A 32P-postlabelling assay for the detection of alkylphosphotriesters in DNA.

Within the group of DNA alkylation products, phosphotriesters (PTE) are among the most stable lesions. Hence, alkyl PTE are attractive biomarkers for DNA alkylation monitoring purposes. We have developed a 32P-postlabelling method for the analysis of both methyl and ethyl PTE in DNA. Since PTE bonds are not cleaved by any known DNA degrading enzyme, they are easily obtainable as PTE dinucleoside monophospates. A purification step, separating the PTE dinucleoside monophosphates from interfering compounds, such as mono- or oligonucleotides resulting from incomplete digestion of DNA, was developed using Waters C18 Sep-Pak cartridges. Phosphotriester dinucleoside monophosphates themselves are not a substrate for phosphorylation by polynucleotide kinase. Polynucleotide kinase probably requires a negative charge on the phosphate closest to the 5'-end. Therefore, prior to the post-labelling step they have to be converted into either phosphodiester dinucleoside monophosphates or 3'-phosphate alkylated mononucleotides by treatment with alkali. For analysis of the labelled compounds we developed a two-step procedure, combining TLC and HPLC, that gave very straightforward information on the composition of the rather complex mixture. The detection limit is approximately fmol PTE.

Chemical properties of the ultimate metabolites of 2-amino-5-phenylpyridine (PHE-P-1) and its ortho-methyl derivative.

The reactivity of the N-acetoxy metabolite of 2-amino-5-phenylpyridine (Phe-P-1), a pyrolysis product of phenylalanine, towards 2'-deoxyguanosine (dG), 2'-deoxyguanosine-3'-monophosphate (dGMP) and DNA was studied and compared with that of the ortho-methyl derivative. Reaction of 2-acetoxyamino-5-phenylpyridine (N-OAc-APP) with dG resulted in substitution at the 8-position of this nucleoside by the ortho carbon of the amine. The major reaction, however, was acetylation of dG. In contrast, 2-acetoxyamino-3-methyl-5-phenyl-pyridine (N-OAc-MeAPP) mainly attacked the 8-position of dG by the exocyclic nitrogen and hardly any acetylation of the nucleoside occurred. The adducts were chromatographically isolated and characterized by their mass and NMR spectra. Upon reaction of N-acetoxy compounds with DNA and dGMP, formation of the same adducts was observed, besides the formation of minor amounts of unidentified compounds, as was established by 32P-postlabelling analysis. The amount of DNA-bound amine, formed by the interaction of N-OAc-APP with DNA, was approximately 15 times smaller than that observed after the reaction with the corresponding ortho-methyl derivative under the same conditions.

Method for the analysis of oxidized nucleosides by gas chromatography/mass spectrometry.

A method for sensitive analysis of the oxidatively modified nucleosides 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU) is described. The method combines acetylation and pentafluorobenzylation of the nucleosides followed by analysis by gas chromatography/electron capture negative ion chemical ionization-mass spectrometry. The detection limits of the method for aqueous standards of HMdU and 8-OHdG were 12 and 18 fmol of starting material (signal-to-noise ratio, 3:1), respectively. The method was linear for 8-hydroxy-2'-deoxyguanosine over five orders of magnitude and gave satisfactory reproducibilities (intraassay RSD < or = 5%) for the analysis of 8-OHdG in both aqueous standards and urine fortified at the level of 35 nM. The limit of detection for the analysis of 8-OHdG in urine was 1.8 pmol, corresponding to a level of 8-OHdG in urine of 35 nM (10 micrograms/liter) at a urine sample volume of 50 microliters. Besides urine the method was applied to the analysis of 5-hydroxy-methyl-2'-deoxyuridine isolated from genomic DNA of Bacillus subtilis bacteriophage H1. Results obtained indicate that the method is potentially suitable for the determination of oxidized nucleosides in biological samples. The selectivity of the method should be enhanced in order to lower the limit of detection in biological samples.

32P-postlabelling analysis of alkylphosphotriesters in DNA.

We have developed a highly sensitive 32P-postlabelling assay for the detection of alkylphosphotriesters in DNA using selective enzymatic DNA hydrolysis, sample preparation by C18 Sep-Pak solid phase extraction and product analysis by HPLC. The assay provides information on the quantity of the alkylphosphotriesters and gives some clues with respect to their identity.

Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

Detection of 8-hydroxyguanine in small amounts of DNA by 32P postlabeling.

A method for the sensitive detection of 8-hydroxyguanine residues in small amounts of DNA (0.2-2 micrograms) was developed. It comprises (i) the enzymatic hydrolysis of DNA to 2'-deoxyribonucleotide 3'-monophosphates, (ii) degradation of the bulk amount of normal purine and pyrimidine deoxyribonucleotides in the DNA digest by treatment with trifluoroacetic acid and hydrazine, respectively, under conditions retaining the structure of d(8-OH-G)p necessary for 5' phosphorylation by T4 polynucleotide kinase (PNK), (iii) 5' phosphorylation of d(8-OH-G)p by T4 PNK-catalyzed transfer of 32P from [gamma-32P]ATP, and (iv) 2D thin-layer chromatography on polyethyleneimine-cellulose sheets to purify and resolve 32P-postlabeled d(8-OH-G)p. Model experiments with mixtures composed of synthesized d(8-OH-G)p and DNA hydrolysate indicate that it is possible to detect one 8-hydroxyguanine residue out of 2 x 10(6) normal bases starting with 1 microgram DNA. The methodology, which allows for a further decrease of this detection limit, might be very useful for the sensitive detection of DNA damage induced by activated oxygen species in small amounts of DNA. We demonstrate the formation of 8-OH-G in DNA in vitro by low doses of 60Co gamma-rays.

A circular dichroism study on the conformation of d(CGT) modified with N-acetyl-2-aminofluorene or 2-aminofluorene.

The trinucleotide d(CGT) was modified by covalent binding of the carcinogen N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) at the C8 position of the guanine base. The conformations of d(CGT)-AAF and -AF were studied by comparing the absorption and circular dichroism properties with those of dCMP + dGMP-AAF or -AF + dTMP in a molar ratio of 1:1:1 and AAF- and AF-containing dGMP. For both AAF- and AF-d(CGT) complexes the results show significant stacking interactions between the fluorene residue and the base(s) and are discussed in terms of the conformation of d(CGT)-AAF and -AF. In d(CGT)-AF we observe a clear interaction between AF and thymine, whereas the C-G stack is still intact. In the case of d(CGT)-AAF the C-G stack is weakened and the glycosidic rotation angle of dGuo-C8-AAF is most probably syn. The specific fluorene-base interactions persist at elevated temperatures. The carcinogen-base interactions are stronger in the AAF-carrying d(CGT) than in the case of the deacetylated complex. This is consistent with the higher mobility of the AF-adduct and its conformationally heterogeneous appearance in DNA.

Solid-phase synthesis of DNA fragments containing the modified base 7-hydro-8-oxo-2'-deoxyguanosine.

The 5'-(4,4'-dimethoxytrityl) protected 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidite of 7-hydro-8-oxo-2'-deoxy-guanosine, the exocyclic amino and lactam functions of which are protected with acetyl and diphenylcarbamoyl groups, respectively, has been prepared from the 8-bromo derivatives of deoxy- and riboguanosine. This synthon, in combination with standard d-nucleoside 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidites, was applied successfully to a solid-phase synthesis. Well-defined oligodeoxyribonucleotides containing a 7-hydro-8-oxo-2'-deoxyguanosine residue at predetermined positions were obtained after deprotection with methanolic ammonia and purification by gel filtration.

Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O4-ethylthymine adducts in HeLa cells.

We developed a simian virus 40 based shuttle vector system to study the molecular consequences of distinct carcinogen-induced DNA lesions in human cells. To establish the mutagenicity of O4-ethylthymine adducts, oligonucleotides carrying a single O4-ethylthymine adduct at a unique position were ligated into the vector molecules. Following replication in HeLa cells on average 23% of the progeny molecules carried a mutation in the region of modification. The vast majority of these mutations represented single T----C transitions at the position of the modified base, most probably as a consequence of mispairing of the O4-ethylthymine residues during replication. To a minor extent the O4-ethylthymine adduct may also induce T----A transversions or double point mutations. The in vivo mutation frequency of the adduct was found to be comparable to that of a C-A mismatch at the same position, but was lower than that expected from in vitro experiments with adducted DNA templates and purified DNA polymerases.

The fluorescence emission properties of dGuo-C8-AAF, dGuo-C8-AF and the imidazole ring-opened products of dGuo-C8-AF indicate a different dynamic structure for the various compounds.

The reactive metabolites of the carcinogenic N-acetyl-2-aminofluorene (AAF) form adducts with the guanine base of DNA. The fluorescence emission characteristics of N-(deoxy-guanosin-8-yl)-N-acetyl-2-aminofluorene (dGuo-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dGuo-C8-AF) and the two N7 = C8 imidazole ring-opened products of dGuo-C8-AF (ro-dGuo-C8-AF I + II) were investigated and related to their conformational properties. The dGuo-C8-AF adduct (phi F congruent to 4-5 X 10(-4) shows a broad and structureless emission band, which is attributed to the formation of an excited-state complex. In contrast, the emission spectra of dGuo-C8-AAF (phi F congruent to 1.10(-4] and both ro-dGuo-C8-AF compounds [phi F(ro-dGuo-C8-AF I) congruent to 4.10(-3); phi F(ro-dGuo-C8-AF II) congruent to 4.10(-4)] are narrow. This indicates that dGuo-C8-AAF and ro-dGuo-C8-AF I + II do not decay into an exciplex as occurs in dGuo-C8-AF. The spectroscopic features are discussed in terms of the differences in the dynamic structure of the various compounds.

Synthesis of heterocyclic N-acetoxyarylamines and their reactivity with DNA.

2-Acetoxyamino-5-phenylpyridine and 2-acetoxyamino-3-methyl-5-phenylpyridine, being proposed ultimate carcinogens of the heterocyclic aromatic amines 2-amino-5-phenylpyridine (APP) and 2-amino-3-methyl-5-phenylpyridine (AMPP), respectively, were synthesized, crystallized and characterized. Using the 32P-postlabelling technique, we show that the total amount of adducts found in DNA after reaction with these N-acetoxyarylamines is at least 30- and 450-fold higher than in DNA reacted with equimolar amounts of the proposed proximate carcinogens 2-hydroxyamino-5-phenylpyridine and 2-hydroxyamino-3-methyl-5-phenylpyridine, respectively. These results support a postulated activation mechanism, in which N-acetoxyarylamines are the ultimate reactive species responsible for DNA modification by carcinogenic aromatic amines in vivo. The possibility to obtain the reactive 0-acetyl derivatives of APP and AMPP in crystalline form makes them unique model compounds for studies on the interaction of ultimate carcinogens of aromatic amines with DNA.

Excited state properties of the N-(deoxyguanosin-8-yl)-2-aminofluorene adducts.

The spectroscopic characteristics of adducts derived from the covalent binding of the carcinogen 2-aminofluorene to the C8 position of deoxyguanosine [N-(deoxyguanosin-8-yl)-2-amino-fluorene, dGuo-C8-AF], and from an adduct of similar structure formed with the synthetic polynucleotide poly(dG-dC).poly(dG-dC), were investigated. At 77 K both adducts are characterized by well-defined and rather narrow fluorescence emission spectra with maxima at 370 and 390 nm characteristic of the aromatic, monomolecular 2-aminofluorene (AF) residue. In contrast, at room temperature, the fluorescence is characterized by a broad, structureless emission band with a maximum at 460 nm in aqueous mixtures, shifting to 415 nm in solvents of lower polarity (100% propanol); the maxima are located at intermediate wavelengths in solutions of different propanol/water compositions, and this emission is attributed to an excited state complex (exciplex). The fluorescence quantum yield decreases when either the solvent polarity or the temperature are increased, varying from 5.4% (100% propanol) to 0.04-0.05% (100% H2O). The fluorescence decay profiles of dGuo-C8-AF adducts (measured at the National Synchrotron Light Source facility at the Brookhaven National Laboratory) can be roughly, but not exactly, modeled in terms of two exponential decay components in the range of about 0.3-1.0 ns with the propanol concentration greater than 60%; at lower propanol concentrations, a single short lifetime is observed and in 100% water solutions its value is 0.08 ns. The shorter lifetime, favored in solvent mixtures of higher polarities, is attributed to an exciplex with significant charge-transfer character.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative carcinogenicity of the food pyrolysis product, 2-amino-5-phenylpyridine, and the known human carcinogen, 4-aminobiphenyl, in the neonatal B6C3F1 mouse.

2-Amino-5-phenylpyridine (2-APP) is a mutagenic heterocyclic aromatic amine that is formed by pyrolysis of phenylalanine in proteins. Since this mutagen is structurally similar to the multipotent carcinogen, 4-aminobiphenyl (4-ABP), we compared their relative tumorigenic activity in the neonatal male B6C3F1 mouse. After determinations of the maximum tolerated dose (MTD), both aromatic amines were administered i.p. at 2 dose levels (MTD and MTD/2) on days 1, 8, 15 and 22 after birth. Groups were killed at 9 and 12 months and examined for histopathologic changes. No treatment-related neoplastic lesions were observed for 2-APP. In contrast, 4-ABP was strongly carcinogenic and induced a high incidence of multiple hepatocellular adenomas and carcinomas.

A spectroscopic study of the conformation of poly d(G-C).poly d(G-C) modified with the carcinogenic 2-aminofluorene.

The conformational properties of both the B- and Z-form helix of the 2-aminofluorene (AF) modified synthetic polynucleotide poly d(G-C).poly d(G-C) were extensively studied with a variety of optical techniques (UV absorbance, linear dichroism, circular dichroism and fluorescence spectroscopy). The spectroscopic results show, that: (i) the presence of the AF adduct in poly d(G-C).poly d(G-C) facilitates the conversion of the right-handed B-form polynucleotide to the left-handed Z-type poly d(G-C).poly d(G-C) duplex; (ii) in B- as well as in Z-form poly d(G-C).poly d(G-C)-AF the carcinogenic residue is situated in a specific and ordered complex with the DNA, in which the AF chromophore is significantly immobilized and the long axis of the fluorene ring system is positioned in a parallel mode to the DNA base planes; (iii) in B-type poly d(G-C).poly d(G-C)-AF the carcinogenic residue exhibits strong stacking interactions with the adjacent DNA bases, which are coupled to an important destabilization of the AF-containing B-duplex; (iv) in contrast to the B-form complex, the AF-containing Z-type DNA is highly stabilized and a remarkable reduction of the AF-base interactions is observed; (v) in Z-form poly d(G-C).poly d(G-C)-AF the AF chromophore resides at a medium-exposed position. The combined data support a conformational model, in which the planar AF is inserted in the B-type polynucleotide helix, while the carcinogenic residue in the Z-form poly d(G-C).poly d(G-C)-AF structure is placed in an outside position.

An optical study of the conformation of the aminofluorene-DNA complex.

Calf thymus DNA was modified with 2-aminofluorene (AF) to different extents by treatment with N-hydroxy-2-aminofluorene. The AF-modified DNAs together with free AF, the AF-modified guanine (Gua-C8-AF) and the AF-modified deoxyguanosine (dGuo-C8-AF) were subsequently studied by u.v. absorbance, linear dichroism and fluorescence spectroscopy. The emission and absorption properties of double-stranded DNA-AF and single-stranded DNA-AF closely resemble those of dGuo-C8-AF. The emission spectra of these three compounds show a broad, red-shifted emission, characteristic for exciplex formation. The linear dichroism and circular dichroism spectra of double-stranded DNA-AF show that the AF moiety forms a well-defined, regular structure. The dichroic ratio in the 310-340 nm region is constant, which indicates the presence of only one type of adduct. The long-wavelength transition moment of this adduct makes an angle of 72-74 degrees with the DNA helix axis. The binding of AF to double-stranded is DNA is accompanied by a destabilization of the DNA helix structure, a strong quenching of the AF emission quantum yield, intense AF circular dichroism and an apparent immobilization of the dGuo-C8-AF complex. In single-stranded DNA-AF, the AF conformation appears more random, although the interactions between AF and the surrounding bases persist. The strong interactions between AF and the surrounding bases which dominate the optical properties of the studied complexes, the significant destabilization of the DNA double helix after modification with AF, and the relatively small angle between AF and the base planes support a model in which the adduct is inserted into the DNA helix.

The biological activity of single-stranded phi X174 DNA, modified by N-hydroxy-2-aminofluorene, is inhibited by guanine imidazole ring-opening of the major, non-lethal aminofluorene-DNA adduct.

The major aminofluorene-DNA derivative, found in the liver of rats after administration of the hepatocarcinogen N-acetyl-2-aminofluorene and identified as N-(deoxyguanosin-8-yl)-2-aminofluorene (dGuo-C8-AF), was introduced in different amounts in single-stranded phi X174 DNA by reacting the DNA with tritium labeled N-hydroxy-2-aminofluorene. The modified DNA was subsequently incubated in 0.1 M NaOH at 37 degrees C for increasing periods of time to convert the dGuo-C8-AF residues into their guanine imidazole ring-opened forms. The degree of conversion was determined by measuring the amount of residual N-(guanin-8-yl)-2-aminofluorene in trifluoroacetic acid hydrolyzates of the alkali-treated DNA by h.p.l.c. In addition, the effect of ring opening on the biological activity of the DNA was monitored by transfecting the DNA to Escherichia coli wild-type spheroplasts. The results indicate that the major aminofluorene-DNA adduct formed initially, which contributes little to inactivation, becomes lethal when its guanine imidazole ring is opened.

By-pass of the major aminofluorene-DNA adduct during in vivo replication of single- and double-stranded phi X174 DNA treated with N-hydroxy-2-aminofluorene.

To examine the effects of aminofluorene-DNA adduct formation on the biological activity of DNA, single-stranded (ss) phi X174 DNA and phi X174 replicative form (RF) DNA were modified to different extents with 3H-labeled N-hydroxy-2-aminofluorene and subsequently transfected to Escherichia coli spheroplasts with different repair capabilities. When the fraction of active ss phi X174 DNA molecules was measured as a function of the mean number of adducts per molecule, exponential survival curves were obtained from which it could be deduced that in wild-type, uvrA- and recA- cells at least 86%, and in uvrC- cells at least 82% of the introduced adducts do not cause inactivation. In the case of RF DNA the survival curves are non-exponential, but they nevertheless show that an exceptionally high number of adducts per RF molecule must be introduced to destroy its biological activity. On average 52 adducts per RF molecule were needed to reduce the survival to 37%, irrespective of whether wild-type, uvrA- or recA- cells were used. On the other hand, the survival of the uvrC- cells was considerably lower, but even in these cells a majority of the adducts is not lethal. By h.p.l.c. analysis of the modified DNA after hydrolysis with trifluoroacetic acid, 81 and 84% of the adducts in ss- and RF DNA, respectively, could be identified as N-(guanin-8-yl)-2-aminofluorene. The results strongly indicate that this type of major modification product is very frequently by-passed during replication of both single- and double-stranded DNA. The results together with the data obtained by sucrose gradient analysis both before and after an alkali treatment and those obtained by h.p.l.c. analysis suggest that inactivation of ssDNA is mainly due to minor modifications such as secondary lesions consisting of chain breaks and alkali-labile sites together with unidentified interaction products.

DNA adducts formed from the probable proximate carcinogen, N-hydroxy-3,2' -dimethyl-4-aminobiphenyl, by acid catalysis or S-acetyl coenzyme A-dependent enzymatic esterification.

The arylamine carcinogen 3,2'-dimethyl-4-aminobiphenyl (DMABP) has been proposed to be metabolically activated to DNA-binding derivatives through the formation of an N-hydroxy intermediate. In this study, the subsequent activation of N-hydroxy-DMABP through acid catalysis or enzymatic esterification was examined. [Ring-3H]N-hydroxy-DMABP was reacted with calf thymus DNA at pH 4.6 for 15 min to yield 370 arylamine residues per 10(6) nucleotides, while at pH 7.4 the binding was only two residues per 10(6) nucleotides. The DNA modified under acidic conditions was enzymatically hydrolyzed and analyzed by h.p.l.c. which indicated the presence of three major adducts. The products were identified by spectral and chemical properties as N-(deoxyguanosin-8-yl)-DMABP (60-70%), 5-(deoxyguanosin-N2-yl)-DMABP (2-3%) and N-(deoxyadenosin-8-yl)-DMABP (1-3%). The same adducts have previously been detected in the liver and colon of rats administered DMABP or its hydroxamic acid. Incubation of rat hepatic or intestinal cytosol at pH 7.4 for 15 min with [ring-3H]N-hydroxy-DMABP in the presence of S-acetyl coenzyme A (AcCoA) and calf thymus DNA resulted in DNA binding at levels of 30-80 arylamine residues per 10(6) nucleotides. H.p.l.c. analysis of the DNA modified in the presence of AcCoA indicated the formation of the same adducts detected in the acid-catalyzed reactions. When arylhydroxamic acid N,O-acyltransferase assays were conducted with rat liver cytosol and N-acetyl-N-hydroxy-DMABP as the substrate, binding to nucleic acids was not observed. Similarly, 3'-phosphoadenosine-5'-phosphosulfate-dependent sulfotransferase-mediated DNA binding could not be demonstrated. These data indicate that in a suitable acidic environment, N-hydroxy-DMABP will react with DNA to yield the same adducts found in vivo. Under neutral conditions, however, N-hydroxy-DMABP appears to undergo AcCoA-dependent transacetylation to an electrophilic acetoxy ester which will spontaneously react with DNA.

Syntheses of 5-phenyl-2-pyridinamine, a possibly carcinogenic pyrolysis product of phenylalanine, and some of its putative metabolites.

5-Phenyl-2-pyridinamine (PPA) is a pyrolysis product of phenylalanine, the presence of which has been demonstrated in broiled sardines. Since PPA is mutagenic in the Ames test and is structurally related to the aminobiphenyls, it has to be considered as potentially carcinogenic. In this study procedures for the synthesis of PPA and its possible metabolites were developed to make them available for biological studies. PPA was synthesized in one step from 2,5-pyridinediamine. However, this method is only suitable for the preparation of small amounts. Larger quantities were synthesized starting from 5-nitro-2-pyridinamine in four steps. PPA was also prepared via a six-step synthesis, starting from 6-amino-3-pyridinecarboxamide. This route was also used for the synthesis of tritiated PPA [( 3H]PPA) and 2-nitro-5-phenylpyridine, the latter being the precursor of the two putative proximate carcinogenic metabolites, viz. the hydroxylamine and the hydroxamic acid of PPA. In the course of these multi-step syntheses a new method for the preparation of unsymmetrical biaryls was worked out. The following possible metabolites were also synthesized: N-(5-phenyl-2-pyridinyl)acetamide in the course of the synthesis of PPA starting from 5-nitro-2-pyridinamine, and both 5-(4-hydroxyphenyl)-2-pyridinamine (4'-OH-PPA) and N-[5-(4-hydroxyphenyl)-2-pyridinyl]acetamide starting from 5-(4-aminophenyl)-2-pyridinamine. After incubation of PPA in suspensions of freshly isolated hepatocytes from rats pretreated with polychlorinated biphenyls (Aroclor 1254) or PPA itself, the presence of 4'-OH-PPA was demonstrated.

Formation of DNA adducts in vivo in rat liver and intestinal epithelium after administration of the carcinogen 3,2'-dimethyl-4-aminobiphenyl and its hydroxamic acid.

Administration of the 3H-labeled colon carcinogen, 3,2'-dimethyl-4-aminobiphenyl (DMABP) and its hydroxamic acid derivative, N-hydroxy-N-acetyl-DMABP, to male F344 rats resulted in high levels of covalent binding to hepatic and intestinal DNA, RNA and protein. For both compounds, binding to hepatic macromolecules was 2-4 times higher than in the intestine. High pressure liquid chromatographic analysis of the enzymatically hydrolyzed DNA from liver and intestinal epithelium indicated the presence of two carcinogen-DNA adducts: 5-(deoxyguanosin-N2-yl)-DMABP (15%), N-(deoxyguanosin-8-yl)-DMABP (50%), and a decomposition product of the latter (15%). N-acetylated adducts were not detected. When measured after 7 days, all adducts in the intestinal DNA had decreased by 70%, while only a 29% decrease had occurred in the hepatic DNA. To determine if the loss of DMABP products was a consequence of cell turnover or repair, rats were treated with [3H]thymidine and DMABP, and the specific activity of hepatic liver and intestinal DNA was measured. Between 1 and 7 days only a slight decrease in [3H]thymidine content occurred in hepatic DNA as compared with a 95% reduction in intestinal DNA. Thus, the higher rate of DNA synthesis in the intestine versus that in the liver may serve to promote fixation of the initiating lesion and account for the preferential induction of intestinal cancer by DMABP. Furthermore, comparison of these data with metabolic activation pathways reported earlier strongly suggest that N-hydroxy-DMABP is the proximate carcinogenic metabolite of both DMABP and N-hydroxy-N-acetyl-DMABP.