,6-benzoflavone activated both DAA and DAPB, DAA being by far the more mutagenic of the two. S-9 preparations from mixed liver, kidney and spleen homogenates from animals pretreated with Aroclor or with phenobarbital and 5,6-benzoflavone were less effective than the liver homogenates. Aroclor-induced S-9 fractions from kidneys slightly activated DAA, but S-9 fractions from spleen were ineffective in all cases. The mutagenicity ranking of the aromatic amines was DAA greater than DAPB greater than DABB. The latter compound caused little or no increase over the control numbers of revertant colonies. The order of effectiveness of inducers was Aroclor 1254 greater than phenobarbital + 5,6-benzoflavone greater than no inducer, and that of preparations from different organs was liver greater than mixture of liver, kidney and spleen greater than kidney greater than spleen.

Mutagenic evaluation of the monocyclic aromatic amine N-methylamino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene in the Salmonella typhimurium/mammalian microsome test.

The hair-dye component N-methylamino-2-nitro-4-N', N'-bis(2-hydroxyethyl) aminobenzene was investigated for mutagenic activity in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The testing was performed in the absence and in the presence of a rat-liver microsomal activation system induced by Aroclor 1254. Our results indicate that N-methylamino-2-nitro-4-N', N'-bis(2-hydroxyethyl)aminobenzene does not induce mutations in Salmonella typhimurium strains, either in the absence or in the presence of the metabolic activation system. The purity of the compound was controlled by utilizing high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC).

Mutagenic evaluation of the hair-dye component 2,4-diaminophenoxyethanol in Salmonella typhimurium/microsome plate test and Saccharomyces cerevisiae strains D4 and XV185-14C.

The hair-dye coupler 2,4-diaminophenoxyethanol was tested for mutagenicity in the histidine-requiring mutants of Salmonella typhimurium (TA1535, TA100, TA1537, TA1538 and TA98). The tests were carried out in the absence and presence of uninduced as well as an Aroclor-1254-induced rat-liver microsomal activation system. In the absence and presence of uninduced S9 this compound was not mutagenic in all strains used. Negative results were also obtained in the presence of an Aroclor-1254-induced rat-liver microsomal activation system. The mutagenic activity of this compound was also investigated in 2 systems of yeast, the gene conversion system with strain D4 and the reversion system with strain XV185-14C. In the absence and presence of metabolic activation, no mutagenic effect was observed.

The nonmutagenicity of purified 4-amino-2-nitrophenol in Salmonella typhimurium.

Two samples of 4-amino-2-nitrophenol were tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100. Significant mutagenic activity was detected with a sample of this compound obtained from Aldrich (technical grade (Aldrich), TGA) both in the presence and in the absence of a rat-liver S9 metabolic activation mixture. When toluene-insoluble contaminants of the TGA product were separated and tested for mutagenicity, they were found to be mutagenic in the same strains as the TGA sample. After removal of these major contaminants from the TGA product its mutagenicity was reduced; a nonmutagenic sample was obtained by further purification. The other sample of 4-amino-2-nitrophenol, which was synthesized and purified in our laboratory, was nonmutagenic. H.p.l.c. profiles of the unpurified TGA sample and the purified samples revealed a large number of contaminants in the TGA product. Likewise, t.l.c. revealed the presence of contaminants in the TGA product.

Mutagenicity of aminonitrophenol compounds in Salmonella typhimurium: a study of structural-activity relationships.

Synopsis In our studies of structure-activity relationships, four aminonitrophenol isomers and eleven derivatives of 3-amino-4-nitrophenol and 4-amino-3-nitrophenol were tested for their ability to induce mutations in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. In the presence of an Aroclor-1254-induced rat-liver microsomal activation system (S9mix), 4-N-beta-hydroxyethylamino-3-nitroanisole and (4-amino-3-nitro) phenoxyethanol were mutagenic in several of these strains. The compounds 3-amino-4-nitrophenol, 3-N-methylamino-4-nitrophenol, 3-N-beta-hydroxyethylamino-4-nitrophenol, 3-amino-4-nitroanisole, 3-N-methylamino-4-nitroanisole, 3-N-beta-hydroxyethylamino-4-nitroanisole, (3-amino-4-nitro)phenoxyethanol, (3-methylamino-4-nitro)phenoxyethanol, (3-N-beta-hydroxyethylamino-4-nitro)phenoxyethanol, 4-amino-3-nitrophenol and 4-N-beta-hydroxyethylamino-3-nitrophenol were inactive, both in the presence and in the absence of S9 mix. In contrast to the results with 3-amino-4-nitrophenol and 4-amino-3-nitrophenol, which were negative, the isomers 2-amino-4-nitrophenol and 2-amino-5-nitrophenol were found to be mutagenic. These results on mutagenic and non-mutagenic aminonitrophenols and their derivatives suggest that the occurrence of mutagenic activity among these compounds depends on the nature of the substituent chemical groups and their position in the molecular structure of the compounds.

Studies on the mutagenicity of resorcinol and hydroxy-3-(p-amino)anilino-6,N-[(p-amino)phenol]benzoquinone-monoimine-1,4 in Salmonella typhimurium.

The hair-dye coupler resorcinol and the oxidation product of p-phenylenediamine d resorcinol, hydroxy-3-(p-amino)anilino-6,N-[(p-amino)phenol]benzoquinonemonoimine-1.4, were tested for mutagenicity in the histidine-requiring mutants of Salmonella typhimurium (TA100, TA1535, TA1537, TA1538 and TA98). The investigations were carried out in the absence and presence of rat-liver homogenate induced by Aroclor 1254 and the components of the NADPH-generating system. There was no indication of mutagenic activity by these 2 compounds at any of the 8 concentrations used. The nuclear magnetic resonance spectrum of the reaction product of p-phenylenediamine and resorcinol was recorded and is in agreement with its chemical structure.

Structure-activity relationship within a series of m-diaminobenzene derivatives.

We investigated the mutagenicity of m-diaminobenzene (m-phenylenediamine) and four 2,4-diaminoalkylbenzenes (methyl, ethyl, isopropyl and n-butyl) in Salmonella typhimurium strains TA100, TA1538 and TA98 in the absence and presence of S9 induced by Acoclor 1254. m-Diaminobenzene was the most active mutagen, followed by 2,4-diaminotoluene and 2,4-diaminoethylbenzene, resp. Negative response was observed for both 2,4-diaminoisopropylbenzene and 2,4-diamino-n-butylbenzene. Thus, depending on the size of the substituting alkyl group at the C1 position of 2,4-diaminoalkylbenzene, a decline and loss of mutagenic activity was observed.