Majdi M. Shahin

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.

Structure-activity relationships within a series of 2,4-diaminoalkoxybenzene compounds

Six 2,4-diaminoalkoxybenzenes were examined for their ability to induce mutation in Salmonella typhimurium. Each compound was tested at 8 concentrations in 5 strains. The mutagenicity was influenced by the size of the alkoxy group substituted at the C1 position of 2,4-diaminobenzene. When S9 induced by Aroclor 1254 was present, 2,4-diaminoanisole (the methoxy derivative) exhibited the highest mutagenic activity. The compounds 2,4-diaminoethoxybenzene, 2,4-diaminoisopropoxybenzene and and 2,4-diamino-n-propoxybenzene were also mutagenic, but were distinctly less active than 2,4-diaminoanisole. With the compounds 2,4-diaminophenoxyethanol and 2,4-diamino-n-butoxybenzene, the increases in numbers of revertant colonies above control levels were slight or absent. The mutagenicity of 2,4-diaminoalkoxybenzenes was detected in strains TA98, TA1538 and in some cases TA1537. None of the compounds was active in strain TA1535. The relative response of the various strains suggests that 2,4-diaminoalkoxybenzenes induce frameshift mutations but not base-pair substitutions. None of the compounds was active without metabolic activation. In addition to conducting the standard plate test, we tested the urine of rats exposed to 2,4-diaminophenoxyethanol and 2,4-diaminoanisole for mutagenicity in Salmonella typhimurium. The rats were exposed by topical application, oral administration or intraperitoneal injection. The results were positive for 2,4-diaminoanisole and negative for 2,4-diaminophenoxyethanol.

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.

Studies on the mutagenicity of p-phenylenediamine in Salmonella typhimurium. Presence of PCB's in rat-liver microsomal fraction induced by Aroclor.

The mutagenicity of fresh solutions of p-phenylenediamine (PPD) and Aroclor 1254 was investigated. The histidine-requiring strains of Salmonella typhimurium were used in the absence and presence of uninduced and/or Aroclor-induced rat-liver homogenate. The presence of polychlorinated biphenyls (PCBs) was also examined by chromatographic methods in Aroclor-induced rat-liver homogenate. In the absence of metabolic activation, as well as in the presence of uninduced rat-liver homogenate, PPD was not mutagenic in the strains used. In the presence of Aroclor-induced S9 a twofold increase (or less) was observed in the number of revertant colonies over those of the controls in TA1538 and TA98. There was no increase in the number of revertant colonies over those of the controls when PPD was dissolved in NH4OH solution and the solution mixed with H2O2 before the addition of S9 mix. Aroclor 1254 was not mutagenic in TA1538 or TA98. However, the presence of PCBs in Aroclor-induced rat-liver homogenate (induced S9) was identified by gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) and gas--liquid chromatography/mass spectrometry (GC/MS).

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.

Mutagenicity evaluation of nitroanilines and nitroaminophenols in Salmonella typhimurium

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.

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

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‐β‐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‐β‐hydroxyethylamino‐4‐nitrophenol, 3‐amino‐4‐nitroanisole, 3‐N‐methylamino‐4‐nitroanisole, 3‐N‐β‐hydroxyethylamino‐4‐nitroanisole, (3‐amino‐4‐nitro)phenoxyethanol, (3‐methylamino‐4‐nitro)phenoxyethanol, (3‐N‐β‐hydroxyethylamino‐4‐nitro)phenoxyethanol, 4‐amino‐3‐nitrophenol and 4‐N‐β‐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.

Relationships between the Chemical Structure and Mutagenic Activity of Monocyclic Aromatic Amines

Hundreds of compounds have now been tested for mutagenic activity in many laboratories throughout the world. Mutagenicity tests are performed to validate a variety of assay systems and to contribute to the toxicological safety evaluation of chemicals. An area of research in genetic toxicology that has important implications for the development of safe chemical products is the study of series of homologous compounds in order to relate chemical structure to genetic activity.

Studies on the mutagenicity of resorcinol and hydroxy-3-(p-amino)anilino-6,N-[(p-amino)phenol]benzoquinonemonoimine-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 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.