Effect of dipyridoimidazole pretreatment on mutagen activation in rats.

Rats were pretreated with a single oral dose of different mutagenic fractions obtained from glutamic acid pyrolysate: Glu-P-2 (2-amino-dipyrido[1,2-a:3',2'-d]imidazole), Glu-P-3 (3-amino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole), the tar residue and a basic extract (B2). The liver S9 fractions of these animals were used to investigate the mutagenic activation of 3 promutagens (2-aminoanthracene, Glu-P-2 and Glu-P-3) in Salmonella typhimurium strain TA1538. Different factors were analyzed; influence of the structure of the compounds administered, doses, time interval between pretreatment and sacrifice and sex of the rats. Interpretation of the hepatic induction effects was complicated, however, by the fact that simple oral pretreatment with the solvents (DMSO or ethanol) enhances the activation of the substrates tested for mutagenicity. A dose-effect relationship was found between 2-AA mutagenic activation and Glu-P-2 pretreatment. Glu-P-3 induced the activation of 2-AA more than did Glu-P-2, in the male as in the female. The mutagenicity of 2-AA activated with S9 from male rats was found to be optimal after 24 h pretreatment with 20 mg Glu-P-2/kg b.w. The mutagenicity of Glu-P-2 was poorly influenced by the different pretreatments applied to either the males or the females, whereas some dose effect was found in the autoinduction of Glu-P-2 mutagenicity. Compared to Glu-P-2, the mutagenicity of Glu-P-3 was increased at higher levels when tested with S9 from males pretreated with the same compound, but no differences were observed between males and females.

Mutation spectrum of the mutagen 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido[1,2-a:3',2'- d]imidazole in Escherichia coli.

The mutation frequency and mutation spectrum resulting from 3-N-acetylamino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole (AGluP3) DNA adducts using a previously developed forward mutation assay were established. AGluP3-induced mutagenesis requires the umuC gene product(s) and exhibits similar amounts of base pair substitution and frameshift mutation. Comparison between these results and those obtained with the isosteric amine 2-N-acetylaminofluorene suggests the involvement of deacetylated adduct in the molecular mechanisms of AGluP3-induced mutagenesis.

Comparison of the reactivity of B-DNA and Z-DNA with two isosteric chemical carcinogens: 2-N,N-acetoxyacetylaminofluorene and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido-[1,2-a:3',2' -d] imidazole.

The reactivity of nucleic acids in various conformations and two isosteric chemical carcinogens 2-N,N-acetoxyacetylaminofluorene (N-AcO-AAF) and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido [1,2-a:3',2'-d] imidazole (N-AcO-AGlu-P-3) have been studied. Both carcinogens bind covalently to poly(dG-dC).poly(dG-dC) (B form) and to poly(dG-br5C).poly(dG-br5dC) (Z form). They also bind covalently to (dC-dG)16 and to (dG-dT)15 sequences inserted in plasmids when the inserts are in the B form but they do not bind to the inserts in the Z form. The reactivity of guanine residues at the B-Z junctions depends upon the superhelical density of the plasmids and upon the base sequences at the junction. The distribution of AGlu-P-3 modified guanines in a restriction fragment of pBR322 is not uniform and is different from that of AAF-modified guanines. The conclusion is that N-AcO-Glu-P-3 as N-AcO-AAF can probe at the nucleotide level the polymorphism of DNA. On the other hand, the non-reactivity of both chemical carcinogens and Z-DNA and the hyperreactivity of some junctions might have some importance in the understanding of chemical carcinogenesis.

In vitro enzymatic methylation of DNA modified with the mutagenic amine: 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido(1,2-a:3'2'-d )imidazole.

Both the initial velocity and the overall methylation of DNA modified by acetylamino-4,6-dimethyldipyrido(1,2-a:3',2'-d)imidazole (A-Glu-P-3) by rat liver DNA-(cytosine-5-)-methyltransferase are decreased as compared to native DNA. A-Glu-P-3 bound to guanine residues may block the movement of the enzyme along the helix. The modified DNA does not inhibit the enzymatic methylation of native DNA. The enzyme has a lower affinity for the modified DNA than for native DNA. The hypomethylation caused by this carcinogen could have a significance in gene activity, cellular differentiation and cancer induction.

Immunological titration of 3-N-acetyl-hydroxyamino-4,6-dimethyldipyrido(1,2-a:3',2'-d) imidazole-rat liver DNA adducts.

Antibodies to N-(guanosin-8-yl)-3-N-acetylamino-4,6-dimethyldipyrido(1,2-a :3',2'-d)imidazole were elicited in rabbits by immunization with a conjugate formed between this compound and bovine serum albumin. The specificity of the antibodies was studied by radioimmunoassay. These antibodies were used to titrate the adducts formed in liver DNA of rats treated with 3-N-acetyl-hydroxyamino-4,6-dimethyldipyrido(1,2-a:3',2'-d)imidazo le,p6 a supposed metabolite of the mutagenic amine 3-amino-4,6-dimethyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-3). It is found that Glu-P-3 residues are covalently bound to the C8 of guanine in acetylated form (70%) and deacetylated form (30%).

Mutagenicity of some derivatives of dipyrido[1,2-a:3',2'-d]imidazoles in Salmonella typhimurium with metabolic activation by rat liver and small intestine subcellular fractions.

The mutagenic effect of 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) was compared with that of the 3-amino, 3-nitro, or 3-N-hydroxylated derivatives of the same base ring with methyl groups at positions 4 and 6 of the molecule. The compounds were tested in Salmonella typhimurium strain TA98 without metabolic activation and in the presence of different concentrations of subcellular fractions from livers or small intestines of rats pretreated with different P448/P450 inducers. The 4,6-dimethyl compounds are always more mutagenic than Glu-P-2. Pretreatment with Aroclor 1254 (ARO) is the most effective inducer in the activation of the 2- and 3-amino compounds by liver S9, whereas the same fraction decreases the mutagenicity of the 3-nitro derivative. S9 from small intestine increased the mutagenic effect of the 3-nitro and 3-N-hydroxylated compounds, but it was unable to activate the amino compounds.

Reaction of DNA with a mutagenic 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido[1,2-a:3',2'- d]imidazole (N-AcO-AGlu-P-3) related to glutamic acid pyrolysates.

3-Amino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-3), an analog amine of the potent genotoxic Glu-P-1 isolated from a glutamic acid pyrolysate, has been chemically synthesized. Glu-P-3 was found much more mutagenic than Glu-P-1 to S. typhimurium TA 98 and TA 100 with S-9 mix. 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido[1,2-a:3',2'-d] imidazole (N-AcO-AGlu-P-3), a possible metabolite of Glu-P-3, binds covalently to the C-8 position of guanine residues in DNA. The binding induces large conformational changes of the macromolecule.

Conformational changes induced in DNA by the in vitro reaction with the mutagenic amine: 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido (1,2-a: 3', 2'-d) imidazole.

The conformation of synthetic or natural DNAs modified in vitro by covalent binding of N-AcO-A-Glu-P-3 was investigated by fluorescence and circular dichroism. In all cases, substitution occurs mainly on the C8 of guanine residues. In modified poly(dG-dC).poly(dG-dC) or poly(dA-dC).poly(dG-dT) in B conformation, A-Glu-P-3 residues interact strongly with the bases whereas in Z conformation these residues are largely exposed to the solvent and interact weakly with the bases. A-Glu-P-3 and N-acetyl-2-aminofluorene (AAF) residues are equally efficient to induce the B-Z transition of poly(dG-dC).poly(dG-dC) and of poly(dA-dC).poly(dG-dT). Modifications of poly(dG).poly(dC) and calf thymus DNA indicate strong interactions between A-Glu-P-3 and the bases.

C-alkylation of N-acetoxy-N-2-acetylaminofluorene: effects on its reaction with DNA in vitro.

In vitro reactions of DNA with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF), N-acetoxy-7-ethyl-N-2-acetylaminofluorene (N-AcO-EtAAF), N-acetoxy-7-n-butyl-N-2-acetylaminofluorene (N-AcO-But-AAF) are compared. C-alkylation of N-AcO-AAF affects the reactivity of the metabolite towards DNA. The electronic effect and the size of alkyl group seem to determine the reactivity of the metabolite. Although the adducts are about the same for the three metabolites, the proportion of guanine-C-8 adducts diminishes with an increase in the size of the alkyl group.

Mutagenicity of several derivatives of dipyrido[1,2-a:2',3'-d]imidazoles.

Different derivatives of dipyrido[1,2-a:2',3'-d]imidazoles have been investigated, as mutagens for Salmonella typhimurium. The nature of different substitution groups and their positions on the base ring influenced markedly the mutagenicity of these compounds. From this structure/effect relationship study, it was demonstrated that the 2 and 3 positions were of special interest. The 3-N-hydroxylated compound was the most active mutagen tested. We also observed that the frequently found frameshift mutagens were responsible for base-pair substitution. Metabolic activation by liver S9 mix increased the reversion rates of the strains tested. The SCE assays correlated poorly with the Salmonella/microsome mutagenicity test.

Biochemical effects of some derivatives of dipyrido[1,2-a:3,'2'-d]imidazole related to protein pyrolysates on rat liver microsomes.

Several derivatives of dipyrido[1,2-a:3',2'-d]imidazole related to protein pyrolysates have been studied for their effects on the P-450 system of hepatic parenchyma and two dependent monoxidase enzymes, zoxazolamine hydroxylase and dimethylnitrosamine-N-demethylase (DMN-d-ase). We found that the compounds can be divided into two groups. The first group consists of compounds which inhibit the production of cytochrome P-450 and zoxazolamine hydroxylase and induce the formation of DMN-d-ase; these compounds are known to be powerful mutagens. The compounds of the second group, non-mutagens or weak mutagens, have an opposite effect: they induce the biosynthesis of cytochrome P-450 and zoxazolamine hydroxylase and repress the production of DMN-d-ase. These findings are of interest since it is known that certain of the studied compounds can be found in human food.

Spectroscopic study of the effects of C-alkylation of N-acetoxy-N-acetyl-2-aminofluorene on some properties of the carcinogen-modified DNA.

Several physicochemical properties of DNA reacted in vitro with the carcinogen metabolite N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) are compared to those of DNA reacted with two C-alkylated derivatives, viz. N-acetoxy-7-ethyl-N-2-acetylaminofluorene (N-AcO-EtAAF) and N-acetoxy-7-n-butyl-N-2-acetylamino-fluorene (N-AcO-ButAAF). Ultraviolet absorption, high resolution derivative melting curves and circular dichroism techniques are employed in the present study. C-alkylation of N-AcO-AAF influences the conformational modifications of DNA induced by the covalent binding of the metabolite. The size of the alkyl group seems to determine the effect on DNA conformation (e.g., the bulkiest N-AcO-ButAAF denatures the most DNA). However, certain similarities between the three metabolites are observed, such as the preferential binding to G-C rich regions and the carcinogen-carcinogen and carcinogen-DNA base stacking interactions.

Reactivity of B and Z-DNA towards N-acetoxy-2-acetylaminofluorene.

Poly d(G-C) d(G-C) in B-form, on one hand, and poly d(G-br5C). poly d(G-br5C) and poly d(G-m5C) . poly d(G-m5C) in Z-form, on another hand, were treated with N-AcO-[3H]AAF and the kinetics of these reactions were followed by radioactivity. Covalent binding of carcinogen to the polymers was evaluated after separation of the reacted polymers from non-reacted carcinogen by thin-layer chromatography. We found that B-form polymer reacts twice faster than the Z-form polymers. Proportions of main adducts in the three polymers are almost the same. Results are discussed in relation to the calculated electrostatic potential minima and steric accessibility at the reactive site (1, 2).

In vitro reaction of the carcinogenic derivative N-acetoxy-7-ethyl-N-2-acetylaminofluorene with DNA. A spectroscopic study.

In vitro reaction of DNA with N-acetoxy-7-ethyl-N-acetylaminofluorene (N-AcO-EtAAF is compared to that with N-acetoxy-N-acetyl-2-aminofluorene. Introduction of an ethyl group at the position 7 of the fluorene ring is found to induce an increase of reactivity of this (model) metabolite of carcinogen N-acetoxy-2-aminofluorene (2-AAF) toward DNA and its constituents. Spectroscopic methods (u.v. absorption and circular dichroism) are used to characterize the DNA reacted with N-AcO-EtAAF (DNA-EtAAF). Our results point out some conformational similarities between native DNA-EtAAF and DNA-AAF such as the presence of destabilized regions around the bound carcinogen and of stacking interactions between these residues and the adjacent nucleotides. Quite important differences seem to exist between adducts or modified sequences in native as compared to denatured DNA (contrary to DNA-AAF and DNA bearing covalently bound-2-aminofluorene.

Genetic effects of chlorinated anilines and azobenzenes on Salmonella typhimurium.

The mutagenicity of 19 herbicide-derived chlorinated azobenzenes and structurally related chlorinated anilines and nitrobenzenes was assayed towards several strains of S. typhimurium, using the plate incorporation method and the fluctuation test, in the presence or in the absence of liver post-mitochondrial fractions, in aerobic and anerobic conditions. Positive results were obtained with 4,4'-dichloroazobenzene, 4,4'-dichloroazoxybene, 3,4-dichloronitrobenzene and, to a much lesser extent, with 3,4,3',4'-tetrachloroazobenzene. No mutagenic effect was observed with 2,3,7,8-tetrachlorodibenzo-p-dioxin in any condition.

Antibodies to N-hydroxy-2-aminofluorene modified DNA as probes in the study of DNA reacted with derivatives of 2-acetylaminofluorene.

Antibodies were elicited in rabbits immunized with a mixture of methylated bovine serum albumin and N-hydroxy-2-aminofluorene reacted DNA (DNA-AF). These antibodies were used in competition radioimmunoassays (RIA) with [3H]DNA-AF as radioactive antigen and different N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) and N-hydroxy-2aminofluorene (N-OH-AF) reacted DNAs, deoxyguanosine, dGMP, dGpA and dApG as inhibitors. Based on the results of RIA it is concluded that the binding sites of the two residues, -AF and -AAF, to guanosine in DNA, are essentially the same. Moreover, the same sites seem to be reactive also in the direct reaction of N-AcO-AAF with dGMP. Circular dichroism measurements of modified DNA confirm the RIA results.

Alkaline stability of guanosine and some of its derivatives modified by the carcinogen N-acetoxyacetylaminofluorene.

The alkaline treatment of Guo, dGuo, dGMP and denatured DNA modified by N-acetoxyacetylaminofluorene (N-AcO-AAF) was performed in 0.1 M NaOH at 40 degrees C. The kinetics of the reaction were followed by ultraviolet absorption and by chromatographic methods and were found different for the four products under study. Circular dichroism spectra show differences in the environment of acetylaminofluorene residue in these products. The alkaline treatment of Guo-AAF (and dGuo-AAF) leads to the formation of three products. These products were separated by thin layer chromatography and by HPLC and were characterized by spectroscopic methods. One is the already known unstable Guo-AF (and respectively dGuo-AF) (1). The other two products are relatively stable products of the transformation of Guo-AF (or dGuo-AF). These last ones present almost identical ultraviolet absorption spectra, but very different circular dichroism spectra.

Conformational changes induced in DNA by in vitro reaction with N-hydroxy-N-2-aminofluorene.

The conformation of DNA modified in vitro by the covalent binding of N-OH-AF was investigated by ultraviolet absorbance, circular dichroism and by radioimmunoassay using specific antibodies against Guo-AAF and nDNA-AAF. The results obtained by both physico-chemical and immunological methods are in agreement with a model involving destabilized regions in the double helical DNA around the carcinogen molecule in which, however, the -AF residues are stacked to the adjacent nucleotides. The RIA results show that the -AF residues are less accessible to antibodies in native than in denatured DNA-AF and thus suggest -AF residues partially buried in the interior of the DNA helix. The present model is compared to the one existing for DNA modified by reaction with N-AcO-AAF (DNA-AAF) (1,2).